AM933172jrzj

RESEARCHARTICLEOpenAccessNextgenerationgenomesequencingrevealsphylogeneticcladeswithdifferentlevelofvirulenceamongSalmonellaTyphimuriumclinicalhumanisolatesinHongKongChiKeungCheng1,ManKitCheung1,WenyanNong1,PatrickTikWanLaw1,JingQin1,JuliaMei-LunLing2,KaiManKam3,4,WilliamManWaiCheung1andHoiShanKwan1*AbstractBackground:SalmonellaTyphimuriumisfrequentlyisolatedfromfoodborneinfectioncasesinHongKong,butthelackofgenomesequenceshashinderedin-depthepidemiologicalandphylogeneticstudies.
Inthisstudy,wesoughttoreconstructthephylogeneticrelationshipandinvestigatethedistributionandmutationpatternsofvirulencedeterminantsamonglocalS.
Typhimuriumclinicalisolatesusingtheirgenomesequences.
Results:Weobtainedgenomesequencesof20S.
Typhimuriumclinicalisolatesfromalocalhospitalclusterusinga454GSFLXTitaniumsequencingplatform.
PhylogeneticanalysiswasperformedbasedonsinglenucleotidepolymorphismpositionsofthecoregenomeagainstthereferencestrainLT2.
Antimicrobialsusceptibilitywasdeterminedusingminimalinhibitoryconcentrationforfiveantimicrobialagentsandanalysesofvirulencedeterminantswereperformedthroughreferencingtovariousdatabases.
Throughphylogeneticanalysis,werevealedtwodistinctcladesofS.
TyphimuriumisolatesandthreeoutliersinHongKong,whichdifferremarkablyinantimicrobialsusceptibilityandpresentationandmutationsofvirulencedeterminants.
ThelocalisolateswerenotcloselyrelatedtomanyofthepreviouslysequencedS.
Typhimuriumisolates,exceptLT2.
Astheisolatesinthetwocladesspannedover10yearsofisolation,theyprobablyrepresentendemicstrains.
TheoutliersarepossiblyintroducedfromoutsideofHongKong.
ThecloserelatednessofmembersinoneofthecladestoLT2andtheJapanesestoolisolateT000240suggeststhepotentialreemergenceofLT2progenyinregionsnearby.
Conclusions:Ourstudydemonstratedtheutilityofnext-generationsequencingcoupledtotraditionalmicrobiologicaltestingmethodinaretrospectiveepidemiologicalstudyinvolvingmultipleclinicalisolates.
Theevolutionofmultidrug-andciprofloxacin-resistantstrainsamongthemorevirulentcladeisalsoanincreasingconcern.
Keywords:Foodborneinfection,Epidemiology,Phylogeny,Virulencedeterminants,Singlenucleotidepolymorphism,Antimicrobialsusceptibility*Correspondence:hoishankwan@cuhk.
edu.
hkEqualcontributors1SchoolofLifeSciences,TheChineseUniversityofHongKong,HongKongSAR,ChinaFulllistofauthorinformationisavailableattheendofthearticle2015Chengetal.
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Chengetal.
BMCGenomics(2015)16:688DOI10.
1186/s12864-015-1900-yBackgroundSalmonellafoodborneinfectionisacommonbutim-portantpublichealthissueworldwide.
Amongthemanyserovars,SalmonellaTyphimuriumisfrequentlyisolatedfromoutbreaksasoneofthecommonbacterialcausa-tiveagents.
TheWorldHealthOrganizationhasalsoemphasizedtherisingconcernofmultidrugresistanceinthisnon-typhoidSalmonellaserovar,whichpotentiallyaccountsforthetransferofantimicrobialresistancetootherhumanpathogens[1].
Withthecontinualreduc-tioninthecostforhigh-throughputgenomesequencing,thousandsofgenomesofpathogenicbacteriahavenowbeensequencedandSalmonellaisofnoexception[2].
Inadditiontotheconventionalanalysisofantimicrobialresistanceprofiles,couplingofgenomesequencingtophylogeneticanalysishasopenednewtrendsofin-depthepidemiologicalstudiesatbothregionalandgloballevels.
