Il microbiota intestinale: composizione e funzione … microbiota intestinale: composizione e funzione Maurizio Sanguinetti Istituto di Microbiologia Università Cattolica del Sacro

Il microbiota intestinale: composizione e funzione

Maurizio Sanguinetti

Istituto di Microbiologia

Università Cattolica del Sacro Cuore - Roma

Human Microbiota

Lee YKL, Science 2010

Humans: Meta-organisms10-fold greater numbers of microbial than human cells,

metabolically and immunologically integrated,

with a biomass >1 Kg

VirusBacteriophages Bacteria Yeast

GUT MICROBIOTA: only bacteria?

Acquiredand

Innate immunity

MucosalBarrier

Epithelial barrier

Vascular and lymphatic systems

Neuroenteric systemDigestive enzymes

Endocrinesystem

COMPOSITION OF THE GUT MICROBIOTA:MOLECULAR APPROACH

Eckburg et al, Science 2005

BACTEROIDETES

FIRMICUTES

Ambient Diet

Vaginal microbiota (mother)Skin

microbiota (mother/father/parent

s/babysitter)

Fecal microbiota (mother)

Native CORE microbiota (4-36 months of life)

At birth the human body is sterile

33 metagenomes (Sanger)

85 metagenomes (Illumina)

154 metagenomes (16S pyroseq.)

Arumugam M. et al & MetaHIT consortium(2011) Enterotypes of the human gutmicrobiome. Nature 473, 174-180

• Multidimensional cluster analysis and principalcomponent analysis (PCA) revealed the forming of three distint clusters thathave been designated as«enterotypes»

• Each enterotype isidentifiable by the variationof one of three genera: Bacteroides, Prevotella and Ruminococcus

• Enterotypes are driven by groups of species thattogether contribute to the preferred community compositions

HUMAN MICROBIOME PROJECTS: 3 main enterotypes

Enterotypes are identifiable by the variation in the levels of one of three genera:ENTEROTYPE 1: BacteroidesENTEROTYPE 2: PrevotellaENTEROTYPE 3: Ruminococcus

From: Arumugam et al., Nature 2011

Drivers of this enterotype seem to derive energy primarily from

carbohydrates and proteins through fermentation, as these closely

related genera have a very broad saccharolytic potential

ENTEROTYPE 1


Degrade mucin glycoproteins

ENTEROTYPE 2


It is the most frequent and comprises species capable of degradingmucin

ENTEROTYPE 3


Functional differences amongenterotypes

These phylogenetic and functional

differences among enterotypes thus

reflect different combinations of

microbial trophic chains with a probable

impact on synergistic interrelations with

the human hosts


But…specific effects in each GI tract!

EFFECTS OF GUT MICROBIOTA ON HOST HEALTH

Barrier effectImmunocompetence/ToleranceSynthesisMetabolic/Trophic functionDrug methabolismBehavior conditioning

Intestinal bacteria confer indirect (immune-mediated) colonizationresistance against enteric pathogens

a. Bacteroides thetaiotaomicron enhances expression of the peptidoglycan-binding C-typelectin regenerating islet-derived protein IIIγ (REGIIIγ), which is an antimicrobial peptide thatprimarily targets and kills Gram-positive bacteria

b. Segmented filamentous bacteria (SFB) closely associate with the intestinal epithelium and enhance IgA production by B cells, serum amyloid A (SAA)-dependent T helper 17 (TH17) cell differentiation, pro-inflammatory cytokine production and epithelial production of antimicrobial peptides. These processes confer protection against Citrobacter rodentium

Intestinal bacteria confer direct colonization resistance againstenteric pathogens

d. B. thetaiotaomicron consumes carbohydrates used by C. rodentium, which contributes to the competitive exclusion of the pathogen from the intestinal lumen

e. Bacteroides thuringiensis secretes a bacteriocin that directly targets spore-forming Bacilli and Clostridia, including Clostridium difficile, through an unknown mechanism of action

f. Gram-negative bacteria, such as Vibrio cholerae, deliver toxic effector proteins directly to Escherichia coli through type VI secretion systems

g. A variety of Bifidobacterium spp. produce organic acids and peptides that impair growth and adhesion of pathogenic E. coli to enterocytes

