Characterization of Vibrio cholerae isolates from 1976 to 2013 in Shandong Province, China

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Medical

Microbiology

Characterization

of

Vibrio

cholerae

isolates

from

1976

to

2013

in

Shandong

Province,

China

Hui

Lü

_,

_Yuqi

_Yuan

_,

_Na

_Sun

_,

_Zhenwang

_Bi

_,

_Bing

_Guan

_,

_Kun

_Shao

_,

Tongzhan

Wang

_,

_Zhenqiang

_Bi

a_{ShandongCenterforDiseaseControlandPrevention,ShandongProvincialKeyLaboratoryofInfectiousDiseaseControlandPrevention,}

Jinan,China

b_{AcademyofPreventiveMedicine,ShandongUniversity,Jinan,China} c_{TheSecondHospitalofShandongUniversity,Jinan,China}

d_{DepartmentofEpidemiologyandHealthStatistics,SchoolofPublicHealth,ShandongUniversity,Jinan,China}

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Articlehistory:

Received7August2015 Accepted9June2016

Availableonline10October2016 AssociateEditor:Elizabethde AndradeMarques Keywords: Vibriocholerae Epidemiology PFGE Virulencegenes

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Choleracontinuestobeaseriouspublichealthissueindevelopingcountries.Weanalyzed theepidemiologicaldataofcholerafrom1976to2013inShandongProvince,aneastern coastalareaofChina.Atotalof250VibriocholeraeisolateswereselectedforPCRanalysis ofvirulencegenesandpulsed-fieldgelelectrophoresis(PFGE).Theanalysisofthevirulence genesshowedthatthepositiveratesfortcpAandtcpIwerethehighestamongstrainsfrom thesouthwestregion,whichhadthehighestincidencerateofcholera.Lowpositiverates fortcpA,tcpIandctxABamongisolatesfromafter2000maybeaninfluencingfactor con-tributingtothecontemporarydeclineincholeraincidencerates.Spatiotemporalserotype shifts(Ogawa,Inaba,Ogawa,InabaandO139)generallycorrelatedwiththevariationsin thePFGEpatterns(PIV,PIIIc,PIa,PIIIb,PIIIa,PIb,andPII).O1strainsfromdifferentyearsor regionsalsohadsimilarPFGEpatterns,whileO139strainsexclusivelyformedonecluster anddifferedfromallotherO1strains.ThesedataindicatethatV.choleraeisolatesin Shan-dongProvincehavecontinuallyundergonespatiotemporalchanges.Theserotypeswitching betweenOgawa andInabaoriginatedfromindigenous strains,whilethe emergenceof serogroupO139appearedtobeunrelatedtoendemicV.choleraeO1strains.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mail:bzq63@163.com(Z.Bi).

1 _{Theseauthorscontributedequallytothiswork.}

http://dx.doi.org/10.1016/j.bjm.2016.09.013

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Vibrio cholerae is a Gram-negative bacterium that causes cholera,anacutelife-threateningdiarrheadisease.Todate, over200serogroupsofV.choleraehavebeenrecognized. How-ever, onlythe O1and O139serogroupsare associatedwith epidemicandpandemiccholerainhumans.Basedon pheno-typicandgenotypicdifferences,theO1serogroupisfurther dividedinto twobiotypes,namely,classical andElTor,and twomajorserotypes,namely,OgawaandInaba.Sevencholera pandemicshaveoccurredgloballysince1817,andthefirstsix pandemicsarebelievedtobecausedbyO1classicalbiotype strains.1_{Itwasnotuntil1961thatO1ElTorstrainsbecame} predominantandinitiatedtheseventhcholerapandemic.In 1992,adistinctpathogenicserogroup,O139,wasdetectedin bothBangladeshandIndia.2_{Recently,atypicalorvariantEl} TorbiotypeswithattributesofboththeclassicalandElTor biotypesemergedandbecamepredominantglobally.The Mat-labvariantswerefirstisolatedinMatlab,Bangladeshbetween 1991and1994.3_{OthervariantElTorisolates,includingaltered} ElTor,MozambiqueElTorandhybridElTorstrains,havebeen identifiedinAsia,AfricaandAmerica.4–7