Overthepastfewyears,hundredsofgenomesofvariousSalmonellaserovars,includingTyphimurium[3],Enteritidis[4],Typhi[5],Newport[6],Heidelberg[7],andPullorum[8],weresequencedtofacilitateevolu-tionarystudies,aswellasepidemiologicalandpathogen-icityinvestigationsinthisimportantpathogen.
DespitetheavailabilityofgenomesequencesforS.
Typhi-muriumisolatesfromallaroundtheworld,theJapanesestrainT000240remainedastheonlysequencedandpub-lishedisolatefromnortheasternAsia[9].
Herewereporttheuseofhigh-throughputgenomesequencing,coupledtotraditionalmicrobiologicaltestingmethod,inaretrospect-ivestudyofSalmonellaTyphimuriumstrainsisolatedfromsubjectshospitalizedinHongKongoverthepasttwode-cades.
Specifically,wereconstructedthephylogeneticrela-tionshipandinvestigatedthedistributionandmutationpatternsofvirulencedeterminantsamong20localisolates.
MethodsBacterialstrainsAtotalof20S.
Typhimuriumisolates(Table1)wereob-tainedfrompatientsadmittedtothehospitalsoftheNewTerritoriesEastClusteroftheHospitalAuthorityinHongKongbetween1993and2007.
Writteninformedconsentforusingthebloodandstoolsamplesinthestudywasobtainedfromallparticipants.
Sevenbloodisolates,threeofwhichisolatedinthemid90'sandtherestisolatedinthemid00's,and13stoolisolateswereobtainedbystand-ardprocedures.
Thebloodisolatesarerepresentativesofthecirculatingclonesduringthesamplingperiodsandactasrepresentativesofsystemicinfectionwhereasthestoolisolateswereusedasthegeneticbackgroundforcomparisonpurpose.
The10-yearspanbetweenisolatecollectionsallowsdeterminationofendemicityoftheselectedstrains.
Theseisolateswereconfirmedbio-chemicallybytheAP120Esystem(bioMérieuxS.
A.
,MontalieuVercieu,France).
GenomesequencinganddenovoassemblyGenomicDNAfromtheisolateswasextractedusingPrepManUltraReagent(AppliedBiosystems)accordingtothemanufacturer'sinstructions.
Whole-genomeshot-gunsequencingwasperformedona454GSFLXTitaniumplatform(RocheDiagnostics)[10].
BasessequencedandcorrespondingqualityvalueswerecalledanddeliveredinstandardformatbyGSFLXfordownstreambioinformaticanalyses.
SequencereadswereassembleddenovousingNewblerassembler(RocheDiagnostics).
SNPsextractionandphylogeneticanalysisAllSNPpositionswereobtainedbyaligningthegenomesequencesofthe20isolateswiththereferencestrainLT2[11]chromosomeusingMauveand454GSReferenceMapper[10].
RawSNPcallswerefilteredtoensurethatatleast90%ofthereadssupporttheSNP.
SNPscalledinphagesequencesandrepetitiveregionsofthereferencegenomewereexcluded.
OnlySNPslocatedintheSalmon-ellacoregenes[12]wereincludedinthephylogeneticanalysis.
AllremainingSNPswereconcatenatedtogener-ateasinglepseudo-sequence.
PhylogeneticanalyseswereconductedinMEGA(version5.
21)[13]andphylogenetictreeswerereconstructedusingtheMaximumParsimony(MP)methodwithaheuristicsearchbasedontheTreeTable1InformationofpatientsandcorrespondingS.
TyphimuriumclinicalisolatesIsolateSourceYearofisolationPatientagePatientsexST728/07Blood20072FST4024/07Blood200754MST4848/06Blood200668MST2850/05Blood200581MST4650/95Blood19954MST6988/94Blood199478FST8493/93Blood199314FST372/06Stool200615MST1660/06Stool20068MST2286/06Stool20061FST486/06Stool20068FST2533/06Stool200618MST1489/06Stool200628FST4650/06Stool20065FST2143/05Stool20052FST4329/05Stool20051FST4038/02Stool200237MST3363/96Stool19967MST3858/96Stool199643FST2287/95Stool199511FChengetal.
