Next-generation sequencing virulome analysis of a Yersinia enterocolitica subsp. palearctica bioserotype 4/O:3 ST18 isolated from human blood in Brazil

brazjinfectdis2017;21(5):550–553

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Next-generation

sequencing

virulome

analysis

of

a

Yersinia

enterocolitica

subsp.

palearctica

bioserotype

4/O:3

ST18

isolated

from

human

blood

in

Brazil

Leonardo

Alves

Rusak

_,

_Ricardo

_Magrani

_Junqueira

_,

_Ernesto

_Hofer

_,

Deyse

Christina

Vallim

_,

_Marise

_Dutra

_Asensi

a_{Fundac¸ãoOswaldoCruz,InstitutoOswaldoCruz,LaboratóriodePesquisaemInfecc¸ãoHospitalar,RiodeJaneiro,RJ,Brazil} b_{Fundac¸ãoOswaldoCruz,InstitutoOswaldoCruz,LaboratóriodeBiologiaComputacionaleSistemas,RiodeJaneiro,RJ,Brazil} c_{Fundac¸ãoOswaldoCruz,InstitutoOswaldoCruz,LaboratóriodeZoonosesBacterianas/SetorListeria,RiodeJaneiro,RJ,Brazil}

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Articlehistory:

Received2December2016 Accepted27April2017 Availableonline30May2017

Keywords: Yersiniaenterocolitica Virulence Brazil Next-generationsequencing

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YersiniaenterocoliticaisawidespreadGram-negativebacteriumthatcausesgastrointestinal diseaseandotherclinicalmanifestationsinhumans.PotentiallypathogenicY.enterocolitica hasbeenisolatedinBrazil,fromhuman,environmental,food,andanimalsources.Herein wereporta genomesequenceofY.enterocoliticasubsp.palearcticastrainYE19,serotype O:3,biotype4,sequencetype18,withvirulencedeterminantsisolatedfromhumanblood inRiodeJaneiroin2005.Theresultscorroborateotherfindingsthatthisstrainharborsa setofvirulencedeterminantsthatcouldplayaroleinhostpathoadaptationandmayalso justifythesuccessfuldisseminationofbioserotype4/O:3inBrazil.Thepresenceofstrains harboringallofthesevirulencegenesinBrazilisapotentialthreattoyoungchildrenand immunocompromisedindividuals,forwhomyersiniosisareasignificantsourceof morbid-ityandmortality.Theresultsofagenomicdataanalysiswillhelpunderstandthevirulence ofBrazilianstrainsandprovidedataforY.enterocoliticastudiesworldwide.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Yersiniaenterocoliticaisanentericpathogenwithawiderange ofclinicalandimmunologicalmanifestations,whichdepend onageandphysicalconditionofthepatient.Responsiblefor intestinaldiseases,themostfrequentoccurrenceininfants and children,including enterocolitis withan inflammatory

∗ _{Correspondingauthor.}

E-mailaddress:rusak@ioc.fiocruz.br(L.A.Rusak).

diarrhea; however, in older children and young adults the symptoms include acute terminal ileitis and mesenteric lymphadenitis(mimickingappendicitis).Ontheotherhand, insomecases,extra-intestinalmanifestationshavealsobeen reported,includingurinarytractandrespiratorytract infec-tion (empyema), osteoarticular infection (reactivearthritis), erythema nodosum, infected mycotic aneurysm, axillary abscesses, and endocarditis.1,2 _{Y. enterocolitica is the major} http://dx.doi.org/10.1016/j.bjid.2017.04.005

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

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causative agent of yersiniosis worldwide and is typically transmittedviathefecal–oralroutetohumansandanimals.3 Y.enterocoliticacanbeclassifiedbybiochemicalcharacteristics andserotypedintosixbiotypes(BT)(1A,1B,2,3,4,and5).1 Strains that are non-pathogenic to humans and animals belong to BT1A, whereas highly pathogenic strains belong toBT1B;BT2–5arelowtomoderate-pathogenicstrains.The serotype O:3 biotype 4 comprises about 80–90%of human isolatesinGermanyandEurope,withrisingglobalrelevance.4 Little attention has been given to this microorganism in Brazil, where it is still poorly studied. Although Brazil has no official data about the incidenceof this pathogen, sporadiccaseshavebeenpublishedandsomestudies demon-stratethepathogenicpotentialofstrainsisolatedfrommany sources,includinghumans,theenvironment,food,and ani-mals.Thebioserotype4/O:3isthemainbioserotypeisolated fromhumanclinicalcases.5,6

Theaimofthisbriefcommunicationwastoanalyzethe virulenceprofileandannouncesthegenomesequenceofY. enterocoliticasubsp.palearctica,strainYE19,serovarO:3, bio-type4isolatedin2005frombloodofahospitalizedpatient inRiodeJaneiro.Itwas depositedinthe Collectionof Lis-teria (CLIST) located at the Bacterial Zoonoses Laboratory (LABZOO/OswaldoCruz Institute/OswaldoCruz Foundation) undertheaccessionnumber CLIST4117.Sequence type18 (ST18)wasdeterminedbymultilocussequencetyping(MLST) andbyuseoftheYersiniaMLSTwebsite.TheST18comprises themainY.enterocoliticaserotypeO:3sequencetypebasedon MLSTdatabase.