During the ongoing seventh cholera pandemic, cholera caused byO1El Torarrived inChina in1964 and reached Shandong Province in that same year. After 1964, cholera sweptthroughtheentireShandongProvince.Unfortunately, thereportsorrecordsfrom1966to1975wereincompletedue toirregularitiesinroutinework.In1997,V.choleraeserogroup O139wasisolatedfromthecityofLaiyangforthefirsttime inShandongProvince.Thereafter,serogroupO139hasbeen thepredominantserogroupdetectedincholeraepidemicsin theprovince.Inthisstudy,epidemiologicaldataofcholerain ShandongProvincefrom1976to2013werecollectedand ana-lyzed,andonthisbasis,representativestrainsofV.cholerae

fromdifferentareasandyearswereselectedtoscreenthe vir-ulencegenesbyPCRamplificationandstudythemolecular subtypingusingpulsed-fieldgelelectrophoresis(PFGE).

Materials

and

methods

AllrecordsorreportsofcholerainShandongProvincesince 1976werecollected,andadatabaseincludingserotype,time, districtandepidemicintensity(sporadicoroutbreak,meaning asinglecaseormultiplecases,respectively,withan epidemi-ologicallinkthatemergedatthemaximumincubationperiod) wasestablished.

Representativestrainsandidentification

Atleastonestrainandnomorethanthreestrainswere cho-senfromeachdistrictwithacholeraoutbreak.Clinicalstrains fromsporadiccasesinvariousdistrictswerealsoincluded. The identification of the isolates was completed. Briefly, lyophilizedV.choleraewereenrichedinalkalinepeptonewater for 6h and then streaked on thiosulfate citrate bile salts sucrose(TCBS)agar (Oxoid Ltd,Hampshire, UK)plates and

Fig.1–GeographicdistributionofcholerainShandong Provincesince1976.Theproportionvalueinthekeyrefers tothepercentageofcholerapatientsineachcity.

incubatedfor24hat37◦C.Typicalgoldenyellowcolonieswere identifiedbybiochemicalreactionandserotyping.

Pulsed-fieldgelelectrophoresis(PFGE)

PFGE was performed according to the PulseNet standard-ized protocol for V. cholerae.8 _{Genomic DNA of V. cholerae} was digested with 50U of the restriction enzyme Not I

(New England Biolabs, Ipswich, MA, USA) at 50◦C for 4h. Electrophoresis was performed using a CHEF-DRIII system (Bio-Rad, USA). Images were captured on a Gel Doc 2000 system(Bio-Rad,USA).Tiffimageswereanalyzedusing BioN-umericsv.6.6software(AppliedMaths).Thebandingsimilarity wasdeterminedbytheDicecoefficientwitha1.0%band posi-tiontolerance,andadendrogramwasconstructedusingthe unweighted-pairgroupmethodwithanarithmeticmean algo-rithm(UPGMA).

Virulencegenesdetecting

Chromosomal DNA was extracted from V. cholerae using a DNeasyBlood&Tissuekit(QIAGEN,Germany)asaDNA tem-plateforPCRassay.Thesixvirulencegenes(ctxAB,tcpA,tcpI, rtxA,hlyA and toxR)were amplifiedusinga S1000Thermal Cycler(Bio-Rad,USA)asdescribedpreviously.9–12

Dataanalysis

Differencesofdistributionsinserotypeandvirulencegenes among V. cholerae were assessed with chi-square analysis usingSASv.10.1software.Differenceswereconsidered signif-icantforpvalues<0.05.

Results

Epidemiologicalcharacteristics

Nearly every district of Shandong Province has suffered cholera epidemics since 1976 (Fig. 1). The predominant serotypesince1976hadchangedfromOgawa(1976–1979)to Inaba(1980–1989),Ogawa(1993–1999),Inaba(2001),andthen O139(1997–2013).Citiesinthesouthwest(Heze,Zaozhuang,

Fig.2–Proportionsofcholerapatientsindifferentyearsin ShandongProvince.

andJining)andthenortheast(Dezhou,BinzhouandQingdao) ofShandongProvincewerethehardesthitareas,whereas con-siderablyfewercholeracasesoccurredincitiesinthecentral region(Jinan,Zibo,Laiwuand Weifang).Moreover,58.1%of thepatientsinfectedwithserotypeOgawalivedinthenorth, while85.8%ofthepatientsinfectedwithserotypeInabalived inthesouthwest(Table1).