BMCGenomics(2015)16:688Page2of8BisectionandReconnection(TBR)approach.
SalmonellaEnteritidisPT4(GenBankAccessionAM933172)andSalmonellaCholeraesuisSC-B67(GenBankAccessionAE017220)wereusedasoutgroups.
Nodalsupportswereinferredfrom500bootstrapreplicates.
AntimicrobialsresistanceprofilingThe20S.
Typhimuriumisolatesweretestedforsuscepti-bilitytoampicillin,gentamicin,chloramphenicol,tri-methoprim,andciprofloxacinbyanagardilutionmethodaccordingtotherecommendationsoftheClinicalandLaboratoryStandardsInstitute(CLSI)[14].
Isolateswithminimalinhibitoryconcentrations(MICs)greaterthanthoseforsusceptiblestrainsassuggestedbyCLSIwereregardedasresistant.
Multidrugresistancewasdefinedasresistanttothreeormoreoftheantimicrobialstested.
VirulencedeterminantsanalysisGenesandmutationsresponsibleforantimicrobialresist-ancewereretrievedfromtheliteratureandcomparedamongthe20isolates.
VirulencefactorsandSalmonellaPathogenicityIslands(SPIs)forSalmonellapathogenicitywereobtainedfromtheVirulenceFactorsDatabase(VFDB)(http://www.
mgc.
ac.
cn/VFs/)andalignedagainsteachoftherespectivegenomesequencesforthedetectionofgeneticvariations[15].
ProphageelementsfortheisolateswereidentifiedbythewebserverPHAgeSearchTool(PHAST)(http://phast.
wishartlab.
com/)[16].
ResultsPhylogenetictreeanalysisrevealedtwomajorphylogeneticcladesinHongKongGenomesof20localS.
Typhimuriumisolateswerese-quencedherewithanaveragedepthof38*(Table2).
TheSNP-basedphylogenetictreesgroupedtheS.
Typhimur-iumisolatesintotwomajorphylogeneticclades(Fig.
1,Additionalfile1,Additionalfile2andAdditionalfile3).
CladeAconsistedof10isolateswithapredominanceofninestoolisolatesandonlyasinglebloodisolate,whereascladeBconsistedofatotalofsevenisolatesincludingthreebloodisolatesandfourstoolisolates.
Theremainingthreeisolatesappearedtobesporadicinfectionsandtheywerealsodistantlyrelatedbythemselves.
Intriguingly,theywereallbloodisolates.
Theyearofisolationdidnotseemtobeanimportantdeterminingfactorinthephyl-ogeny,asisolatesretrievedfromthe90'sand00'swerebothfoundineachoftheclades.
ContrastingantimicrobialsresistanceprofilesamongphylogeneticcladesThe20isolatesweretestedfortheirsusceptibilitytofiveantimicrobialsfromdifferentclasses(Table3).
FifteenofTable2Statisticsforthe20sequencedS.
TyphimuriumgenomesIsolateTotallength(bp)Readno.
Contigno.
N50(bp)FoldcoverageGenBankaccessionST728/074,674,705587,02251297,45854JRYT00000000ST4024/074,710,407410,92342324,70738JRYU00000000ST4848/064,835,9481,116,35133412,17687AUXE00000000ST2850/054,823,833370,56349324,99633JRZV00000000ST4650/954,839,422364,94832413,04331JRZX00000000ST6988/944,821,910570,72749311,18547JRZW00000000ST8493/934,821,249214,09658149,90218JRZU00000000ST372/064,812,346158,27282100,49214JRZT00000000ST1660/064,817,227689,23133412,26955JRZS00000000ST2286/064,680,742274,34464226,04725JRZR00000000ST486/064,694,852797,42890297,45771JRZQ00000000ST2533/064,671,245209,11664197,51218JRZP00000000ST1489/064,667,695240,65057225,75918JRZO00000000ST4650/064,667,933230,84156226,04817JRZN00000000ST2143/054,856,278775,03437458,26768JRZM00000000ST4329/054,667,023216,06659192,71116JRZL00000000ST4038/024,675,722301,88259223,18725JRZK00000000ST3363/964,675,601653,49346324,53753JRZJ00000000ST3858/964,672,420341,17245423,11132JRZI00000000ST2287/954,846,386371,97136412,23833JRZH00000000Chengetal.
