Upload
lyque
View
238
Download
2
Embed Size (px)
Citation preview
Turkiyersquode Mikrobiyota CalısmalarındaKullanılan Laboratuvar Yontemleri
Meltem Yalınay MD PhD
Gazi Uumlniversitesi Tıp Fakuumlltesi
Tıbbi Mikrobiyoloji AD
10122016
Mikrobiyota Analiz Youmlntemleri
Mikroskobi
Kuumlltuumlr
Kuumlltuumlre Dayalı Molekuumller Youmlntemler
Kuumlltuumlr Bağımsız Molekuumller Youmlntemler
Dizi Analizi
Yeni Nesil DizilemeBiyoinformatik
Analiz
Mikrobiyota Analiz Youmlntemleri-I
GaitaDNA
İzolasyonu
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Mikrobiyota Analiz Youmlntemleri
Molekuler Yontemler
Kuumlltuumlre Dayalı Molekuumller Youmlntemler
Kuumlltuumlr Bağımsız Molekuumller Youmlntemler
Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri
FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme
Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj
qPZR
Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık
PZR bias Bilinmeyen tuumlrlere
uygulanamaz Tek hedef
DGGETGGE
bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin
kullanımına olanak sağlar
Filogenetiktanımlama yapamaz
PZR bias
T-RFLP Hızlı Yarı kantitatif Maliyet etkin
Filogenetiktanımlama yapamaz
PZR bias
16S rRNA Filogenetik tanımlama Kantitatif
PZR bias Zahmetli Pahalı Klonlama bias
Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj
Pirodizileme
(454 GS FLX+ Roche)
Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla
oumlrneği analiz etme imkanı Koloni biası yoktur
Yuumlksek maliyet Homopolimerlerde
yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik
analize ihtiyaccedil duyar
Geri doumlnuumlşuumlmluuml terminatoumlr
dizileme
(HiSeq20002500 Illumina)
Etkili Okuma suumlrelerini iyileştiren
suumlreklilik Yuumlksek verimlilik Manuel iş azlığı
Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme
Ligasyon
(5500xl SOLiD Life
Technologies)
Duumlşuumlk hata oranı Yuumlksek verimlilik
Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi
Gerccedilek zamanlı sekans (PacBio
RS Pacific Biosceince)
Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu
Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota Analiz Youmlntemleri
Mikroskobi
Kuumlltuumlr
Kuumlltuumlre Dayalı Molekuumller Youmlntemler
Kuumlltuumlr Bağımsız Molekuumller Youmlntemler
Dizi Analizi
Yeni Nesil DizilemeBiyoinformatik
Analiz
Mikrobiyota Analiz Youmlntemleri-I
GaitaDNA
İzolasyonu
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Mikrobiyota Analiz Youmlntemleri
Molekuler Yontemler
Kuumlltuumlre Dayalı Molekuumller Youmlntemler
Kuumlltuumlr Bağımsız Molekuumller Youmlntemler
Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri
FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme
Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj
qPZR
Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık
PZR bias Bilinmeyen tuumlrlere
uygulanamaz Tek hedef
DGGETGGE
bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin
kullanımına olanak sağlar
Filogenetiktanımlama yapamaz
PZR bias
T-RFLP Hızlı Yarı kantitatif Maliyet etkin
Filogenetiktanımlama yapamaz
PZR bias
16S rRNA Filogenetik tanımlama Kantitatif
PZR bias Zahmetli Pahalı Klonlama bias
Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj
Pirodizileme
(454 GS FLX+ Roche)
Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla
oumlrneği analiz etme imkanı Koloni biası yoktur
Yuumlksek maliyet Homopolimerlerde
yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik
analize ihtiyaccedil duyar
Geri doumlnuumlşuumlmluuml terminatoumlr
dizileme
(HiSeq20002500 Illumina)
Etkili Okuma suumlrelerini iyileştiren
suumlreklilik Yuumlksek verimlilik Manuel iş azlığı
Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme
Ligasyon
(5500xl SOLiD Life
Technologies)
Duumlşuumlk hata oranı Yuumlksek verimlilik
Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi
Gerccedilek zamanlı sekans (PacBio
RS Pacific Biosceince)
Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu
Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota Analiz Youmlntemleri-I
GaitaDNA
İzolasyonu
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Mikrobiyota Analiz Youmlntemleri
Molekuler Yontemler
Kuumlltuumlre Dayalı Molekuumller Youmlntemler
Kuumlltuumlr Bağımsız Molekuumller Youmlntemler
Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri
FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme
Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj
qPZR
Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık
PZR bias Bilinmeyen tuumlrlere
uygulanamaz Tek hedef
DGGETGGE
bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin
kullanımına olanak sağlar
Filogenetiktanımlama yapamaz
PZR bias
T-RFLP Hızlı Yarı kantitatif Maliyet etkin
Filogenetiktanımlama yapamaz
PZR bias
16S rRNA Filogenetik tanımlama Kantitatif
PZR bias Zahmetli Pahalı Klonlama bias
Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj
Pirodizileme
(454 GS FLX+ Roche)
Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla
oumlrneği analiz etme imkanı Koloni biası yoktur
Yuumlksek maliyet Homopolimerlerde
yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik
analize ihtiyaccedil duyar
Geri doumlnuumlşuumlmluuml terminatoumlr
dizileme
(HiSeq20002500 Illumina)
Etkili Okuma suumlrelerini iyileştiren
suumlreklilik Yuumlksek verimlilik Manuel iş azlığı
Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme
Ligasyon
(5500xl SOLiD Life
Technologies)
Duumlşuumlk hata oranı Yuumlksek verimlilik
Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi
Gerccedilek zamanlı sekans (PacBio
RS Pacific Biosceince)
Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu
Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota Analiz Youmlntemleri
Molekuler Yontemler
Kuumlltuumlre Dayalı Molekuumller Youmlntemler
Kuumlltuumlr Bağımsız Molekuumller Youmlntemler
Fenotipik Parmak İzi Analiz YoumlntemleriGenotipik Parmak Analizi Youmlntemleri
FISHKantitatif Dot BlotRAPDPZR-DGGE PZR-TGGET-RFLPAPMikroarray16S rRNArecA Gen AnaliziqPZRDNA dizilemeYeni nesil dizileme
Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj
qPZR
Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık
PZR bias Bilinmeyen tuumlrlere
uygulanamaz Tek hedef
DGGETGGE
bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin
kullanımına olanak sağlar
Filogenetiktanımlama yapamaz
PZR bias
T-RFLP Hızlı Yarı kantitatif Maliyet etkin
Filogenetiktanımlama yapamaz
PZR bias
16S rRNA Filogenetik tanımlama Kantitatif
PZR bias Zahmetli Pahalı Klonlama bias
Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj
Pirodizileme
(454 GS FLX+ Roche)
Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla
oumlrneği analiz etme imkanı Koloni biası yoktur
Yuumlksek maliyet Homopolimerlerde
yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik
analize ihtiyaccedil duyar
Geri doumlnuumlşuumlmluuml terminatoumlr
dizileme
(HiSeq20002500 Illumina)
Etkili Okuma suumlrelerini iyileştiren
suumlreklilik Yuumlksek verimlilik Manuel iş azlığı
Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme
Ligasyon
(5500xl SOLiD Life
Technologies)
Duumlşuumlk hata oranı Yuumlksek verimlilik
Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi
Gerccedilek zamanlı sekans (PacBio
RS Pacific Biosceince)
Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu
Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota Analiz YoumlntemleriYontem Avantaj Dezavantaj
qPZR
Filogenetik tanımlama Kantitatif Hızlı Yuumlksek duyarlılık
PZR bias Bilinmeyen tuumlrlere
uygulanamaz Tek hedef
DGGETGGE
bull Hızlı bull Yarı kantitatif bull Oumlrneklerin ileri testler iccedilin
kullanımına olanak sağlar
Filogenetiktanımlama yapamaz
PZR bias
T-RFLP Hızlı Yarı kantitatif Maliyet etkin
Filogenetiktanımlama yapamaz
PZR bias
16S rRNA Filogenetik tanımlama Kantitatif
PZR bias Zahmetli Pahalı Klonlama bias
Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj
Pirodizileme
(454 GS FLX+ Roche)
Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla
oumlrneği analiz etme imkanı Koloni biası yoktur
Yuumlksek maliyet Homopolimerlerde
yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik
analize ihtiyaccedil duyar
Geri doumlnuumlşuumlmluuml terminatoumlr
dizileme
(HiSeq20002500 Illumina)
Etkili Okuma suumlrelerini iyileştiren
suumlreklilik Yuumlksek verimlilik Manuel iş azlığı
Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme
Ligasyon
(5500xl SOLiD Life
Technologies)
Duumlşuumlk hata oranı Yuumlksek verimlilik
Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi
Gerccedilek zamanlı sekans (PacBio
RS Pacific Biosceince)
Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu
Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota Analiz YoumlntemleriYontem (Uumlretici firma) Avantaj Dezavantaj
Pirodizileme
(454 GS FLX+ Roche)
Okuma suumlresi uzundur Yuumlksek verimlilik Duyarlı Aynı zamanda birden fazla
oumlrneği analiz etme imkanı Koloni biası yoktur
Yuumlksek maliyet Homopolimerlerde
yuumlksek hata oranı Kısa sekans okuma Kapsamlı biyoinformatik
analize ihtiyaccedil duyar
Geri doumlnuumlşuumlmluuml terminatoumlr
dizileme
(HiSeq20002500 Illumina)
Etkili Okuma suumlrelerini iyileştiren
suumlreklilik Yuumlksek verimlilik Manuel iş azlığı
Uzun ccedilalışma suumlresi Kısa okuma uzunlukları Yuumlkseltme geliştirilme
Ligasyon
(5500xl SOLiD Life
Technologies)
Duumlşuumlk hata oranı Yuumlksek verimlilik
Ccedilok kısa uzunluklar Uzun ccedilalışma suumlresi
Gerccedilek zamanlı sekans (PacBio
RS Pacific Biosceince)
Oumlrnek hazırlaması kolay Reaktif maliyeti duumlşuumlk Ccedilok uzun okuma uzunluğu
Hata oranı yuumlksek Sistem maliyeti yuumlksek Sistem kurulumu zordur
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota Analiz Youmlntemleri
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Bioresour Technol 2015 Sep192735-40 doi 101016jbiortech201505086 Epub 2015 Jun 9Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinationsAydin S1 Shahi A2 Ozbayram EG2 Ince B3 Ince O2Author information
AbstractAs it is currently often not know how anaerobic bioreactors eg for biogas production react if the substrate is contaminated by toxic compounds like antibiotics This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions According to PCR-DGGE results the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure especially derived from Gram-negative bacteria through bioaugmentation to successful for antibiotic biodegradationCopyright copy 2015 Elsevier Ltd All rights reserved
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Int J Food Microbiol 2012 Feb 15153(3)428-35 doi 101016jijfoodmicro201112008 Epub 2011 Dec 13
Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucukKesmen Z1 Yetiman AE Gulluce A Kacmaz N Sagdic O Cetin B Adiguzel A Sahin F Yetim HAbstractIn this study the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage On the one hand the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples On the other hand rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region As a result of the PCR-DGGE analysis of all the samples total 8 different lactic acid bacteria were identified and Lactobacillus sakei Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb sakei Lactobacillus plantarum Lb curvatus Lactobacillus brevis Lactobacillus farciminisand Lactobacillus alimentarius However Leuconostoc and Weisella were also detected as minor genera Again Lactococcus piscium Weissella halotolerans Staphylococcus succinus and the comigratedStaphylococcus piscifermentansStaphylococcus condimentiStaphylococcus carnosus group were detected only with the culture-independent method while Lb plantarum Leuconostoc mesenteroidesand Leuconostoc citreum were identified only by using the culture-dependent method In the results it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Folia Microbiol (Praha) 2013 May58(3)201-10 doi 101007s12223-012-0199-1 Epub 2012 Oct 4
Culturable bacterial microbiota of Plagiodera versicolora (L) (Coleoptera Chrysomelidae) and virulence of the isolated strainsDemirci M1 Sevim E Demir İ Sevim AAuthor information
AbstractPlagiodera versicolora (Laicharting 1781) (Coleoptera Chrysomelidae) is an important forest pest which damages many trees such as willow poplar and hazelnut In order to find new microbes that can be utilized as a possible microbial control agent against this pest we investigated the culturablebacterial flora of it and tested the isolated bacteria against P versicolora larvae and adults We were able to isolate nine bacteria from larvae and adults The isolates were characterized using a combination of morphological biochemical and physiological methods Additionally we sequenced the partial sequence of the 16S rRNA gene to verify conventional identification results Based on characterization studies the isolates were identified as Staphylococcus sp Pv1 Rahnella sp Pv2 Rahnella sp Pv3 Rahnella sp Pv4 Rahnella sp Pv5 Pantoea agglomerans Pv6 Staphylococcus sp Pv7 Micrococcus luteus Pv8 and Rahnella sp Pv9 The highest insecticidal activity against larvae and adults was obtained from M luteus Pv8 with 50 and 40 mortalities within 10 days after treatment respectively Extracellular enzyme activity of the bacterial isolates such as amylase proteinase lipase cellulose and chitinase was also determined Consequently our results show that M luteus Pv8 might be a good candidate as a possible microbial control agent against P versicolora and were discussed with respect to biocontrol potential of the bacterial isolates
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
J Pediatr Gastroenterol Nutr 2013 Mar56(3)328-32 doi 101097MPG0b013e31827a964bBreast milk jaundice effect of bacteria present in breast milk and infant fecesTuzun F1 Kumral A Duman N Ozkan HAuthor information
AbstractOBJECTIVEBreast milk is an important source of bacteria in establishing the infantile intestinal microbiota that appear to influence the enterohepatic circulation of bilirubin The aim of the present study was to evaluate the effect of breast milks microbiological content on the development of breast milk jaundice (BMJ)METHODSA total number of 60 mother-infant pairs enrolled to the study Two groups were defined BMJ group (n=30) full-term otherwise healthy newborns who were considered BMJ control group (n=30) full-term healthy newborns without jaundice All newborns in the study were exclusively breast-fed The breast milk samples and the feces of infants were evaluated for content of selected bacterial populations (Bifidobacterium Lactobacillus Clostridium Staphylococcus and Streptococcus species) with real-time polymerase chain reactionRESULTSBifidobacterium bifidum content in the breast milk and B adolescentis B bifidum and B longum content in the fecal samples were higher in the control group than in the BMJ group The milk and fecal concentrations of B bifidum were significantly correlated The concentrations of breast milk B bifidum and fecal B bifidum B adolescentis and B longum were found to be negatively correlated with bilirubin levelsCONCLUSIONSOur results suggest that Bifidobacterium species in breast milk may protect against BMJ
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Mikrobiyota ccedilalışmalarında molekuumlleryoumlntemler
1 PCR (qPCR ile 16S rRNA kantitasyonu)
2 DNA Parmak izi analizleri
1 Denaturating gradient jel elektroforezi
2 SSCP
3 T-RFLP
3 Gen kuumltuumlphanesi oluşturulması ve 16S rRNA sekans
1 Amplifikasyon uumlruumlnlerinin plazmid aracılığı iletransformasyonu dizi analizi
2 Yeni Nesil Dizileme
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
OumlZEL TEŞEKKUumlR
CEREN ERDOĞDU
SERAP SUumlZUumlK
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Helicobacter pylori Tedavisinin Bağırsak Mikrobiyota Uumlzerine Etkisi
Serap Suzuk1 Meltem Yalınay2 Tarkan Karakan3
1 THSK Mikrobiyoloji referans Laboratuvarı DB2 GUumlTF Tıbbi Mikrobiyoloji Anabilim Dalı