Shandong Provincehas witnessedtwo peaks incholera epidemicssince1976(Fig.2).One epidemicwascaused by serotypeInabainthesouthwesternregionduring1983–1984, andtheotherwascausedbyserotypeOgawalocatedmainly in the northern districts in 1994. Unlike explosive epi-demicscausedbyserogroupO1priorto2001,choleracaused byserogroup O139during 1997–2013 was usuallysporadic. Accordingly,thenumberofcholeracasesdecreased signifi-cantlyinthe21stcentury.

Representativestrains

A total of 218 representative clinical strains were selected between1976and2013from17districtsinShandongProvince. Wealsoincluded32environmentalstrainsascontrols.Among these250isolates,121strainsbelongedtoserotypeOgawa,116 strainsbelongedtoserotypeInaba,and13strainsbelonged toserogroupO139.Fig.3showsthelocationsof representa-tiveV.choleraestrainswithdifferentserotypesfromdifferent decades.

Distributionofvirulencegenes

All250isolateswereanalyzedusingPCRtodetectthe pres-ence of ctxAB,tcpA, tcpI, toxR, rtxA and hlyA (Table 1).The positiveratesofctxAB,tcpIandtcpAamongclinicalisolates (76.6%,90.4%and92.2%,respectively)weremuchhigherthan thoseamongenvironmentalstrains(21.9%,56.3%and56.3%, respectively).NearlyallthetestedstrainscarriedthetoxR,rtxA

andhlyAvirulencegenes.Manymoreofthestrainsfrom out-breakspossessedthetcpIandtcpAvirulencegenescompared to thestrains from sporadiccases. However,no significant differencewasobservedinthedistributionofctxABbetween outbreakstrainsandsporadicisolates.Inaddition,the posi-tiveratesfortcpAandtcpIwerehighestinthesouthwestern region.Intheclinicalstrains,thedetectionratesofctxAB,tcpA

andtcpIincreasedfrom1976to2000anddecreasedafter2000.

Fig.3–LocationsofrepresentativeV.choleraestrainswithdifferentserotypescategorizedbydecadesinShandongProvince: (A)representativestrainsfrom1976to1979weremainlyserotypeOgawaandlocatedinthesouthandnorthofShandong Province;(B)representativestrainsfrom1980to1989weremainlyserotypeInabaandlocatedinthesouthwesternregions; (C)representativestrainsfrom1990to1999weremainlyserotypeOgawaandspreadacrosstheentireprovince;and(D) representativestrainsfromthe21stcenturyweremainlygeographicallyfragmentedO139strains.

70 PFGE-Not1 80 90 87.6 86.0 89.2 85.1 85.8 88.6 78.6 75.5 71.7 P III P II P I P IV 100 V. cholerae O1 serotype inaba clinical isolates V. cholerae O139 isolates V. cholerae O1 serotype ogawa clinical isolates Enviromental isolates and a few

clinical strains

Fig.4–OveralldendrogramofV.CholeraeinShandong Province.

Interestingly,theprevalencesofthesixvirulencegeneswere unrelatedtoserotype.

Clusteringanalysisandgeneticrelatedness

All250isolatesweredifferentiatedinto179pulsotypesusing PFGEwithNotI-digestedgenomicDNA.UPGMAclusteredthe pulsotypesinto 4majorgroups, namely,PI,PII,PIIIandPIV (Fig.4).AgeneralassociationbetweenserotypeandPFGEtypes wasobserved,andtheserotypeswitchedaccordingtoyearand regionofisolation(Fig.5).

From1976to1979,thepredominantserotypeOgawawas distributedinthenortheasterndistricts.Mostclinicalstrains fromnorthernregionsin1976and1977andeasternpartsin 1979clusteredintogroupPIV,whichwasalsotruefor84.3% oftheenvironmentalisolates. Allthe isolatesingroupPIV wereabsentofctxABandtcpAandshowed67.95%similarity. Theclinicalisolatesfromthenorthernareasin1978harbored

ctxABandtcpAandformedsubgroupPIIIc.

Choleraepidemicsduring1980–1989were locatedinthe southwesterndistrictswiththepredominantserotypeInaba. Duringthe1980s,89%oftheclinicalstrainsandone environ-mentalstrainclusteredintosubgroupPIawith69pulsotypes. Strainscollectedfromthesamedistrictindifferentyearsor fromadjoining districtsatthe sametime typicallygavean identicalpulsotype(PIa1–PIa12).Additionally,theoverall sim-ilarityofthisclusterwas93.3%.