BMCGenomics(2015)16:688Page3of8theisolates(75%)wereresistanttoatleastoneanti-microbialclass.
Morethanhalfofthestrainswerere-sistanttoampicillin(70%),trimethoprim(60%)andchloramphenicol(55%),whilelesswasresistanttocip-rofloxacin(25%)andgentamicin(20%).
Exceptgenta-micin,theproportionofresistancetoampicillin,trimethoprim,chloramphenicol,andciprofloxacinwashigherinthebloodisolates(100,86,71and29%)thanthestoolisolates(54,46,46and23%).
Amongthe20isolates,11(55%)weremultidrug-resistant,inwhichfivewerebloodisolates.
Intriguingly,withrespecttothephylogenetictreeabove,allsevenisolateswithincladeBweremultidrug-resistant,withallofthemresistanttotheolderantimicrobialsampicillinandchloramphenicolandsixofthemresistanttotrimethoprim.
Incontrast,amongthe10isolatesfromcladeA,onlytwoofthemweremultidrug-resistant,withatleasthalfofthemstillsusceptibletotheseolderantimicrobials.
LossofvirulencedeterminantsincladeAisolatesGenomesequenceanalysisrevealedtheabsenceofthevirulenceplasmidpSLT,a~90kbplasmidofLT2whichFig.
1Maximum-parsimonyphylogenetictreeof47S.
TyphimuriumgenomesbasedonSNPsidentifiedbymappingtotheLT2referencegenome.
OnlySNPsinthe"core"geneswereincluded.
ThetreewasrootedusingSalmonellaEnteritidisPT4(GenBankAccessionAM933172)andSalmonellaCholeraesuisSC-B67(GenBankAccessionAE017220).
Redisolates:localbloodisolates;Blueisolates:localstoolisolates;Blackisolates:referenceGenBankisolates.
ThenumberoneachbranchisthenumberofSNPdifferences.
ThescalebarrepresentsthenumberofSNPsChengetal.
BMCGenomics(2015)16:688Page4of8harborsmanyimportantvirulencefactorsincludingthespvlocus,pef(plasmid-encodedfimbriae)locusandthecomplementresistancegenerck[17],inthe10cladeAisolates.
TheSalmonellaPathogenicityIslands(SPIs),whichen-codetwotypeIIIsecretionsystems(T3SS)andanumberofvirulenceeffectors,representanothercategoryofimportantvirulencefactors.
GenomesequencesrevealedthatSPI1-5werepresentinall20isolatesandwerehighlyconservedinsequence.
However,anumberofSNPswerefoundinSPIsinisolatesfromcladeA,forinstance,fhlA(nucleotideposition1916)inSPI1,orf242(pos.
541)andsseC(pos.
1272)inSPI2aswellassugR(pos.
183)andmgtB(pos.
351)inSPI3(Additionalfile4).
TheSNPinsseC,whichwasshowntobeanim-portanteffectorproteintoalterhostcellphysiologyandpromotebacterialsurvival[18],resultedinaprevi-ouslyundescribedGlu424>Aspaminoacidchange.
AnothereffectorproteinsseI/srfH,whichlieswithintheGifsy-2prophage,alsoshowedaSNPatnucleotideposition139andresultedinaAla47>Thraminoacidchange.
Nevertheless,othereffectorproteins,includingthoseencodedoutsideofSPI1andSPI2suchassopBandsopE2,didnotshowanysequencevariation.
ApartfromthepSLTplasmidandSNPsintheSPIs,isolatesincladeAalsocontainedlessgeneticmaterialsarisenfromprophages.
AllisolatesfromcladeBcon-tainedacompletecopyoftheSalmonellaprophagesGifsy-1andGifsy-2[19,20],whereasisolatesfromcladeAcontainedonly~39and~68%geneticmaterialsfromtherespectiveprophages.
