Genetic analysis of the first mcr-1 positive Escherichia coli isolate collected from an outpatient in Chile

brazjinfectdis2019;23(3):203–206

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Genetic

analysis

of

the

first

mcr-1

positive

Escherichia

coli

isolate

collected

from

an

outpatient

in

Chile

Camila

Gutiérrez

_,

_Javier

_Zenis

_,

_Paulette

_Legarraga

_,

_Jaime

_R.

_{Cabrera-Pardo}

_,

Patricia

García

_,

_Helia

_Bello-Toledo

_,

_Andrés

_{Opazo-Capurro}

_,

Gerardo

González-Rocha

a_{UniversidaddeConcepción,FacultaddeCienciasBiológicas,DepartamentodeMicrobiología,Concepción,Chile} b_{PontificiaUniversidadCatólicadeChile,EscueladeMedicina,DepartamentodeLaboratoriosClínicos,Santiago,Chile} c_{UniversidaddelBio-Bio,FacultaddeCiencias,DepartamentodeQuímica,Concepción,Chile}

d_{MilleniumNucleusonInterdisciplinaryApproachtoAntimicrobialResistance(MICROB-R),Chile}

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Articlehistory:

Received3January2019 Accepted19May2019 Availableonline20June2019

Keywords: Colistin-resistance mcr-1 Escherichiacoli Chile

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Globaldisseminationofmcr-likegenesrepresentsaseriousthreattopublichealthsince itjeopardizestheeffectivenessofcolistin,anantibioticusedas alast-resorttreatment againsthighlyantibiotic-resistantbacteria.In2017,amcr-1-positiveisolateofEscherichiacoli

wasfoundinChileforthefirsttime.Hereinwereportthegeneticfeaturesofthisstrain (UCO-457)bywhole-genomesequencing(WGS)andconjugationexperiments.The UCO-457strainbelongedtoST4204andcarrieda285kbIncI2-typeplasmidcontainingthemcr-1

gene.Moreover,thisplasmidwastransferredbyconjugationtoanE.coliJ53strainathigh fre-quency.Theisolateharboredthecma,iroN,andissvirulencegenesanddidcarryresistance genestotrimethoprim/sulfamethoxazoleandfluoroquinolones.Otherantibioticresistance determinantssuchas␤-lactamases-encodinggeneswerenotdetected,makingtheisolate highlysusceptibletotheseantibiotics.Ourresultsrevealedthatsuchsusceptibleisolates couldbeactingasplatformstodisseminateplasmid-mediatedcolistinresistance.Basedon thisevidence,weconsiderthatmcr-likeprevalencedeservesurgentattentionandshouldbe examinednotonlyinhighlyresistantbacteriabutalsoinsusceptibleisolates.

©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Colistin represents the last resort to treat serious infec-tions caused byhighly resistant Gram-negative pathogens, such as extensively drug-resistant (XDR) Enterobacteriaceae

∗ _{Correspondingauthor.}

E-mailaddress:andopazo@udec.cl(A.Opazo-Capurro).

and non-fermentingbacilli.1_{Inlate2015,Liuetal.founda}

plasmid-bornemcr-1gene,whichconferscolistinresistance, inEscherichiacoliandKlebsiellapneumoniaefromdiversesources inChinaincludingfoodproducts,livestock,andhumans.1,2

Thiswasthefirstdescriptionofacolistinresistancetrait har-boredbyamobilegeneticelement,thusimplicatingthelikely

https://doi.org/10.1016/j.bjid.2019.05.008

1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

204

horizontal spread of colistin resistance. Indeed, mcr genes havesincebeen describedacross the worldfroma myriad ofsources including food products, animals, humans, and environmentalsamples.1 _{InSouth America (SA),mcr-1 has}

beenidentifiedinEnterobacteriaceaeisolatedfromArgentina, Ecuador,Colombia,Bolivia,Venezuela,andBrazil.Inaddition,

mcr-5hasbeenreportedinBrazil.3,4 _{Sofar,noothermcr-like}

geneshavebeenidentifiedinSA.