From: Balfour and Sartor, Gastroenterology 2008, modified

Luminal PAMPs LPS (Gram -) UPEC/Profilin Flagellin Peptidoglican/lipopeptide Bacterial lipopeptide ds RNA Fibronectina (many bacteria) Lipothecoic acid (Gram +) Lipooligosaccharide

Specific PRRsTLR-4TLR-11TLR-5TLR-1 e TLR-2TLR-2 e TLR-6TLR-3α5β1 integrinTLR-2PAF

Endosomial PAMPs ss RNA CpG DNA

Specific PRRsTLR-6 e TLR-7TLR-9

Microbiota stimulates IMMUNITY throught PRRs

Gastro-intestinal diseases associatedto an unbalanced GUT Microbiota

1. Gastrointestinal infections2. Inflammatory Bowel Diseases3. GI Cancer4. Irritable Bowel Syndrome 5. Intestinal Bacterial Overgrowth6. Food Intolerance/Allergy7. Obesity and Metabolic Syndrome8. Liver diseases9. …

J48 decision tree describing the patterns of colonisation associated with presence or absence ofC. difficile. Decision tree is showing only routine sample sets. The decision tree showscombinations of microbial groups that predict the outcome regarding C. difficile colonisation.

• We performed a nested case–control study including 25 CDI cases and 25 matched controls. Fecal specimens collected prior to disease onset were evaluated by 16S rRNAgene amplification and pyrosequencing to determine the composition of the intestinal microbiota during the at-risk period.

• The diversity of the intestinal microbiota was significantly reduced prior to an episode of CDI. Sequences corresponding to the phylum Bacteroidetes and to the families Bacteroidaceaeand Clostridiales Incertae Sedis XI were depleted in CDI patients compared to controls, whereas sequences corresponding to the family Enterococcaceae were enriched.

• In multivariable analyses, cephalosporin and fluoroquinolone use, as well as a decrease in the abundance of Clostridiales Incertae Sedis XI were significantly and independently associated with CDI development.

• This study shows that a reduction in the abundance of a specific bacterial family –Clostridiales Incertae Sedis XI - is associated with risk of nosocomial CDI and may represent a target for novel strategies to prevent this life-threatening infection.

From: Frank et al, PNAS 2007

Bacterial variety in IBD human mucosa

From: Junjie Qin et al, Nature 2010

Bacterial diversity in IBD human mucosa

From: Round and Mazmanian, Nat Rev Immunol 2009

Some bacteria of microbiota have a positive effect on experimental IBD: beneficial Microbiota

PSA uptake by intestinal DCs

PSAPresentation

to naïve CD4+

cells

Induction of IL-10-

producing Treg cells

Bacteroides fragilis-PSA

Fecal Microbiota Transplantation

Increasing evidence supports amicrobial influence in the pathogenesisof IBD, likely due to an inappropriateimmune response toward a componentor components of microbiota

From: Anderson, Aliment. Pharmacol. Ther., 2012

From 5320 articles, SELECTED only 9 case series/case formanagement of IBD, and 8 for treatment of infectious diarrhoea(TOTAL 41 patients with IBD, 27 UC, 12 CD, 2 unclassified)

Follow-up between 2 weeks and 13 years

FMT was administered via colonoscopy/enema (26/33) or via enteraltube (7/33)

Reduction of IBD symptoms (19/25)Cessation of IBD medications (13/17) and disease remission (15/24)Resolution of C. difficile infection in all those treated for such (15/15).

FMT: Ulcerative Colitis

From: Kunde, J Pediatr Gastroenterol Nutr. 2013

GI Cancer associated to an unbalanced GUT Microbiota

1.Stomach2.Pancreas3.Biliary tract4.Liver5.Small Bowel6.Colon

The bacterial microbiome promotes carcinogenesis through several mechanisms.a) Changes in the microbiome and host defences may favour increased bacterial translocation, leading to

increased inflammation. These effects may occur locally or through long-distance effects in other organs. b) Genotoxic effects are mediated by bacterial genotoxins — such as colibactin and cytolethal distending

toxin (CDT) — that, after being delivered to the nucleus of host cells, actively induce DNA damage in organs that are in direct contact with the microbiome

c) Metabolic actions of the microbiome may result in the activation of genotoxins such as acetaldehyde, dietary nitrosamine and other carcinogens, in the metabolism of hormones such as oestrogen and testosterone, in the metabolism of bile acids and in alterations of energy harvest. The microbiota also mediates tumour suppressive effects (shown in green) through inactivation of carcinogens, through the generation of short-chain fatty acids such as butyrate and through the biological activation of cancer-preventing phytochemicals. From: Schwabe and Jobin, Nat Rev Cancer, 2013

