Vaksin Typhoid

8/18/2019 Vaksin Typhoid

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Antimicrobial Resistance, Virulence Profles andMolecular Subtypes o SalmonellaentericaSerovars Typhi and ParatyphiA Blood Isolates rom ol!ata, India durin" #$$%&#$'(

Shanta )utta'*, Suro+it )as', tpala Mitra', Priyan!a -ain', Indranil Roy#, Shelley S. /an"uly(,++0ayini Ray1, Phal"uni )utta', )ilip umar Paul2

' 3ational Institute o 4holera and 5nteric )iseases, ol!ata, India, # 4alcutta Medical Research Institute, ol!ata, India, ( Advance Medical

Research Institute, Salt 6a!e,

ol!ata, India, 1 Apollo /lenea"les 7ospitals, ol!ata, India, 2 )r. B. 4. Roy Post /raduate Institute o Pediatric Sciences, ol!ata, India

Abstract

5nteric ever, caused by Salmonella enterica, remains an unresolved public health problem in Indiaand antimicrobial therapy is the main mode o treatment. The ob+ective o this study 0as tocharacteri8e the Salmonella enterica isolates rom ol!ata 0ith respect to their antimicrobial

resistance 9AMR:, virulence profles and molecular subtypes. Salmonella enterica blood isolates 0ere

collected rom clinically suspected enteric ever patients attendin" various hospitals in ol!ata, Indiarom -anuary #$$% to -une #$'( and 0ere tested or AMR profles by standard protocols; or resistance"ene transer by con+u"ation; or resistance and virulence "enes profles by P4R; and or molecular

subtypes by Pulsed


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4haracteri8ation o S Typhi and Paratyphi A, ol!ata, India

of India limiting the use of these drugs in the treatment of typhoid

6$7. 0ubse8uently ciproflo4acin as used as the drug of choice

in adults. 9ut fre8uent use of ciproflo4acin led to the global

emergence of nalidi4ic acid resistant S. Typhi and S. Paratyphi Aassociated ith decreased susceptibility to ciproflo4acin$ causing

increased treatment failure cases #$:,"!. Thereafter emergence

of ciproflo4acin resistant S. Typhi and S. Paratyphi A further decreased the number of treatment options "&$"+$"'. 0poradic

reports of Salmonella enterica isolates resistant to higher generation cephalosporins like cefta5idime$ cefota4ime$

cefuro4ime erased even the last option for treatment of enteric

fever "%, "6. A5ithromycin has been used as an effective

alternative in uncomplicated cases$ but resistance to this antibiotic

has been reported from India and other countries "7 ":$ !&.

noledge on antimicrobial resistance (A13) profiles of

Salmonella enterica blood isolates is important for predictingthe best possible treatment option for enteric fever patients of any

region. 1any genes involved in A13 are found on mobile

elements !"$!!$ and are therefore likely to be transferred

beteen bacterial pathogens$ hich has a significant public health

impact.

S. Typhi and S. Paratyphi A are intracellular pathogens

possessing a myriad of virulence factors hich contribute indisease progression and severity. The virulence genes are

generally distributed on Salmonella pathogenicity islands(0PIs)$ hich are large genomic regions of +,"%& kb ac8uired

hori5ontally !+. ;irulence gene profiles of the local isolates

could shed light on the virulence types of the organisms and

predict clinical se8uels of the disease.

1olecular typing of any organism is necessary to determine the

relatedness among the isolates and to study the molecular

epidemiology of the organism. Pulsed


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staining the gel ith ethidium bromide (&.% mg*ml). Plasmid

incompatibility typing of S. Typhi isolates as performed by P?3 using published primers of the "7 major incompatibility groups

(MA genes for b-lactamaseN cat gene for chloramphenicol resistanceN sul"$ sul!$sul+ and dr genes for sulphonamide and trimethoprimresistanceN tetA and tet9 for tetracycline resistanceN aadA$strA and str9 genes for streptomycin resistance and integraseint" gene for the presence of class " integron. The gene cassetteithin the variable region of class " integron and the presence of

ac52" gene at +: end of the conserved segment ere alsodetermined !!$++$+'. Alleles of blaTE1$ cat, dr, aadA genesand the resistance gene cassettes of class " integron ere

determined by direct se8uencing of the P?3

Table '. Primers list or detection o resistance "enes in S. Typhi ol!ata isolates.