Itiscrucialtotrackthepresenceanddisseminationof mcr-likegenessincedecreasingtheeffectivenessofcolistinleaves notherapeuticoptiontotacklehighlyresistantGram-negative bacteria. Unfortunately, data concerning colistin-resistance andprevalenceofmcr-likegenesinSA,andChileinparticular, arescant. Todate,Legarragaetal.5 _{publishedthe firstand}

onlyclinicaldescriptionofmcr-1inChile,whichwasfoundin anE.coliisolate(UCO-457)recoveredfromaurinaryinfection in an outpatient of a hospital located in Santiago, Chile.5

The authors further described that this isolate was resis-tant to ciprofloxacin and trimethoprim/sulfamethoxazole, and susceptible to ␤-lactams, aminoglycosides and nitrofurantoin.

Inordertoachieveafullercharacterizationofthefirst mcr-1positiveE.colistrain,weaimedtocarryoutwhole-genome sequencing (WGS) of the UCO-457 isolate and study the transferabilityofmcr-1byconjugation.Antimicrobial suscep-tibilityprofilesweredeterminedbythediscdiffusionmethod followingtheCLSIguidelines.6_{Theminimuminhibitory}

con-centration(MIC)tocolistin(CST)wasdeterminedbythebroth dilutiontechnique,andtheresultswereinterpretedaccording totheEuropeanCommitteeonAntimicrobialSusceptibility Testing(EUCAST;http://www.eucast.org/clinicalbreakpoints) guidelines. WGS was performed using the Illumina MiSeq platform with a coverage of 30× with 2×250bp paired-end reads. De novo assemblies were carried out bySPAdes v3.7 yielding 61 contigs (≥1000bp). Species identification, sequence type(ST), serotype, Fim type, and plasmid repli-con typing content were identified in silico using tools available at the Center for Genomic Epidemiology (CGE) server (http://www.genomicepidemiology.org/). Virulence genes were investigatedusing the virulence finder tool of the CGE server and the Pathosystems Resources Integra-tion Center (PATRIC) toolkit (https://www.patricbrc.org/). Antibiotic-resistance genes (ARGs) were screened using ResFinder (http://cge.cbs.dtu.dk/services/ResFinder/) and the Comprehensive Antibiotic Resistance Database (CARD) (http://arpcard.mcmaster.ca).

Thetotalgenomelengthwas4,947,619bp,withaN50contig

sizeof210,597bpandG+Ccontentof50.54%,consistentwith other speciesintheEscherichia genus. ThisWholeGenome Shotgun projecthas been deposited atDDBJ/ENA/GenBank undertheaccessionRXIT00000000.Theversiondescribedin thismanuscriptisversionRXIT00000000.1.

FromtheWGSdata,wecorroboratedtheidentificationof theisolateasE.coliusingtheSpeciesFindertoolontheCGE website.TheisolatecorrespondedtoST4204(CC10) accord-ingtotheWarwickMLSTscheme.Interestingly,mcr-1-positive

isolatescollectedfromurinarytractinfectionsinChinahave alsobeenassociatedtothisST.7,8_{Theplasmidcontainingthe}

mcrgeneintheseisolatesalsocarriedtheextended-spectrum ␤-lactamasegeneblaCTX-M-55,inwhichbothresistancegenes

were co-transferredbyconjugation.7,8 _Thestrainwas

nega-tiveforblaCTX-M,consistentwithitsobservedsusceptibilityto

␤-lactams.5

Likewise, in silico typing revealed that UCO-457 cor-responded to the serotype O6:H10 and belonged to the subclone fimH24.Thisserotypehasbeen previouslydefined aspathogenic,sinceittypicallyproducestoxins.9_However,we

didnotdetectanytoxin-relatedgenes,althoughwedidfind diversevirulencegenessuchascma(accessionnoFJ664752),

iroN(accessionnoAF449498)andiss(accessionnoDQ381420). ThecmageneencodesacolicinMprotein,whichcorresponds toabacteriocin,10_{whileIroNcorrespondstoaenterobactin}

siderophore receptor protein, which is considered to be a urovirulence factor.11 _{Moreover,iss encodes a protein that}

increasestheserumsurvivalofE.coli,thusconferring resis-tancetothecomplementsystem.12