Anext-generationsequencing(NGS)techniquewasused to analyze the virulome of YE 19 strain. Genomic DNA was extracted using DNeasy® Blood & Tissue Kit (Qiagen, Germany) and the DNA library wasprepared withNextera XT (Illumina, San Diego, CA). Sequencing was performed on an Illumina HiSeq 2500 platform (Illumina Inc., USA) generating100bppaired-endreads.Thesequencedatawere processedusingTrimmomatic(v0.35)and paired-endreads weremergedusingFLASh(v1.2.11).7_{Denovoassemblywas} performedusingSPAdes(v3.7.1).8_Ragout(v1.2)9_wasutilized forreferenceassisted scaffoldingofcontigs againstY. ente-rocoliticasubsp.palearcticaY11genome(NC017564.1).Sealer (v1.9.0)10_{wasusedforgapfillingandPilon(v1.16)}11_performed polishing.TheresultingassemblywasevaluatedusingREAPR (v1.0.18).12_{TheNCBIProkaryoticGenomeAnnotationPipeline} (released 2013) annotated the final genome model com-prising19 scaffolds witha totallengthof 4,550,059bpand 47% GC.The summaryof genomic statistics isreported in Table 1. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession LYBL00000000.Theversiondescribedinthispaperisversion LYBL01000000.

Thepresenceofvirulencefactorswasinvestigatedusing SRST2program(v0.1.7)againsttheVirulenceFactorDatabase (VFDB).Thedetailsofallvirulencegenesfoundarereported insupplementaryfile 1.Fig. 1isbuilttoshow whereeach virulencegeneislocatedonYE19genome.

Atotalof103virulencegeneshavebeenidentifiedfrom theVFDB.Thesevirulencegenesarerelatedtomotility,the secretionsystem,toxins,theOantigen(lipopolysaccharide– LPS),heminuptake,invasion,andadherence.

Table1–Genomefeatures.

Property YE19 Totallength(bp) 4550059 Scaffolds 19 Contigs 135 N50 620817 GC(%) 47 Genes(total) 4138 CDS(total) 4034 Genes(coding) 3857 CDS(coding) 3857 Genes(RNA) 104 rRNAs 8,7,7(5S,16S,23S) tRNAs 73 ncRNAs 9

PseudoGenes(total) 177

Wedetectedvirulencedeterminantsofmotilitycontrolled byflhE,fliNfliL,flgA,fliD,fliC,fliP,flgD,flhA,fliS,flhB,flgK,fliF, flgB,flgJ,flgC,flgL,flgN,flgM, flhC,fliE, fliO,fliH,flgE,fliB,fliA, fliK, fliI, flgF, flgI, fliG, fliR, flgH, flhD,flgG, fliT, fliM, fliZ, and fliQ.Thesegenesactinotherprocesses,suchascellular inva-sion,biofilmformation,andthesecretionsystem(thetypeIII secretion/flagellabiosynthesisapparatus).13,14_Wefoundthe cheA, cheB,cheD, cheR, cheW,cheY, cheZgenes, which actin thechemotaxismechanism,bywhichbacteriaefficientlyand rapidlyrespondtochanges inthe chemicalcompositionof theirenvironment.Thisbehaviormodulatesthedirectionof flagellarrotation.15_{Finally,wefoundthemotAandmotBgenes,} whichencodeflagellarmotorproteinMotAandMotB, respec-tively,andbuildtheflagellar-motorsupramolecularcomplex stator.15

The yst1L, yst1J, yst1K, yst1F, yst1E, yst1G, yst1C and YE105C0912genesencodeproteinsthatbuildthetypeII secre-tionsystem(T2SS).AllYersiniaspecies,non-pathogenicaswell aspathogenic,possessatleastoneofthetwoT2SS.Theone T2SSthathassofar beenstudiedistermedYts1,whichis involvedindisseminationandcolonizationofdeepertissues likeliverandspleeninmouseinfectionexperiments. Further-more,theYts1OgeneencodestheYts1Oprotein,whichisa putativeprepilin-likepeptidase.16