Resistance /ene Eli"onucleotide seuences 92:R(::

bla T5M TTTT4/T/T4/444TTATT44 4/TT4AT44ATA/TT/44T/A4T4

blaEJA A4A4AATA4ATAT4AA4TT4/4 A/T/T/TTTA/AAT//T/AT4

blaS7V 4A4T4AA//AT/TATT/T/ TTA/4/TT/44A/T/4T4/

cat T444AAT//4AT4/TAAA/AA4 T4/T//TATT4A4T44A/A/4/

sul' T//T/A4//T/TT4//4ATT4 /4/A///TTT44/A/AA//T/

sul# 4//4AT4/T4AA4ATAA44 /T/T/4//AT/AA/T4A/

sul( 4ATT4TA/AAAA4A/T4/TA/TT4/ 4AT4T/4A/4TAA44TA///4TTT//A

dr /T/AAA4TAT4A4TAAT// TTAA444TTTT/44A/ATTT

tetA /TAATT4T/A/4A4T/T4/4 4T/44T//A4AA4ATT/4TT

tetB 4T4A/TATT44AA/44TTT/ 4TAA/4A4TT/T4T44T/TT

strA 4TT//T/ATAA4//4AATT4 44AAT4/4A/ATA/AA//4

strB AT4/T4AA///ATT/AAA44 //AT4/TA/AA4ATATT//4

aadA /T//AT//4//44T/AA/44 ATT/444A/T4//4A/4/

intI' ///T4AA//AT4T//ATTT4/ A4AT///T/TAAAT4AT4/T4

intI' variable re"ion //4AT44AA/4A/4AA/ AA/4A/A4TT/A44T/A

ac52' //4T//4TTTTT4TT/TTAT4/ T/A/4444ATA44TA4AAA/4

doiD'$.'(='F+ournal.pone.$'$'(1=.t$$'

products using a +6+& 2@A analy5er (Applied 9iosystem$


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M)R,multidr u"resistant9resistancetoampicillin,chloramphenicol,co

b

3aR,nalidi>icacidr esistant.

c

)4S,decreasedsusceptibilitytociproo>acin.

d

4iR,cipr oo>acinr esistant.

Alldataareinno.9@:.

doiD'$.'(=' F +our nal.pone.$'$'(1=.t$$#


D0A) for preparing solid agarose plugs. The plugs ere treated

ith % ml cell lysis buffer (%& m1 Tris$ %& m1 E2TA$ pF 7.&$

"/ 0arcosyl) and !% ml proteinase (!& mg*ml) and incubated at

%'u? for ! h in a shaking ater bath. The plugs ere ashed

thoroughly ith pre-armed ater for to times and prearmed

Tris-E2TA buffer (pF 7.&) for four times at "& mins interval at

%&u?. The 2@A plugs ere digested ith '& D of JbaI (@eEngland 9iolabs$ 1A) at +6u? for "7 h. The digested 2@A as

run on "/ pulsed field certified agarose gel (9io-3ad$ Fercules$?alif.) prepared in &.%4 T9E buffer (0igma) using ?FE< 23III