Thisapparentreductionofgenomiccontenthadresultedinthelossofseveralgenespreviouslyimplicatedtoinvolveinlong-termsystemicinfectioninmice(STM2585,2586,2596,2597,2635[Gifsy-1])andreplicationinmacrophages(STM1031,1033,1041[Gifsy-1],2585,2589,2595,2599,2603,2605[Gifsy-2])[21,22].
WhilefiveoutofthesevenisolatesfromcladeBcontainedacompletecopyofFels-2,isolatesfromcladeAandthethreespor-adicisolatesdidnotharborthisSalmonellaprophage.
Inaddition,isolatesincladeAhadalsolostatotalof~20%ofgeneticmaterialsfromphageST104comparedtocladeBisolates,whereassporadicisolatesST2850/05andST8493/93didnotharborST104.
Instead,thesetwoisolatescontainedacompletecopyofthephageST64B,whichisalsoidentifiedinmanypreviouslyse-quencedSalmonellaisolatesbutnotinisolatesincladeA,B,LT2andtheJapaneseisolateT000240[9].
Table3Susceptibilitytofiveantimicrobialsforthe20S.
TyphimuriumisolatesIsolateSourceCladeYearofisolationAMPGENCHLTRICIPTotal#ofresistanceST728/07BloodA2007RSSRS2ST4024/07Blood–2007RSRRR4ST4848/06BloodB2006RRRRR5ST2850/05Blood–2005RSSSS1ST4650/95BloodB1995RSRRS3ST6988/94BloodB1994RSRRS3ST8493/93Blood–1993RSRRS3ST372/06StoolB2006RRRRR5ST1660/06StoolB2006RSRRR4ST2286/06StoolA2006RSSRS2ST486/06StoolA2006SSSSS0ST2533/06StoolA2006RRSRS3ST1489/06StoolA2006SSRSS1ST4650/06StoolA2006SSSSS0ST2143/05StoolB2005RSRSR3ST4329/05StoolA2005SSSSS0ST4038/02StoolA2002SSSSS0ST3363/96StoolA1996RSRRS3ST3858/96StoolA1996SSSSS0ST2287/95StoolB1995RRRRS4AMPAmpicillin,GENGentamicin,CHLChloramphenicol,TRITrimethoprim,CIPCiprofloxacinChengetal.
BMCGenomics(2015)16:688Page5of8DiscussionThepotentialreemergenceofLT2progenySalmonellaTyphimuriumisoneofthemostcommonbacterialcausesoffoodborneinfectionsinHongKong,with150–200reportedcaseseachyear.
Nevertheless,genomesoftheseclinicalisolateshaveseldombeense-quenced.
Inthisreport,wepresentgenomesequencesof20S.
TyphimuriumclinicalisolatesinHongKongthroughout1993–2007.
Phylogeneticanalysisindicatedthattwomajorphylogeneticclades(representedbycladeAandBinFig.
1)hadbeencirculatinginHongKongforalmostthepasttwodecades,withsomespor-adicinfectionscausedbyphylogeneticallydistinctisolates.
Weshowedthatseveralofthepreviouslyse-quencedS.
Typhimuriumisolates,includingthehumanisolatesDT104[23]andD23580[24],didnotshowhighphylogeneticrelatednesstoisolateseitherincladeAorB(Fig.
1).
Notably,isolatesfromcladeBshowedre-markablegeneticrelatednesstothelaboratoryreferencestrainLT2,whichwasoriginallyisolatedinthe1940s.
ComparativegenomicanalysisalsoindicatedthattheJapaneseisolateT000240displayshighsimilaritytoiso-latesincladeB.
Izumiyaetal.
[9]commentedthatmultidrug-resistantprogenyofLT2mightbereemer-gingalongsideDT104andotherdefinitivephage-typestrains,andourdatasuggestedthatsuchprogenyofLT2mighthavealreadyreemergedinregionsnearbyJapanoveratleastthepasttwodecades.