3 GUumlTF Gastroenteroloji Bilim Dalı
3 KLİMUD Kongresi Antalya 19-22 Kasım 2015
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Ccedilalışmanın Amacı
bull Helicobacter pylori (HP) tedavisi alan goumlnuumllluumllerde antibiyotik kullanımının bağırsak mikrobiyotası uumlzerindeki etkilerini qPZRyoumlntemi ile goumlstermek
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Ccedilalışmanın Dizaynı
Etik Kurul Onayı Alınması
Goumlnuumllluumllerin Seccedililmesi
Antibiyotik kullanım oumlncesi gaita oumlrneğinin
alınması
10 guumlnluumlk PPI+TET+MET+BİZ
Tedavi bittikten 6 hafta sonra
Antibiyotik kullanım sonrası gaita oumlrneğinin
alınması
İki grup gaita oumlrneğinden qPZR ile seccedililen
bakterilerin kantitasyonunun
yapılması
İstatistiksel Analiz
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Materyal Metod
bull Bu ccedilalışmada kullanılan standart suşlar
ndash Bifidobacterium breve ATCC 15700
ndash Bacteroides fragilis ATCC 25285
ndash Lactobacillus acidophilus ATCC 4356
ndash Akkermansia muciniphila ATCC BAA-835
ndash Faecalibacterium prausnitzii ATCC 27766
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Materyal Metod
ATCC suşlarındankuumlltuumlr yapıldı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Gram Boyama
A260dalga boyunda 3 okumaDNA kopya sayısı hesaplandı
Standart Eğriler oluşturuldu (110
1100 11000 110000 1100000 diluumlsyonlar )
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Materyal Metod
39 Goumlnuumllluumlden Gaita Oumlrneği Alındı Tedaviden 6 Hafta Sonra
Goumlnuumllluumllerin 18rsquoinden Antibiyotik
Kullanım Sonrası Gaita Oumlrneği Alındı
200 mg gaita-80degC saklandı
200 mg gaita-80degC saklandı
Ekstraksiyon(QIAmp DNA StoolMiniKit Qiagen Almanya)
-20degC saklandı
Ekstraksiyon(QIAmp DNA StoolMini Kit Qiagen Almanya)
-20degC saklandı
ordmC
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
Flu
ore
scence
7
6
5
4
3
2
1
ordmC
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
dF
dT
10
08
06
04
02
00
Bif
Threshold
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Materyal Metod
bull Verilerin tuumlmuuml Microsoft Excele girilerek logbakteriyel miktarı hesaplandı
bull Veriler SPSS 220 paket programı ile analiz edildi
bull Antibiyotik kullanım oumlncesi ve antibiyotik kullanım sonrası karşılaştırmalar Mann-Whitney U Testi ile değerlendirildi
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Bulgular
Bakteriler
Antibiyotik Kullanım
Oumlncesi OrtalamaplusmnSS
(log10g)
Antibiyotik Kullanım
Sonrası OrtalamaplusmnSS
(log10g)
p
Bifidobacterium spp 781plusmn112 611plusmn274 0000
B fragilis 824plusmn119 569plusmn289 0000
Lactobacillus spp 739plusmn061 632plusmn174 0000
A mucinophilia 808plusmn060 610plusmn086 0000
F prausnitzii 921plusmn209 632plusmn159 0000
SS Standart Sapma
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Nonalkolik Karaciğer Yağlanması Olan Hastalar ve Sağlıklı Kontrollerde
Bağırsak Mikrobiyotasının MolekuumllerYoumlntemlerle Karşılaştırılması
Ceren ErdoğduDanışman ProfDr Meltem Yalınay
9 Eyluumll 2016Ankara
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
İntestinal mikrobiyota ve NAFLD ilişkisi
Lİ et al 2013 Journal of Parenteral and Enteral Nutrition
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Hipotez
bull NAFLD ve kontrol mikrobiyotası arasındafarklılıklar bulunmaktadır
ndash Mikrobiyotadaki farklılıklar barsaktan dolaşımageccedilen endotoksin miktarını arttırabilir
ndash İnflamasyon artabilir
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Metod
bull 52 hasta (46rsquosı karaciğer biyopsisi ile doğrulanmış)
bull 38 sağlıklı kontrol
ge18 yaşında olan
artmış serum aminotransferaz seviyeleri goumlzlenen
alkol tuumlketimi lt20 grhafta olan
son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik tedavisi almamış olan
eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Metod
bull US ve artmış karaciğer enzimleri
bull Brunt skorlama sistemibull NAFLD tanısında steatoz inflamasyon ve fibrozis derecesi goumlz
oumlnuumlnde bulundurulmuşturbull Hastalarda fibrozis derecesi F0-F1 orta dereceli fibrozis Fge2 şiddetli
fibrozis olarak değerlendirilmiştir
bull Hasta Bilgi Formundash cinsiyet yaş kilo sigara kullanımı ilaccedil kullanımı vsndash Laboratuvar bulguları (Accedillık glukoz trigliserid HDL LDL ALT AST ALP)
Brunt EM 1999 Liver
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
qPCR analizleri iccedilin standartlarınhazırlanması
Akkermansia muciniphila ATCC BAA-835 Faecalibacterium prausnitzii ATCC 27766Bifidobacterium breve ATCC 15700 Lactobacillus acidophilus ATCC 4356Bacteroides fragilis ATCC 25285Escherichia coli ATCC 25922
Kuumlltuumlr05 Mc Farland Suumlspansiyon
cfu sayım
Ekstraksiyon
Nanodrop Oumllccediluumlm
110 diluumlsyon
Standart eğri
0
5
10
15
20
25
30
35
1E+02 1E+04 1E+06 1E+08
Cro
ssin
g-p
oin
t (C
p)
valu
e
DNA copy number microL
A muciniphila
B fragilis group
Bifidobacterium spp
F prausnitzii
Enterobacteriaceae
Lactobacillus spp
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Akkermansia muciniphila ATCC BAA-835 kuumlltuumlruuml
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Faecalibacterium prausnitzii ATCC 27766 kuumlltuumlruuml
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Dışkı oumlrneklerinden qPCR analizleri
Dışkı oumlrnekleri
200 mg Ekstraksiyon Nanodrop Oumllccediluumlm