(9io-3ad) apparatus ith an initial sitch time of !.! sec and a

final sitch time of #+.7 sec at # ;*cm for !' h. The gel as

stained ith ethidium bromide (" mg*ml$ 0igma)$ destained ith

deioni5ed ater and Pverall around !&/ isolates of both the serovars

ere resistant to ciproflo4acin. ne isolate in each

serovar (S. Typhi and S. Paratyphi A) as susceptible to all thedrugs tested. 0trikingly different resistance profiles ere observed

among S. Typhi and S. Paratyphi A serovars ith respect to the


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a


ne smaller plasmid of %& kb si5e

and Inc@ type as found in one S. Typhi isolate$ hich asresistant to tetracycline$ co-trimo4a5ole$ streptomycin$ and

8uinolones (Table %). Plasmid as not found in @a3 S. Typhiisolates. 0i4 S. Paratyphi A isolates harbored single plasmid (of either !.% or +.% kb si5e).

Transer o antimicrobial resistance "enemar!ers

The "7& kb plasmid of S. Typhi olkata isolates as nottransferable$ but the %& kb plasmid present in one S. Typhi isolate(>C ++) as transferable by conjugation e4periment (Table %).

The respective 5. coli transconjugant as resistant totetracycline (1I? "# mg*ml) and co-trimo4a5ole (1I? G+!

mg*ml) but not to streptomycin (1I? "! mg*ml) and 8uinolones

by disc diffusion and E-test. The transconjugant as positive for

tetA$ sul"$ drA"% and aadA" genes by P?3.

)etermination o resistance and virulence"ene mar!ers by P4R

Table % shos distribution of A13 gene alleles and presence of

integrons among ampicillin$ chloramphenicol$ co-trimo4a5ole and

tetracycline resistant S. Typhi olkata isolates. ?lass " integrons

present in S. Typhi study isolates had typical +: conservedsegment comprising of acE2" and sul" genes. A total of %% S.Typhi and !" S. Paratyphi A isolates ere screened for virulencegenes by P?3. The majority of S. Typhi and S. Paratyphi Aisolates possessed all virulence markers tested indicating that

these strains ere likely to be virulent. The functional aspects of

the genes$ primer se8uences$ amplicon si5es and P?3 positivity

among study isolates are shon in Table #.

P


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(!%/) in this study as in tandem ith the result of an earlier

study from @e 2elhi hich reported +'/ isolation of 123 S.Typhi in "::: and ##/ in !&&% +:. 9ut other studies from

olkata$ Pondicherry$ 2elhi and >rissa shoed decline in 123

S. Typhi isolation during late nineties +$:$"&$""$'&. @o 123 S.Paratyphi A as isolated in this study$ although increased

isolation of 123 S. Paratyphi A ranging from "".#/ to "%.#!/has been

P6ES E35 000.plosone.or" C Au"ust #$'1 Volume % Issue ? e'$'(1=


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Table 1.Antimicrobial resistance profles o S.Typhi andS.Paratyphi A ol!ata isolates rom #$$%K#$'(.

Antimicrobial resistance profle S . Typhi 9n==: S . Paratyphi A 9n#2:

3aa 1292?.1: '$91$:

3aF4iFEF6e '(9'C.%: $

AF4FLFSF3aFAca $=9%.':

AF4FLFSF3aa $C9=.?:

4FLF3aa $(9(.%:

AF4FLFSF3aF4iFAc '9'.(: $ TFLF3aF4iFEF6e '9'.(: $

3aFE a $ 29#$:

3aFEFA8a $ 19'C:

3aF4iFE $ (9'#:

#9?:3aF4iFEFA8 $

All data are in no. 9@:; Abbreviations usedD A, ampicillin; 4, chloramphenicol; L, co&trimo>a8ole; T, tetracycline; S, streptomycin; 3a, naliacid; 4i, ciproo>acin; E,oo>acin; 6e, levoo>acin; A8, a8ithromycin; Ac, amo>icillinFclavulanic acid.

aassociated 0ith decreased susceptibility to ciproo>acin 9)4S:; Ene isolate in each serovar 0as susceptible to all antimicrobials tested

doiD'$.'(='F+ournal.pone.$'$'(1=.t$$1



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spi1) TypeI secretionproteinnecessary orthe secretion o Spi15