SerotypeOgawareplacedserotypeInabaandprevailedin the northeastofShandong Provincefrom 1993to1999. All thestrainsfrom1993to1997inthenortheasternareasand 2strainsin1986clusteredintosubgroupPIIIb.However,inthe followingtwoyears,choleracasesweredistributed through-outtheentireprovinceandallthestrainsformedsubgroup PIIIa.

SerotypeInabastrainsreemergedinthecentraland north-ern districts in 2001. All the strains from 2001 and one environmentalstrainfrom1994weregroupedtogether.The O139strainsfrom1997to2013formedsubgroupPIIa,inwhich 2clinicalstrainscollectedfromZiboin2004sharedthesame pulsotypewithoneclinicalisolatecollectedfromJiningin2005 (PIIa1).Additionally,strainsisolatedfromonepatientin2013 andfromthecookingutensilsusedbythispatientshowedthe samePFGEpattern(PIIb1).

Discussion

From epidemiological data, 2 different multi-year cholera epidemics were distinguished in Shandong Province since 1976(Fig.2),whichsharedthesamepatternwiththeentire nation.OneepidemicwasmainlycausedbyserotypeInaba inthesouthwesternregionsfrom1980to1989,andtheother epidemicwascausedbyserotypeOgawamainlyinthe north-ern districts from 1993 to 1999. Poor economic level and sanitaryconditionmayhaveresultedinthe2multi-year epi-demicsthatcorrespondedwiththecharacteristicgeographic distribution.13 _{V. cholerae exists within an aquatic natural} reservoir.Environmentalfactors,suchasclimateandweather, playanimportantroleintheevolutionofV.choleraeandare probablyinvolvedinthedifferentgeographicaldistributions betweenserotypeOgawaandInabainShandongProvince.14

Inclinicalstrainscollectedpriorto2000,thedetectionrates ofctxAB,tcpAand tcpIincreasedover time,suggestingthat selectivepressuresoftheenvironmentmayexistinShandong Province.15LargernumbersofclinicalstrainscarriedthectxAB, tcpIandtcpAgenescomparedtoenvironmentalstrains, sup-portingthefactthatcholeratoxin(CTX)andtoxincoregulated pilus(TCP)aretheprincipalvirulencefactorsofV.cholerae.16 Therefore, the lowpositive rates forctxAB (64.3%)and tcpI

(71.4%)intheisolatesfromaftertheyear2000wouldlikelybe aninfluencingfactorinthedeclineincholeraincidencerates (Table1).However,23.4%oftheclinicalisolatesweredevoid ofctxAB,indicatingthatotherpathogenicmechanisms,such ashlyAandRTXgenesencodinghemolysinorcytolysin,may playsomeroleinthediseaseprocess.17,18_{Threegenes,toxR,}

rtxAandhlyA,werecarriedinnearlyallthetestedstrains, sug-gestingthattheyareessentialforpathogenicity,especiallyin strainsthatlackctxAB.19

Thepositiverates fortcpAandtcpIamongthe outbreak strains were higherthan those from the sporadic isolates. Meanwhile,thepositiveratesfortcpAandtcpIwerehighestin thesouthwesternregion,whichhadsufferedthemostsevere epidemicscomparedtotherestoftheprovince.This distribu-tionpatternprovedthatthevirulencegenestcpAandtcpIwere associatedwiththeepidemicintensityofcholerainShandong Province.Overall,thedetectionratesforctxAB,tcpAandtcpI

95 100 80 90 100 90 Pla1 PlIIa1 PlIIa2 PlIIb1 PlIIb2 PlIIb3 PlIIb4 PlIIb5 PlIIb6 PlIIc1 PlVc1 P lII a P lII b P lII c P lV a P lV b P lV c P lV d Pla2 Pla3 Pla4 Pla5 Pla6 Pla7 Plb1 Plla1 Pllb1 P l b P ll a P ll b P l P lI P Il 95 100 P lV P l a Pla8 Pla9 Pla10 Pla11 Pla12 100

Fig.5–DendrogramofV.choleraefromeachPFGEgroupinShandongProvince.

whichrelatestothespatiotemporalcharacteristicsofcholera inShandongProvince.