From: Schwabe and Jobin, NatRev Cancer, 2013

Evidence for tumour-promoting

effects of the bacterial microbiota

Murine studies

Evidence for tumour-promoting effects of the bacterial microbiota

Human studies

From: Schwabe and Jobin, Nat Rev Cancer, 2013

IPSID, immunoproliferative small intestinal disease; MALT, mucosa-associated lymphoid tissue;

From: Schwabe and Jobin, Nat Rev Cancer, 2013

GUT MICROBIOTA and REGULATION OF ENERGY STORAGE

• Wild Type (WT) mice have 42% more total body fat and 47% more gonadal fat than germ-free (GF) mice

• Colonisation of GF mice with microbiota from WT produces a 60% increase in body fat mass, associated with increased insulin resistance

From: Backhed et al. PNAS, 2004

Gut microbiota has a role in obesity

Changes in gut microbial ecology• Reduction in Bacteroidetes and proportional increase in Firmicutes• Dramatic fall of overall diversity• Bloom of a single class of Firmicutes: the Mollicutes

Alteration of metabolic potential• Enrichment for phosphotransferase systems• Enrichment for genes encoding beta-fructosidases Consequences• Increased capacity to import “Western-diet”-typical carbohydrates • Increased capacity to metabolize imported sugars

From: Tilg et al., Gastroenterology, 2009


18 treatment-naive male subjects with metabolic syndrome

Random assignment to either an allogenic (from lean maledonors with a body mass index 23 kg/m2; n 9) or autologous gutmicrobiota infusion (reinfusion of own collected feces; n 9)

Large (fecal samples) and small- (duodenal biopsies) intestine gutmicrobiota composition and fecal short-chain fatty acids weremeasured at baseline and 6 weeks after infusion

Insulin sensitivity was measured before and 6 weeks after gutmicrobiota infusion

Improvement in peripheral insulin sensitivity 6 weeksafter allogenic gut microbiota infusion (median rate ofglucose disappearance, Rd: 26.2 -45.3 mol/kg/min; P<0.05)


Tutto ciò può essere traslato nella «real life»?L’esperienza dell’UCSC

• Valutazione del microbiota di biopsie gastriche in

pazienti con infezione da Helicobacter pylori in corso

di trattamento con inibitori di pompa protonica (PPI)

• Valutazione del microbiota in pazienti con colite da

Clostridium difficile e trattati con trapianto di

microbiota (fecale)

• Valutazione del microbiota in pazienti con GvHD

intestinale cronica e potenzialmente trattabili con

trapianto di microbiota

Helicobacterpylori

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1 2 3 4 5 6 7 8 9 10 11 12 13

PPI NO PPI

Veillonella sp

Streptococcaceae

Prevotella sp

Porphyromonas sp

Microbiota gastrico di pazienti con infezione da H. pylori in corso di trattamento con inibitori di pompa protonica (PPI)

Duodenal Infusion of Donor Fecesfor Recurrent Clostridium difficile

E. van Nood, A. Vrieze, M. Nieuwdorp, S. Fuentes, E.G. Zoetendal, W.M. de Vos, C.E. Visser, E.J. Kuijper, J.F.W. M. Bartelsman, J.G.P. Tijssen, P. Speelman, M.G.W. Dijkgraaf, and J.J. Keller.