AnalysisofantimicrobialresistancedeterminantsWealsoshowedthatthecladeAandBisolatesdif-feredremarkablyintheirlevelofvirulenceintermsofantimicrobialsresistance,presenceofvirulenceplasmidsandprophageelements.
NotonlydidcladeBcompriseahigherproportionofbloodisolates,allisolateswithinthecladewerealsomultidrug-resistant.
Genomesequencealignmentrevealedthatnoneofour20localisolatesharboracompletecopyoftheSalmonellaGenomicIsland1(SGI1)foundintheDT104lineage[25].
Specifically,allisolatesincladeAdidnotharboranyofthegenomicfragmentsfromSGI1.
Intriguingly,isolatesincladeBharboranapproximately5.
2kb-fragmentoriginatedfromSGI1(Fig.
2a),whichisrepresentedbyaclass1integronconsistingofOXA-1beta-lactamaseblaoxa-30,aminoglycosideresistancepro-teinaadA1,asmallmultidrugresistanceproteinqacEΔ1andsulfonamideresistancegenesul1.
Thisclass1integronisinturnlocatedwithinthepreviouslycharacterized82kbGI-DT12genomicislandinT000240.
Togetherwiththechloramphenicolacetyl-transferaseandtetracyclineresistanceproteintetAgenes[26]located6.
7and10.
8kbupstream,respect-ively,totheclass1integron,thisgenomicislandcon-fersresistancetoanumberofantimicrobialsincludingampicillin,kanamycin,chloramphenicol,tetracycline,sulfonamidedrugs,andquaternaryammoniumcompounds.
Fig.
2BRIGdiagramsshowinganoverviewofthegenomicrelationshipbetweenthesevensequencedgroupBS.
TyphimuriumisolatesandatheSGI1genomicislandinDT104(GenBankAccessionAF261825)andbtheT000240plasmidpSTMDT12_L(GenBankAccessionNC_016861)[29].
Theinnermostrings(inred)representthereferencesequences,andtheouterringsshowBLASTNcomparisonsofthegroupBgenomesagainstthereferencesusinganE-valuecut-offof0.
001.
KnowngenesofSGI1inDT104[25]andknownproteinproductsofopenreadingframesintheresistanceislandofpSTMDT12_L[9]aremarkedontheoutermostringsin(a)and(b),respectivelyChengetal.
BMCGenomics(2015)16:688Page6of8ThelargeplasmidpSTMDT12_LidentifiedinT000240isalsoexclusivelyfoundinthecladeBisolates(Fig.
2b).
Nevertheless,duetothepresenceoffourtransposases(threeofwhichareIS26)andarecombinationproteinintheplasmid,theresistanceislandshowedstructuralvari-ation,whichresultsintheexistenceoftheaminoglycoside3-N-acetyltransferasegene(aac(3),forgentamicinresist-ance)inonlyfouroftheisolatesandthedihydrofolatereductasegene(dfrA1,fortrimethoprimresistance)inonlythreeoftheisolates.
CiprofloxacinresistanceinSalmonellaTyphimuriumaswellasotherserovars,mostnotablyTyphiandPara-typhi,hasbecomeaglobalconcerninrecentyears[27].
Fiveoutofour20isolates(ST4806/06,ST372/06,ST1660/06,ST2143/05,andST4024/07)wereshowntobeciprofloxacin-resistant.
ThefirstfourisolateswerefromcladeB,andtheydemonstratedsimilarmutationpatternsinthequinoloneresistance-determiningre-gions(QRDRs)intheDNAgyraseA(gyrA)andDNAtopoisomeraseIVsubunitAandB(parCandparE)genes[28].
GenomicsequencesrevealedaSer83>PhemutationingyrAforallthefourisolates,butataminoacid87,itwasAsp87>AsnforST4806/06andAsp87>Glyfortheremainingthree.
MutationforparCwasaconsistentSer80>Arg,butfortheparEgeneitwastherarelydescribedLeu416>PheforST4806/06andthemorecommonSer458>Profortherest.
NomutationswereidentifiedinthegyrBgene.