Roche LightCycler 20
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Baskın Lactobacillus ve Bifidobacterium tuumlrlerinin belirlenmesi
Hasta (n=49) Kontrol (n=35)
Her oumlrnekten 50 ngmicroL havuzlandı
LacF ve LacF primerleri ile PCR g-Bifid-F ve g-Bifid-R primerleri ile PCR
Agaroz Jelde Goumlruumlntuumlleme
Purifikasyon
TA Klonlama vektoumlruuml (TA Cloningreg Kit with pCRtrade21)
kullanarak amplifikasyon uumlruumlnlerinin ligasyonu
Ecoli JM 109 kullanarak transformasyon
Ampisilin iccedileren LB besiyerlerine kuumlltuumlr
Lactobacillus gen kuumltuumlphanesi Bifidobacterium gen kuumltuumlphanesi
HastaKontr
olHasta
Kontr
ol
Lac-F Lac-R Koloni PCR
Agaroz jelde goumlruumlntuumlleme
Purifikasyon
g-Bifid-F g-Bifid-R koloni PCR
Sekans PubMed Blast 97 seq similarity
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
İnflamasyon Belirteccedilleri
İnflamauvar sitokinler TNF-α IL-6 hazır ticari ELISA kitikullanıldı
hs-CRPbull Karaciğerden sentezlenen akut faz proteinibull Normal kons 01 mgdLbull Lazer nefelometri ile oumllccediluumlluumlyorbull Karaciğer patolojisinin şiddetibull İnflamasyon belirteci
Yoneda et al 2007 J Gastroenterol
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD (n=52) KONTROL (n=38)
Dışkı
ELISA (TNF-α IL-6)Hs-CRP
DNA
qPCR
Kromojenik LAL Testi(Endotoksin)
A muciniphilaF prausnitziiLactobacillus sppBifidobacterium sppB fragilis groupEnterobacteriaceae
Lactobacillus Bifidobacterium spp plazmid gen kuumltuumlphanesi
Kan
Serum
Metod
Dizi analizi
Rutin Biyokimyasaltetkikler
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol mikrobiyotasının kantitatif karşılaştırılması
bull Ccediloklu lineer regresyon analizi - A muciniphila ve Enterobacteriaceaersquonin NAFLD ve kontrol grupları arasında anlamlı olarak farklı kaldığı (p=00148 ve p=00172)
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
dış
kı
Kontrol
NAFLDp=0003
p=0001 plt0001
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Fibrosis derecesine goumlre mikrobiyotanın karşılaştırılması
A m
ucinip
hilia
F pra
usnitz
ii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
dış
kı
F0-F1
F2
plt0001
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Enterobacteriaceae ile BKİ arasındakipozitif korelasyon
20 30 400
5
10
15
BMI
En
tero
ba
cte
ria
ce
ae
lo
g1
0g
r d
ışk
ı
BKİ ve Enterobacteriaceae sayısı arasında anlamlı olarak pozitif bir korelasyon goumlzlendi (Pearson r=0282 p=0021)
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
-hs-CRP ozellikle karaciğerdeki inflamasyon icin yuumlksek duyarlı bir belirteccedil-Hasta grubunda artmış inflamasyona bağlı yuumlksek serum hs-CRP seviyeleri (plt0001)
Kontr
ol
NAFLD
-05
00
05
10
15
20
mg
dL
Kontrol
NAFLD
hs-CRP
NAFLD ve kontrol grubu hs-CRP seviyeleri
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
-Hasta grubunda yuumlksek Enterobacteriaceae duumlzeyi ile uyumlu olarak yuumlksekendotoksin seviyesi (plt0001)
Kontr
ol
NAFLD
-20
0
20
40
60E
Um
L
Endotoxin
NAFLD ve kontrol grubu serum endotoksin duumlzeyleri
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol grubu sitokin duumlzeyleri
bull TNF-α ve IL-6 seviyeleri bakımından fark yok
bull Serum yarı omuumlrleri kisa olduğu iccedilin T huumlcre reseptoumlrleri ccedilalışılabilir
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Pat
ient
0
20
40
60
80
IL-6
pg
mL
Control
Patient
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol grubundaLactobacillus tuumlrleri
L delbrueckii 9
Lruminis50
Lmucosae2
Lacidophilus4
Lgastricus2
Lsalivarius4
Uncultured Lactobacillus
spp18
L helveticus7
Lsakei4
Kontrol
L delbrueckii Lruminis
Lmucosae Lacidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L helveticus
Lsakei
Ldelbrueckii 3
Lruminis39
Lmucosae9
L acidophilus11
Lgastricus6
Lsalivarius3
Uncultured Lactobacillus
spp5
L Johsonii5
Lreuteri19
NAFLD
Ldelbrueckii Lruminis
Lmucosae L acidophilus
Lgastricus Lsalivarius
Uncultured Lactobacillus spp L Johsonii
Lreuteri
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol grubundaBifidobacterium tuumlrleri
B adolescentis55
Blongum15
Bbifidum6
Bkashiwanohense10
Bcatenulatum4
Bruminatium2
Uncultured Bifidobacterium
spp Clone8
Kontrol
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Bcatenulatum
Bruminatium
Uncultured Bifidobacterium spp Clone
B adolescentis42
Blongum36
Bbifidum2
Bkashiwanohense2
Binfantis8
Uncultured Bifidobacterium
spp clone10
NAFLD
B adolescentis
Blongum
Bbifidum
Bkashiwanohense
Binfantis
Uncultured Bifidobacterium spp clone
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Sonuccedil
NAFLD olan hastalar ve sağlıklı kontrollerde barsak mikrobiyotasikantitatif olarak farklidir
A muciniphila NAFLD grubunda daha duumlşuumlk orandadır
Barsaktaki endotoksinin birincil kaynagi olan Enterobacteriaceae hasta grubunda beklenildiği gibi daha yuksektir
B fragilis kilo durumuna bağımlı bir değişken
Hasta grubunda beklenildigi gibi serum endotoksin seviyeleri dahayuksektir
Inflamasyon belirteccedillerinden biri olan hs-CRP duzeyleri hasta grubunda daha yuksektir
F prausnitzii mikrobiyotanın dominant bir uumlyesi
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Sonuccedil
bull L reuteri hasta grubunda baskın ancakkontrolde saptanmadı
bull L sakei ve L helveticus kontrolde var ancakhasta grubunda saptanmadı