E.coliST131isagloballydisseminatedmultidrug-resistant clone involvedin urinary tract and bloodstream infections which possesses the type 1 fimbriae fimH30 allele.13 _As

described above,UCO-457harboredthefimH24subtypeand thus is notrelated to the uropathogenicclone ST131. Itis important to strengthen the national surveillance in Chile sincetherearenotdataaboutthefimsubtypescirculatingin thecountry.Forinstance,thefimH24subtype,whichislargely unknowninChile,hasbeenassociatedtonationaloutbreaks inDenmark.14

Asexpected,theWGSdataanalysisrevealedthepresence of thecolistin-resistance genemcr-1. Specifically, this gene wasdetectedinacontigof64,036bpwhichdidnotcontain anyadditionalARGs.Withinthecontig,mcr-1wasdelimited upstreambyagenethatencodesaPAP2-familyproteinand downstreambyarelaxase-encodinggene(Fig.1).Thisgenetic environmentdiffersfromthatcommonlyassociatedto mcr-1inwhichtheISApl1-transposonislinkedtothisgeneand mediatesitsdissemination.15_{WedetectedtheIncI2-replicon}

sequence inthe same 64,036bpcontig withmcr-1, but did notdetectany ISApI1elementssurroundingthemcr-1gene (Fig.1).Thisarrangementisreminiscentofthemcr-carrying E.colifromArgentinaandCanadacharacterizedbyTijetetal. wheretheyfoundthatthesegmentcontainingthemcr-1gene had the pap2andrelaxasegenes,16 _{asfoundinourisolate}

(Fig.1).Moreover,someofthecolistin-resistantE.coliisolates analyzedintheirstudyalsolackedtheISApI1element.This couldbeexplainedbythemobilizationoftheISresultingin thetransferofthemcr-1-pap2arrangementtoaconjugative plasmidwithconsequentlossofISApI1,16_{aprocessthatcould}

alsohaveoccurredinUCO-457.

AmongEnterobacteriaceae,mcr-1hasbeenassociatedwith many different incompatibility plasmid groups including IncI2,IncX4,IncHI1,IncHI2,IncFI,IncFII,IncP,IncI2-FIB, IncX1-X2, and IncX3-X4.17 _{Basedon phylogenetic analysis, it has}

beensuggestedthatIncI2plasmidscanmigrateamong dif-ferent bacterial species with E. coli acting as a carrier of

mcr-1betweenthem.18 _{Interestingly,thepap2-mcr-1element}

hasbeen previouslydetectedinIncI2-plasmids.16 _Although

IncI2plasmidsarefrequentlyassociatedwithblaCTX-M-55and

blaKPC-3,18thesegeneswerenotdetectedinUCO-457.These

intriguing patternswarrantfurtherstudyoftheprevalence andpotentialroleofthisplasmidgroupinthedissemination ofmcr-1.

brazj infect dis.2019;23(3):203–206

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Fig.1–Geneticenvironmentofmcr-1inE.coliUCO-457.Genesandtheirtranscriptionalorientationsareindicatedbygreen arrows.Thisdiagramwasdesignedutilizingthe64,036bpcontiginwhichmcr-1wasdetected.

In addition to mcr-1, the sul2 and dfrA14 genes were identifiedexplainingtheresistancephenotypeto trimetho-prim/sulfamethoxazole. We found that the IntI1 gene was in the same contig as dfrA14, suggesting this gene could beassociated to a class1 integron. This finding was con-firmedbytheNCBIProkaryoticGenomeAnnotationPipeline (PGAP),19_{inwhichthedfrA14genewaslocatedimmediately}

downstream the integrase gene (GenBank accession num-berRXIT01000051.1). Wedid notfindany genes associated withthe3conservedregionofthesegeneticelements,such astheqacE1andsul120_{elements,indicatingthatsome}

re-arrangementscouldbepresentinthestructureofthedetected integron.