The NEW ENGLAND JOURNAL of MEDICINEJanuary 16, 2013

From:the Departments of Internal Medicine (E.N., A.V., M.N., P.S.),

Microbiology (C.E.V.), Gastroenterology ( J.F.W.M.B., J.J.K.),

and Cardiology ( J.G.P.T.) and the Clinical Research Unit (M.G.W.D.),

Academic Medical Center, University of Amsterdam, Amsterdam; the Laboratory of Microbiology, Wageningen University, Wageningen (S.F., E.G.Z., W.M.V.);

the Department of Experimental and Medical Microbiology, Leiden University Medical Center, Leiden (E.J.K.); and the Department of Gastroenterology, Hagaziekenhuis, The Hague ( J.J.K.)

- all in the Netherlands --

and the Department of Bacteriology and Immunology, Medical Faculty, University of Helsinki, Helsinki (W.M.V.).

Rates of Cure without Relapse for Recurrent Clostridium difficile Infection

Five weeks after the initiation of therapy, there was a recurrence of infection in 1 of 16 patients (6%) in the infusion group, 8 of 13 (62%) in the vancomycin-alone group, and 7 of 13 (54%) in the group receiving vancomycin with bowel lavage.

Of 16 patients in the infusion group, 13 (81%) were cured after the first infusion of donor feces. The 3 remaining patients received a second infusion with feces from a different donor; of these patients, 2 were subsequently cured. Overall, donor feces cured 15 of 16 patients (94%). Resolution of infection occurred in 4 of 13 patients (31%) in the vancomycin-alone group and in 3 of 13 patients (23%) in the group receiving vancomycin with bowel lavage. The overall cure rate ratio of donor-feces infusion was 3.05 as compared with vancomycin alone (99.9% confidence interval [CI], 1.08 to 290.05)and 4.05 as compared with vancomycin with bowel lavage

Il trapianto fecale nel trattamento della ricorrenza dell’infezione da Clostridium

difficile

Call for patients!

Fecal transplantation for recurrent Clostridum difficile infection2013, first experience in Italy...

Gender Age(yrs)

Number ofCDrecurrences

CDcolture/toxinbeforetreatment

Donor Route ofinfusion

Fecalquantity(gr)/volume ofinfusion(mL)

Resolutionofsymptoms/FMT relatedside effect

CDtoxinaftertreatment

Follow-uptime

Case 1 M 82 5 Positive/positive

Daughter Colonoscopy 200/400 Yes /none Negative

4 months

Case 2 M 75 2 Positive/positive

Son Colonoscopy 180/420 Yes /none Negative

4 months

Case 3 F 24 1 Positive/positive

Brother Colonoscopy 180/500 Yes /none Negative

3 months

Case 4 F 84 3 Positive/positive

Son Colonoscopy 200(420 Yes /none 1 week

Cammarota et al. Dig. Liv. Dis., 2014

• Here, we demonstrate in murine and human recipients of allogeneic bone marrow transplantation (BMT) that intestinal inflammation secondary to graft-versus-host disease (GVHD) is associated with major shifts in the composition of the intestinal microbiota.

• In mouse models of GVHD, we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. In addition, the antibiotic exposure before BMT, which occurs commonly in patients with hematologic malignancies, may be a risk factor for subsequent intestinal GVHD.

• We then characterized gut flora of patients during onset of intestinal inflammation caused by GVHD and found patterns mirroring those in mice. Together, these data demonstrate regulation of flora by intestinal inflammation and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allogeneic BMT recipients.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1_PRE 1_POST 2_PRE 2_POST

Klebsiella sp.0,5%98,6%

Lactobacillus sp.0,3%58,6%

Ruminococcaceae

Rikenellaceae

Bacteroides sp

Bacteroides sp

Ruminococcaceae

Pseudomonadaceae

Microbiota in pazienti con GvHD intestinale cronica

Conclusioni

• Lo studio del microbioma umano rappresenta uno degli argomenti di ricerca attualmente più stimolanti e significativi

• Nei prossimi anni l’associazione di specifici tipi di microbioma a determinati quadri patologici consentirà di intervenire in modo mirato per ripristinare la corretta «flora microbica»

• La sfida per al Microbiologia Medica sarà quella di mettere a punto test diagnostici sufficientemente sensibili, specifici ed economici per venire incontro a tali necessità

Documents

Il microbiota intestinale: composizione e funzione … microbiota intestinale: composizione e funzione Maurizio Sanguinetti Istituto di Microbiologia Università Cattolica del Sacro