/AATA/AA/A4AAA/4/AT

4AT4

/4TTT/T44A4/44TTT4AT4

'#(' 2( '% N(CO

sopB Mediates

inammation and

uid secretion in

intestinal mucosa

AT/4T4A//T4AA/4A/4

A44/T//A4AT44A4AAA

#'1 22 #' This study

sci3 Type VI secretion

lipoprotein

4/AAT4A4A/4/A4T4/A

T4ATTTAT444/TT44/TAA

1(( 22 #' This study

saB Periplasmicfmbrial

chaperone protein

T44/TA4T//T//T/A4AT

///ATATA4T4AA/444T/TA

A

('C 22 #' This study

viaB Vi polysaccharide

biosynthesis protein

TT//4T44//4TTATTA/AA

T/4AAA4A4AT4A/4/TA4A

1C$ 22 3) N#(O

stn 5nteroto>in TT/T4T4/4TAT4A4T//4AA4

4

ATT4/TAA444/4T4T4/T44

C'= 2# #' N(CO

pltA )elivery o 4dtBrom intracellularcompartment totar"et cells

A44TAT4T//AAA4/4AA// T//TATA4/T4A/4T4TTT/444T4

/AT

(1( 22 #' This study

3), not done.

doiD'$.'(='F+ournal.pone.$'$'(1=.t$$C

2?0 (Table '). A multi-centric study from India also reported

increased isolation of @a3 S. Typhi (7+/) and S. Paratyphi A(:+/)$ although 2?0 as not reported #. 0imilar observation

as documented globally from other countries as ell "'$!&. In

this study ciproflo4acin resistance as observed among ":.%/

("%*66) S. Typhi (ma4imum 1I? being G+! mg*ml) and !&/ (%*!%) S. Paratyphi A (ma4imum 1I? being &.6% mg*ml) olkataisolates (


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9Tol:. Pan&susceptible, susceptible to all '= dru"s tested; A, ampicillin; Ac, amo>icillinFclavulanic acid; 4, chloramphenicol;L, co&trimo>a8ole; T, tetracycline; S, streptomycin; 3a, nalidi>ic

acid; 4i, ciproo>acin; E, oo>acin; 6e, levoo>acin.doiD'$.'(='F+ournal.pone.$'$'(1=."$$(

2istribution of 1I?s of nly one S. Typhi isolate (non-123) harboring aselftransmissible (conjugative) %& kb plasmid of Inc@ type

shoed transfer of resistance to tetracycline and co-trimo4a5ole

(Table %). 0imilar transferable %& kb plasmid in S. Typhi has beenreported from 2elhi '6. S. Paratyphi A study isolates (!'/$#*!%) harbored single plasmid of either !.% or +.% kb si5e. 0ingle

small plasmid of si5e range ".6 to +.7 12a in S. Paratyphi A asreported from Bapan earlier '7.


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Prgan 7! +'#,+%+.

!. >chiai 3C$ Acosta ?B$ 2anovaro-Folliday 1? (!&&7) A study of typhoidfeverin five Asian countries disease burden and implications for control. 9ullorld Fealth >rgan 7# !#&,!#7.

+. 9hattacharya 00$ 2as D$ ?houdhury 9 (!&"") >ccurrence QantibiogramSalmonella Typhi Q S. Paratyphi A isolated from 3ourkela$>rissa. Ind B 1ed 1icrobiol "++ '+",'++.

'. ;erma 0$ Thakur 0$ anga A$ 0ingh =$ =upta P (!&"&) Emerging SalmonellaParatyphi A enteric fever and changing trends in antimicrobial resistance patternof Salmonella in 0himla. Indian B 1ed 1icrobiol !7 %",%+.

%. u $ ang F$ Cu B$ u B (!&"&) =enetic diversity of Salmonella Typhi andParatyphi in 0hen5hen$ ?hina from !&&! to !&&6. 91? 1icrobiol "& +!,+7.