bull L reuteri obezite ile ilişkili mikrobiyota uumlyesi
Million M et al Obesity-associate d gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii Int J Obesity 2012 36 817-25
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
CEREN ERDOĞDU
Meltem Yalınay
Tarkan Karakan
Martin J Blaser
Barış Otlu
Doruk Engin
Sinem Solmaz
Berrin Oumlztuumlrk
GUuml Endoskopi Uumlnitesi
GUuml Biyokimya Laboratuvarı (Oumlzlem Guumllbahar)
NYU Biyoistatistik Departmanı (Huilin Li)
Ankara Uumlniversitesi Veterinerlik Fakuumlltesi (Mehmet Şahal)
Roche Diagnostics teknik ekibi
Teşekkuumlrler
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Ceren Erdogdu17112016
Nonalkolik Karaciğer Yağlanması OlanHastalar ve Sağlıklı Kontrollerde Bağırsak
Mikrobiyotasının Yeni Nesil Dizi Analizi İle Karşılaştırılması
Erdogdu C Yalinay M Karakan T Battaglia T Blaser MJ
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Hipotez ndash Araştırma sorusubull NAFLD ve sağlıklı kontroller arasında bağırsak
mikrobiyotası iccedilerik ve yapı bakımından farklıdır
ndash Mikrobiyal ccedileşitlilik iki grupta farklı mı
ndash Mikrobiyota yapısı karaciğer hasarının şiddetine goumlre değişiyor mu
ndash Hastalık ve sağlığa etkisi olan spesifik gruplar hangileri
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Metod
bull NAFLD - biyopsi ile doğrulanmış hastalar ge18 yaşında olan artmış serum aminotransferaz seviyeleri goumlzlenen alkol tuumlketimi lt20 grhafta olan son 6 ay iccedilerisinde herhangi bir kortikosteroid antibiyotik prebiyotik probiyotik
tedavisi almamış olan eş zamanlı başka bir hastalığı olmayan hastalar dahil edilmiştir
bull Fibrozis skorubull F0-F1 orta dereceli fibrozisbull F2 şiddetli fibrozis
bull 16S gen kuumltuumlphanesi-Illumina Miseq sekans
NAFLD (n=43) KONTROL (n=23)
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Alfa ve Beta Cesitlilik (Uzaklık) Oumllccediluleri
Mikrobiyom toplulukları iccedilerisinde ve birbirleriarasındaki uzaklığı oumllccedilmek iccedilin
kullanılan metriklerdir
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Gruplararası alfa ccedileşitlilik (goumlzlenen OTU- operasyonel taksonomik uumlnitedağılım)
F0-F1
F2
Kontrol
Hasta
Alfa Cesitlilik (Alpha Diversity)Bir mikrobiyom verisinde hangi tuumlr canlılar vardır ve ne kadarı oumllccedilmek iccedilinkullanılır Yani bir oumlrnek kendi iccedilinde ne kadar farklıdır sorusuna cevap verir
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol grubunda beta ccedileşitlilik
Kontrol (n=23)
NAFLD (n=43)
P=0004 method ANOSIM
Beta ccedilesitlilik(Beta Diversity)Oumlrneklerinbirbirinden ne kadarfarklı olduğunacevap verir Oumlrnekler arasındakarşılaştırma yapar Oumlrnekteki geneldeğişimi oumllccediler
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Fibrozis derecelerine goumlre beta ccedileşitlilik
F0-F1 (mild fibrosis) n=23
F2 (severe fibrosis) n=20
P=0005 method ANOSIM
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol grubunda relatif dağılımlar
Kontrol NAFLD
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
NAFLD ve kontrol grubuna anlamlı olarak etki edenbakteri grupları
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Sonuccedil
bull Metagenomik youmlntemler mikrobiyomccedilalışmaları iccedilin altın standarttır
bull Karaciğer patolojisi mikrobiyotada dağılım vezenginlik olarak farklılığa neden olmuyor ancakmikrobiyota kompozisyonu gruplar arasındafarklı
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Blaser Lab
NYU Langone Biyoistatistik Boumluumlmuuml
NYU Langone Genom Merkezi
Teşekkuumlrler
Funding
T-RO1DK090989NIH
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Gut Microbiota in Patients with Non-Alcoholic Fatty Liver Disease
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
Total of 52 NAFLD subjects and 38 healthy controls were included Within theNAFLD group 46 of the patients were diagnosed with NASH by biopsy accordingto the Brunt criteria NASH patients classified into two groups according to thefibrosis degree as follows F0-F1 mild fibrosis (n=23) F2 significant fibrosis(n=23) Stool and blood samples were collected at the same day Quantificationof the fecal microbiota was performed by Real-Time PCR Specific primers usedto determine Akkermansia muciniphila Faecalibacterium prausnitziiBacteroides fragilis group Bifidobacterium spp Lactobacillus spp andEnterobacteriaceae In addition to the routine biochemical tests inflammatorycytokines (IL-6 and TNF-alpha) hs-CRP levels and endotoxin levels were alsodetermined in serum Standard statistical methods were used for the calculationof means and standard deviations Independent t-test was used in order tocompare continuous variables For categorical variables chi-square test wasused Multiple linear regression analysis was performed in order to adjust forthe variables that are significant between the groups A p value of lt 005 wasused to establish significance
p values were determined by t-test except male which was determined by chi square test
Figure 1 Comparison of log10gram wet feces levels of bacterial groupsbetween patients with NAFLD and healthy controls
FRIDAY-662 Hacettepe Universitycerenozkulhacettepeedutr
Characteristic
Patients
(n=52)
MeanplusmnSD
Control
(n=38)
MeanplusmnSD
p
Male () 478 324 0100
Age (years) 48 plusmn 12 36 plusmn 10 lt0001