YE105C0309, YE105C0310, YE105C0312, YE105C0313, YE105C0314, YE105C0323, YE105C0324, YE105C0325, YE105C0326, YE105C0327, YE105C0328, YE105C0332, YE105C0333, YE105C0334, YE105C0335, YE105C0336, YE105C0338, YE105C0339, YE105C0340, YE105C0341 are a set of genes that received the name ofthe strain from wheretheywererecognizedforthefirsttime. Thesegenes encodeproteinsthatbuildtheYsctypeIIIsecretionsystem (pYV-encoded T3SS/TTSS). The T3SS is the mechanism by which pathogenic Yersinia injectsproteins directlyinto the eukaryotic cell cytosol.3 _{Y. enterocolitica has three different} typeIIIsecretionsystems,Ysc(pYV-encoded),Ysa(exclusive to biovar 1B), and the flagellar.2,3,13 _{Surprisingly, the genes} foundonYE19genomewererelatedtotheproteins(YscC,R, andU)thatbuilttheYscT3SS17_{(Supplementaryfile1).These} findingsreinforcethehypothesisthatthisstrainalreadyhad thepYVand,somehow,thesegenescouldbeintegratedinto thechromosome.Meanwhile,furtherstudiesareneededto confirmthispossibility.

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Fig.1–YE19strainvirulome.CircularmapoftheYE19strainchromosomeisplayingthedistributionoftheidentified virulencegenes.

WefoundtheYersiniastabletoxinystAgene,whichencodes the production of a heat-stable enterotoxin that causes diarrhea.2

ThepresenceofYE105C0173,YE105C1175,YE105C1176, YE105C1177, YE105C1178, YE105C1179, YE105C1180, YE105C1181, YE105C1182 genes related to the O antigen werefound.TheOantigenorOpolysaccharidechainis asso-ciatedwithantigenicpropertiesandisinvolvedininvasion and colonization processes. In addition, the O antigen is requiredforproperfunctionalityorexpressionofotherouter membranevirulencefactors.2

Wefoundthe hemP,hemR, hemS,hemT, hemU and hmuV genesassociatedwithheminuptake.Thissetofgenes per-mitsthestraintousetheheme-containingcompoundsasa sourceofiron.18_{However,thesegenesarenotthesamegenes} foundonyersiniabactin.2

Thecommon virulencegenesinv(invasivegene)andail (attachment invasionlocus)were present. They encode an invasin,whichdirectlyinitiatescellpenetration,andanouter membrane proteincalled Ail, which encodes a factor that alsopromotestheinvasionofepithelial cells,respectively.1 ThepsaA(myfA)geneisrelatedtoadhesiontothehostcell

andits pH6antigenhomologinY.pseudotuberculosisandY. pestis.19_{ThemyfB,myfC,myfEandmyfFgenesareencodedby} themyfoperon,knownasthemucoidYersiniafactor.These genesencodeproteinsthatconstituteafibrillarstructurethat actsduringadhesion.2_{TheYplageneencodesphospholipase} A that stimulates the acute inflammatoryresponse of the hostandisrequiredforsurvivalofY.enterocoliticainPeyer’s patches.3 _{Finally,theplagene,whichhasbeendescribedin} theY.enterocoliticaW22703strain,20_{encodesapeptidaseA26} omptinproteinthathasproteolyticandadhesionfunctions.19 ABlastsearchonGenbankforthisplageneshowedonly30% nucleotide identity with the plagene from Y.pestis (strain I-3190plasmidpPst);thus,furtherstudiesareneededto deter-minetherelationshipbetweenthesetwogenes.

TheseresultscorroboratethefindingsofBatzillaetal.and suggest thatthis strainharbors asetofvirulence determi-nantsthatmayplayroleinhostpathoadaptation.Thiscould alsojustifythesuccessfuldisseminationofbioserotype4/O:3 inBrazilandworldwide.AsthepYVvirulenceplasmid1_was notfound, wecannotrule out thepossibility thatit might havebeen lostduringstorage.Thepresenceofstrains har-boring all of these virulence genes inBrazil is a potential

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threattoyoungchildrenandimmunocompromised individ-uals,forwhomyersiniosiscanrepresentasignificantsourceof morbidityandmortality.3_{Wehopethisgenomicdataanalysis} helpsunderstandingthevirulenceissueofBrazilianstrains andcontributesforY.enterocoliticastudiesworldwide.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WethankFundac¸ãodeAmparoàPesquisadoEstadodoRiode Janeiro(FAPERJ)andConselhoNacionaldeDesenvolvimento CientíficoeTecnológico(CNPq)forfinancialsupport.

Appendix

A.

Supplementary

data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.bjid.2017.04.005.

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YersiniaMLSTwebsite(availablefrom:

http://pubmlst.org/yersinia/)developedbyKeithJolleyandsited attheUniversityofOxford(Jolley&Maiden2010,BMC

Bioinformatics,11:595)[accessedin08.29.16]. TheVirulenceFactordatabase(VFDB)(availablefrom: http://www.mgc.ac.cn/VFs/)[accessedin08.29.16].