PFGEanalysisdivided V.choleraeisolatesfromShandong Provinceintofourgroupsbasedonserotypeandexpressionof virulencegenes(Fig.4).ThePFGEgroupingpatternchanged with time and space, which was in agreement with the serotypetransitionofV.cholerae.Thenon-toxigenicisolates, containingthemajorityoftheenvironmentalstrainsand a fewoftheclinicalstrains,exhibitedgreaterdiversitythanthe toxigenicisolates. However, the environmentalstrain from 1981,possessingctxABandtcpA,hadthesamepulsotypeas oneclinicalstrainfrom1983,indicatingthatV.choleraestrains wereautochthonoustotheaquaticenvironment.Theclinical Ogawastrainfrom1993showed98%similaritywith2strains from 1986, revealing their clonal relation. A close genetic relationship(96.8%similarity)wasalsoobservedbetweena serotypeInabastrainthatremergedin2001andone environ-mentalstrainfromthe1990s.Theseresultsdemonstratedthat theevolutionofindigenousV.choleraeledtoashiftbetween serotypesInabaandOgawa.

Serogroup O139 first appearedin Shandong Province in 1997andhasreplacedO1strainsinsporadiccasessince2002. ThePFGEpatternsoftheO139strainsdifferedfromthe pro-filesdisplayedbytheO1strainsofclinicalandenvironmental origin,whichagreedwiththepresumptionthatthe progeni-torisolateofserogroupO139inChinawasintroducedfrom India.20 _{Further research, suchas wholegenome} sequenc-ing,mayhelpinunderstandingtheoriginoftheO139strains from Shandong Province.21 _{The O139 strains collected in} 2013 were separated from those collected in the past ten years witha unique pulsotypepredominating inChinafor the most recent two years,which indicated that the O139 strains from 2013 were imported from other provinces of China.

Insummary,the distributionsofserotype andvirulence genesinV.choleraeisolatescollectedfromShandongProvince, whichwereassociatedwithmolecularPFGEtyping,have alter-natedcontinuallysince1976.Thisinformationcouldbeuseful forunderstandingtheevolutionofV.choleraeandfor control-lingnewpotentiallyepidemicclones.

Table1–DistributionsofserotypesandvirulencegenesamongV.choleraeisolatesfromShandongProvince.

Factor Percentageofisolatescarryingvirulencegene Percentageofserotype

hlyA ctxAB toxR tcpI rtxA tcpA Ogawa Inaba O139

Source Patient 100 76.6a _{100 90.4}a _{99.1 92.2}a Environment 100 21.9 100 56.3 100 56.3 Serotype Inaba 100 70.4 100 92.6 99.1 95.4 Ogawa 100 83.2 100 89.1 99 89.1 O139 100 77.8 100 77.8 100 88.9 Temporaldistribution 1976–1979 100 55.6a _{100 61.1}a _{100 61.1}a _10.9a _0.8a ₀a 1980–1989 100 68.8 100 92.7 99.1 93.6 51.3 96.2 0 1990–1999 100 94.8 100 97.4 98.7 97.4 37.5 0.8 7.1 After2000 100 64.3 100 71.4 100 92.9 0.2 2.3 92.9 Spatialdistribution East 100 84.6 100 82.1a _{100 84.6}a _14.8a _3.4a _21.4a West 100 73.5 100 98.5 100 98.5 4.4 49.2 14.3 South 100 76.7 100 91.7 96.7 93.3 12.1 36.6 35.7 North 100 70 100 85 100 90 58.1 4.2 0 Central 100 77.4 100 83.9 100 87.1 10.7 6.5 28.6 Epidemicpatterns Sporadic 100 77 100 86.1a _{99.2 88.5}a ₃₉a _19.4a ₁₀₀a Outbreak 100 76 100 95.8 99 96.9 62 80.6 0

TheeasternregionofShandongProvinceincludesQingdao,Yantai,WeihaiandRizhao;thewestincludesLiaochengandHeze;thesouth includesJining,ZaozhuangandLinyi;thenorthincludesDezhou,BinzhouandDongying;andthecentralregionincludesJinan,Zibo,Laiwu, TaianandWeifang.

a _{Representsastatisticallysignificantdifferenceamongvaluesinthecorrespondinggroup(p<0.05).}

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

This work was supported by Grant No. 81202260 of the National NaturalScienceFoundationofChina(http://www. nsfc.gov.cn), and Grant Nos. 2012ZX10004-201-002 and 2012ZX10004215 of the National Science and Technology MajorProjectofChina(http://www.nmp.gov.cn).

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