TheUCO-457isolatehadalsobeenreportedtobe suscepti-bletoamikacinandgentamicinandresistanttostreptomycin. Wedetectedthe aph(3)-Ibgene in the genome. Thisgene (alsonamedasstrA)mediatesresistancespecificallyto strep-tomycin, explaining the observed phenotype.21 _{Finally, in}

additiontoacquiredresistancegenes,wedetectedtwo-point mutations,S83LandD87NingyrA,aswellasasinglepoint mutationS80I inparC, which are involved inresistance to ciprofloxacin.22

IncomplementtotheWGSanalysis,theplasmidcontent ofUCO-457wasdeterminedbythealkalinelysismethod fol-lowedby pulsed-fieldgel electrophoresis (PFGE).Pure DNA plasmidextracts wereused toexaminewhethermcr-1was contained inthis mobile element by PCR. Moreover, mcr-1

transferabilitywas studied byconjugation using the E. coli

J53strain(sodiumazideresistantandcolistinsusceptible)as therecipientstrain.Susceptibilitiesto␤-lactams, aminoglyco-sides,nitrofurantoinandCSTinadditiontotheirisogenicity andthepresenceofmcr-1andIncI2-repliconweredetermined by the disk diffusion test, broth microdilution, ERIC-PCR andconventional PCR,respectively. From thesestudies,we detecteda largeplasmidofca. 285kb whichcontainedthe

mcr-1andthe IncI2sequence inUCO-457, whichwere also detectedinthetransconjugantE.coliJ53-MCR1.Additionally, wedeterminedthattheplasmidtransferoccurredwithahigh frequency of 2.8×10−4 transconjugants per recipient. It is importanttonotethattheIlluminaplatformgeneratesshort reads,makingitdifficulttodeterminetheentiresequenceof plasmids23_{.Duetothegenerationoftheseshortreads,the}

useofIlluminatopredictplasmids(i.e.,byPlasmidFinder)is limited,beingonlypossibletosearchfortheincompatibility groupsinthedraftgenomedatageneratedfromWGS.23_Asa

consequence,itisnecessarytoisolateandsequencethe plas-midcontainingthemcr-1inUCO-457inordertodetermineits completesequence.

MIC to CST of E. coli J53 varied from 2␮g/mL (J53) to 8␮g/mL(J53-MCR1)after thematingexperiments, reaching thesamevalueasthedonorstrain.Notably,the susceptibil-itypatternofJ53-MCR1tootherantibioticswasnotaltered, demonstratingthatonlymcr-1wastransferredduringthis pro-cess.Thisfindingconfirmsthatthemcr-1genewastheonly antibiotic-resistance gene transferable ata high frequency. Similar resultswere obtainedbyTijet et al.,in whichthey describedthatcolistinwastheonlyantibioticforwhichthe resultingtransformantwasresistantafterconjugationfrom

mcr-1-carryingE.coliasadonor.16_{AstheUCO-457isolatewas}

recoveredfromaurinarytractinfectionfromanoutpatient,it isdifficulttotrackthefurtherspreadofmcr-1fromthiscase.To theauthors’knowledge,therehavebeennoadditionalreports ofmcr-likegenesinChiletodate.

Inconclusion,wecharacterizedthefirstmcr-1-positiveE. coliisolatedinChileandfoundittobepartoftheCC10 identi-fiedinChina.Themcr-1genewasdetectedinanIncI2-plasmid, whichisknowntobepromiscuous.Therefore,further investi-gationiswarrantedinordertodeterminetheprevalenceand disseminationofthismcr-carryingplasmid.Finally,theisolate inwhichmcr-1wasdetectedwashighlysusceptibletoother antibiotics,highlightingthepotentialroleforsuchisolatesto actasplatformsforthedisseminationofmcrgenes.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Funding

source

AttractionandInsertionofAdvancedHumanCapitalProgram (PAI),CONICYT,Chile,undertheprojectPAI79170082.

Acknowledgments

TheauthorswanttothanktotheNationalCommissionfor ScientificandTechnologicalResearchofChile(CONICYT),for supportingthisstudy.WethankProf.EllenLefflerfor

proof-206

readingthismanuscript.JRC–PthanksthesupportofFondecyt regular(No1190652).

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