BMI (kgm2) 29 plusmn 4 22 plusmn 2 lt0001
Waist circumference [46] 94 plusmn 9 83 plusmn 6 0001
ALT (UL) 50 plusmn 41 20 plusmn 11 lt0001
AST (UL) 36 plusmn 18 21 plusmn 4 lt0001
ALP (UL) 105 plusmn 38 62 plusmn 18 lt0001
Total cholesterol (mgdL) 202 plusmn 43 207 plusmn 43 0613
HDL (mgdL) 45 plusmn 9 57 plusmn 10 lt0001
LDL (mgdL) 127 plusmn 39 140 plusmn 38 0065
Triglyceride (mgdL) 184 plusmn 80 96 plusmn 46 lt0001
Glucose (fasting mgdL) 102 plusmn 22 87 plusmn 12 lt0001
Table 1 Demographic and Laboratory Results of the Participants
AbstractIn the whole study group decreased levels of Akkermansia muciniphila (954plusmn192vs 1086plusmn214 log 10gr feces p=0003) and B fragilis group (671plusmn138 vs781plusmn150 log 10gr feces p=0001) were observed Patient group has increasedlevels of Enterobacteriaceae (803plusmn114 vs 689plusmn112 log 10gr feces plt0001)After adjusting for BMI and age B fragilis group was no longer significant NASHsubgroup were evaluated according to the fibrosis stage F2 fibrosis stage hadsignificantly higher Enterobacteriaceae levels as compared to F0-F1 fibrosis stage(plt0001) Supporting the finding of increased abundance of Enterobacteriaceaein patient group serum endotoxin levels were also significantly elevated ascompared to the controls (plt0001) Patients have 3 fold higher hs-CRP levels(012plusmn002 mgdL vs 037plusmn006 mgdL plt0001) which is a promising marker ofinflammation in patients with NAFLD
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us sp
p
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae
0
5
10
15
log
10
gr
fec
es
Control
Patient
A m
ucinip
hilia
F pra
usnitzii
Lacto
bacill
us spp
Bifi
dobacte
rium
spp
B f
ragili
s gro
up
Entero
bacte
riace
ae0
5
10
15
log
10
gr
fec
es
F0-F1
F2
Background The effect of the gut microbiota on several diseases is becomingincreasingly important issue Since there is a close relationship between gut andliver via portal vein the liver is consistently exposed to bacterial products suchas endotoxin The composition and quantification of microbiota members maybe important for gut dysbiosis and subsequent bacterial translocation which maybe the major cause of non-alcoholic fatty liver disease (NAFLD) In order tosupport this hyphothesis we performed qPCR analysis in stool of patients withNAFLD and healthy controlsMethods The stool samples from 52 patients with NAFLD and 38 healthycontrols have been collected NAFLD has been proven by biopsy in 46 of thepatients 16S rRNA qPCR assay has been performed for quantification ofAkkermansia muciniphila Faecalibacterium prausnitzii Bifidobacterium sppLactobacillus spp Bacteroides fragilis group and Enterobacteriaceae Serum IL-6TNF-α hs-CRP and endotoxin levels were assessedResults A muciniphila and B fragilis group were found significantly lower inpatients with NAFLD (p=0003 and p=0001 respectively) As expected theEnterobacteriaceae family members were found to be significantly higher inpatients group (plt0001) In consistent with the higher Enterobacteriaceaeabundance in NAFLD patients elevated serum endotoxin levels were alsodetermined TNF-α and IL-6 levels were not significantly different howeverpatients have 3 fold higher hs-CRP which is a well-known marker ofinflammation Multiple regression analysis has performed in order to adjust forBMI and gender A still significantly lower and Enterobacteriaceae levels weresignificantly higher in patients group after adjusting for BMI and gender(p=00221 p=00186 respectively)Conclusion NAFLD patients were characterized with higher Enterobacteriaceaelevels lower A muciniphila and B fragilis group levels in our study cohortElevated endotoxin levels and inflammation are also supporting our hypothesis
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferase ALPalkalinephosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test except male which was determined by chi square test
Figure 2 Comparison of log10gram wet feces levels of bacterial groups between fibrosis stages in patients diagnosed with NASH
Figure 3 Serum levels of endotoxin (A) hs-CRP (B) TNF-α (C) and IL-6 (D) between patients and controls
Contr
ol
Patie
nt-20
0
20
40
60
EU
mL
Control
Patient
Endotoxin
Contr
ol
Patie
nt0
20
40
60
80
TNF-alpha
pg
mL
Control
Patient
Contr
ol
Patie
nt-05
00
05
10
15
20
mg
dL
hs-CRP
Contr
ol
Patie
nt0
20
40
60
80
IL-6
pg
mL
Control
Patient
BA
C D
bull Patients with NAFLD has significantly decreased fecal A muciniphila levelsand increased Enterobacteriaceae levels
bull Within the NASH subgroup patients with significant fibrosis (Fge2) hassignificantly higher fecal Enterobacteriaceae levels suggesting that thedifferent fibrosis stages of NASH should be considered in future studies
bull Elevated serum endotoxin levels were observed in the patient group inaccordance with the higher Enterobacteriaceae levels
bull Serum hs-CRP levels found to be higher in patients group while nodifference was observed in terms of IL-6 and TNF-alpha
Results
Methods
Conclusions
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Increased Akkermansia muciniphila and Bacteroides fragilis group
Abundance After Islamic Fasting
Ceren Erdogdu1 Meltem Yalinay Cirak2 Tarkan Karakan3
Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Clinical Microbiology Ankara Turkey