#. orld Fealth >rgani5ation Indian @etork for 0urveillance of

Antimicrobial3esistance (!&"!) Antibiogram of Salmonella entericaserovar Typhi and Salmonella enterica serovar Paratyphi A a multi-centrestudy from India. F> 0outh-East Asia B Public Fealth " "7!,"77.

6. 0aha 13$ 2utta P$ 1itra D$ 9hattacharya 0$ 3asaily 3$ et al. ("::!)>ccurrence of multi-drug resistant Salmonella Typhi in ?alcutta. Indian B1ed 3es :% "6:,"7&.

7. Pillai P$ Prakash ("::+) ?urrent status of drug resistance and phage typesof Salmonella Typhi in India. Indian B 1ed 3es :6 "%',"%7.

:. 1ohanty 0$ 3enuka $ 0ood 0$ 2as 9$ apil A (!&) Antibiogram patternand seasonality of Salmonella serotypes in a @orth Indian tertiary carehospital. Epidemiol Infect "+' :#",:##.

"&. 1ene5es =A$ Farish 9@$ han 1A$ =oessens F$ Fays BP (!&"")

Antimicrobial resistance trends in blood culture positive Salmonella Typhiisolates from Pondicherry$ India$ !&&%,!&&:. ?lin 1icrobiol Infect "7(+) !+:, !'%.

"". ?handel 20$ ?haudhry 3 (!&&") Enteric fever treatment failures A globalconcern. Emerg Infect 2is 6 6#!,6#+.

"!. ?rump BA$ retsinger $ =ay $ Foekstra 31$ ;ugia 2B$ et al. (!&&7) ?linicalresponse and outcome of infection ith 0almonella enterica serotype Typhi ithdecreased susceptibility to fluoro8uinolones a Dnited 0tates


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":. 3ai 0$ Bain 0$ Prasad @$ =hosal D$ 2hole T@ (!&"!) 3ationale of a5ithromycin prescribing practices for enteric fever in India. Indian B 1ed 3es+&(") +&,++.

!&. ;lieghe E3$ Phe T$ 2e 0met 9$ ;eng ?F$ ham ? (!&"!) A5ithromycin andciproflo4acin resistance in Salmonella bloodstream infections in ?ambodianadults. PCo0 @egl Trop 2is #("!) e":++. doi"&."+6"*journal.pntd.&&&":++.

!". Ploy 1?$ ?hainier 2$ Tran Thi @F$ Poilane I$ ?ruaud P$ et al. (!&&+)

Integron-associated antibiotic resistance in Salmonella enterica serovar Typhi from Asia. Antimicrob Agents ?hemother '6 "'!6,"'!:.

!!. ain B$ 2iem @ga CT$ idgell ?$ Bames $ ?22*7+.+. orld Fealth >rgani5ation$ =eneva$ 0it5erland.

!:. ?C0I (!&"+) Performance 0tandards for Antimicrobial 0usceptibility TestingNTenty-Third Informational 0upplement. ?C0I document 1"&&-0!+. ayne$PA ?linical and Caboratory 0tandards Institute.

+&. 0jolund-arlsson 1$ Boyce $ 9lickenstaff $ 9all T$ Faro B$ et al. (!&"")Antimicrobial susceptibility to a5ithromycin among Salmonella entericaisolates from the Dnited 0tates. Antimicrob Agents ?hemother %% +:7%,+:7:.

+". ado ?I$ Ciu 0C (":7") 3apid procedure for detection and isolation of large andsmall plasmids. B 9acteriol "'% "+#%,"+6+.

+!. ?arattoli A$ 9ertini A$ ;illa C$ neri Availablehttp**.pulsenetinternational.org*0ite?ollection2ocuments* pfge*%/!&"S%/!&!S%/!&'[email protected]. Accessed!&"+ 1ar !'.

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