Interestingly,theonlyciprofloxacin-resistantstrainoutsidecladeB,ST4024/07,showedonlyasinglemutation(Asp87>Tyr)with-outanyadditionalmutationineithergyrB,parCorparEgenes.
ThissuggeststhatonlyasinglemutationintheQRDRofgyrAissufficienttoconferresistancetociprofloxacin.
Evolutionofciprofloxacin-resistantstrainsAsnotedabove,ciprofloxacinresistancewasonlyidenti-fiedinstrainsisolatedinthe00'sandnotnotedinthe90's.
Inparticular,fouroftheseisolateswerefromthemorevirulentcladeB.
Despiteadditionalantimicrobialshasnotbeentested,resistancetociprofloxacinhasoftenbeenassociatedwithquinolonesresistance.
Suchcombin-ationofmultidrugandapotentialquinoloneresistancehaspromptedclinicianstopayattentiontothespreadofprogeniesfromS.
TyphimuriumstrainsincladeB.
ConclusionsOurstudyrevealedtheexistenceoftwomajorphylogen-eticcladesofSalmonellaTyphimuriumclinicalisolatescirculatinginHongKongoverthepasttwodecades.
Thetwocladesdifferremarkablyinantimicrobialsusceptibil-ity,presentationandmutationsofvirulencedeterminantsandmembersinoneofthecladesareshowntobecloserelativesandlikelyprogeniesofthelaboratoryreferencestrainLT2.
Suchpotentialdisseminationofthismultidrug-resistantgroupofS.
TyphimuriuminthenortheastAsiashoulddeservemoreattention.
AvailabilityofsupportingdataThewholegenomeshotgundatasetsgeneratedinthisstudyhavebeendepositedatDDBJ/EMBL/GenBankundertheaccessionsJRYT00000000,JRYU00000000,AUXE00000000,JRZV00000000,JRZX00000000,JRZW00000000,JRZU00000000,JRZT00000000,JZS00000000,JRZR00000000,JRZQ00000000,JRZP00000000,JRZO00000000,JRZN00000000,JRZM00000000,JRZL00000000,JRZK00000000,JRZJ00000000,JRZI00000000,andJRZH00000000.
AdditionalfilesAdditionalfile1:Maximum-parsimonyphylogenetictreeof47S.
Typhimuriumgenomeswithbootstrapvaluesreportedonnodes.
OnlySNPsinthe"core"geneswereincluded.
ThetreewasrootedusingSalmonellaEnteritidisPT4(GenBankAccessionAM933172)andSalmonellaCholeraesuisSC-B67(GenBankAccessionAE017220).
Redisolates:localbloodisolates;Blueisolates:localstoolisolates;Blackisolates:referenceGenBankisolates.
Thenumberateachnodeisthesupportvalueinferredfrom500bootstrapreplicates.
BootstrapvaluesCompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterestsAuthors'contributionsCKCperformedtheexperiments,analyzedthedataanddraftedthemanuscript.
MKCperformedthephylogeneticanalysesandBRIGanalysesandrevisedthemanuscript.
WNperformedgenomeassemblyandSNPcalling.
PTWLcarriedoutgenomesequencing.
JQassisteddataanalysis.
JMLLcollectedandcharacterizedthebacterialstrains.
HSKconceivedanddesignedthestudy.
KMKandWMWCassistedindesignofthestudy.
Allauthorshavereadandapprovedthefinalmanuscript.
Chengetal.
BMCGenomics(2015)16:688Page7of8AcknowledgementsThisworkwassupportedbytheResearchFundfortheControlofInfectiousDiseases(CHP-PH-06)fromtheFoodandHealthBureauofHongKongSAR,China.
Authordetails1SchoolofLifeSciences,TheChineseUniversityofHongKong,HongKongSAR,China.
2DepartmentofMicrobiology,TheChineseUniversityofHongKong,PrinceofWalesHospital,HongKongSAR,China.
3CentreforHealthProtection,DepartmentofHealth,HongKongSAR,China.
4Currentaddress:StanleyHoCentreforEmergingInfectiousDiseases,JCSchoolofPublicHealth,FacultyofMedicine,TheChineseUniversityofHongKong,HongKongSAR,China.
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