Gazi University Medical Faculty Department of Internal Medicine Division of Gastroenterology Ankara Turkey
FRIDAY Hacettepe Universitycerenozkulhacettepeedutr
Table 2 Laboratory Data of the participants measured before andafter Islamic fasting
Abstract
Background Metabolic disorders such as obesity is often associatedwith the changes in microbiota composition and quantity It has beenwell known that dietary habits also effects intestinal microbiota Herewe hypothesized that long-term fasting may have a distinct effect onintestinal microbiota Ramadan fasting is an excellent model of howlong-term fasting may affect the microbiota composition Total of 9subjects were included in this study during Ramadan between thedates June 18 and July 16 2015 which was approximately 17 hours offasting per day during a 29 day periodMethod The stool samples from 9 volunteers were collected the daybefore the beginning of Islamic fasting and the day end of theRamadan 16S rRNA qPCR assay has been performed for quantificationof Akkermansia muciniphila Faecalibacterium prausnitziiBifidobacterium spp Lactobacillus spp Bacteroides fragilis groupEnterobacteriaceae Blood samples were also collected to test formetabolic and nutritional parameters The before and after Islamicfasting results were statistically analyzed using Wilcoxon Signed-RanktestResults All of the subjects were normal weight according to thecalculated BMI The overall bacterial count did not significantly changeover time in none of the subjects However significantly increasedabundance of A muciniphila and B fragilis group was observed in allsubjects after Islamic fasting when compared to the baseline levels(p=00039 and 00078 respectively) Serum fasting glucose and totalcholesterol levels in all subjects is also significantly reduced in all ofthe subjects (plt001 and p=0009 respectively)Conclusion Islamic fasting which has been approximately 17 hours offasting for each day lead to a change in microbiota members
BMIBody mass index ALTAlanine transaminase ASTaspartate aminotransferaseALPalkaline phosphatase HDLhigh-density lipoprotein LDLlow-density lipoproteinp values were determined by t-test
Figure 2 Quantities of fecal A muciniphila and B fragilis groupmembers for each participant determined at baseline and after theend of Islamic fastingData was statistically analyzed using Wilcoxon Rank Sum test pvalues for A muciniphila and B fragilis group is 00039 and 00078respectively
bull Ramadan fasting is an excellent model of how long-term fastingmay affect the microbiota composition
bull Significantly lower fasting glucose and total cholesterol levelswere determined at the end of the Islamic fasting whencompared to the baseline values
bull Significantly higher fecal A muciniphila and B fragilis groupmembers were determined in the study participants who fastedfor an average of 17 hours during Ramadan
bull This early results suggest that long-term fasting may have adistinct effect on microbiota composition Metagenomicapproaches should be performed in order to better understandthe effect of fasting on microbiota composition
bull
A mucin
iphila
F pra
usnitzii
Lactobacill
us spp
Bifidobacte
rium
spp
B fra
gilis g
roup
Entero
bacteria
ceae0
5
10
15
log1
0gr
fec
es
BF
AF
Characteristic
Before Fasting
(n=9)
MeanplusmnSD
After Fasting
(n=9)
MeanplusmnSD
p
BMI (kgm2) 229 plusmn 11 228 plusmn 11 0810
Waist circumference (19) 830 plusmn 52 824 plusmn 56 0821
ALT (UL) 246 plusmn 124 256 plusmn 112 0868
AST (UL) 228 plusmn 48 233 plusmn 48 0837
ALP (UL) 793 plusmn 228 779 plusmn 226 0904
Total cholesterol (mgdL) 2315 plusmn 622 2175 plusmn 579 0009
HDL (mgdL) 529 plusmn 117 520 plusmn 103 0876
LDL (mgdL) 1574 plusmn 510 1546 plusmn 486 0914
Triglyceride (mgdL) 1105 plusmn 529 1064 plusmn 536 0879
Glucose (fasting mgdL) 878 plusmn 208 738 plusmn 158 0006
Bacterium Standard strain Primer Seq (5rsquo-3rsquo) bp Primer Annealing
(oC) Tm
Bifidobacterium
spp
B breve ATCC
15700
CTCCTGGAAACGGGTGG 550
g-Bifid-F 56 90
GGTGTTCTTCCCGATATCTACA g-Bifid-R
B fragilis group B fragilis ATCC
25285
ATAGCCTTTCGAAAGRAAGAT
495
g-Bfra-F
50 86 CCAGTATCAACTGCAATTTTA g-Bfra-R
Lactobacillus spp L acidophilus
ATCC 4356
AGCAGTAGGGAATCTTCCA 341
Lact-F 55 86
CACCGCTACACATGGAG Lact-R
Amuciniphila Amuciniphila
ATCC BAA-835
CAGCACGTGAAGGTGGGGAC
327
AM-1
60 90 CCTTGCGGTTGGCTTCAGAT AM-2
F prausnitzii F prausnitzii
ATCC 27766
GATGGCCTCGCGTCCGATTAG 199
Fprau223F 60 88
CCGAAGACCTTCTTCCTCC Fprau420R
Enterobacteriaceae E coli ATCC
25922
CATTGACGTTACCCGCAGAAGAAGC
195
Eco1457F
63 87 CTCTACGAGACTCAAGCTTGC Eco1652R
Table 1 Specific primers used to determine 16S rRNA region of thebacteria and standard strains used to construction of PCR standards
Before IF After IF0
5
10
15
A muciniphila
log
10
gr
fec
es
Before IF After IF0
2
4
6
8
10
B fragilis
log
10
gr
fec
es
Results
Methods Conclusions
Figure 1 Log10gr wet feces level means measured before Islamicfasting (baseline) and after Islamic fastingBF Before fasting AF After fasting
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması
Ceren Erdoğdu Meltem Yalınay Tarkan Karakan Thomas Battaglia Martin J Blaser
Nonalkolik Karaciğer Yağlanması Olan Hastalarda Barsak Mikrobiyotasının Yeni Nesil Dizi
Analizi İle Karşılaştırılması