A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying bla(SHV-12) from horses in Sweden

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Short

Communication

A

link

between

the

newly

described

colistin

resistance

gene

mcr-9

and

clinical

Enterobacteriaceae

isolates

carrying

bla

SHV-12

from

horses

in

Sweden

Stefan

Börjesson

a,b,

*

,

Christina

Greko

a

,

Mattias

Myrenås

a

,

Annica

Landén

a

,

Oskar

Nilsson

a

,

Karl

Pedersen

a

DepartmentofAnimalHealthandAntimicrobialStrategies,NationalVeterinaryInstitute(SVA),Uppsala,Sweden

b

DepartmentofBiomedicalandClinicalSciences,LinköpingUniversity,Linköping,Sweden

ARTICLE INFO Articlehistory: Received28June2019

Receivedinrevisedform9August2019 Accepted12August2019

Availableonline5September2019 Keywords: Colistinresistance mcr-9 ESBL Horses Plasmid blaSHV-12 ABSTRACT

Objectives:Theaimofthisstudywastoinvestigatetheoccurrenceofthenewlydescribedtransferable colistin resistance gene mcr-9 in extended-spectrum β-lactamase (ESBL)-producing clinical Enter-obacteriaceaeisolatesfromhorsesinSweden.

Methods: A totalof 56whole-genome sequenced ESBL-producingEnterobacteriaceae isolatesfrom horsesweresubjectedtoinsilicodetectionofantimicrobialresistancegenesandidentiﬁcationof plasmidrepliconstypes.Thecolistinminimuminhibitoryconcentration(MIC)formcr-positiveisolates wasdeterminedbybrothmicrodilution.RelatednessbetweenEnterobacteriaceaecarryingmcrgenes wasdeterminedbymultilocussequencetyping(MLST)andcoregenomeMLST.

Results:ThirtyESBL-producingEnterobacteriaceaeisolatesfromhorseswerepositiveforthecolistin resistancegenemcr-9.TheseisolatesincludedEnterobactercloacae,Escherichiacoli,Klebsiellaoxytocaand CitrobacterfreundiiandbelongedtodiverseMLSTsequencetypeswithineachspecies.Twoofthe mcr-9-containingisolatesoriginatedfromthesamehorse.Allmcr-9-positiveisolateshadcolistinMICsbelowor equaltotheEUCASTepidemiologicalcut-offvalueof2mg/Landwerenegativeforthetwopotential regulatorygenesqseB-likeandqseC-likeformcr-9.ExceptforoneisolatecarryingonlyblaTEM-1B,allofthe

isolatescarriedblaSHV-12andblaTEM-1B,andwereallconsideredmultidrug-resistantastheyharboured

genesencodingresistancetoaminoglycosides,chloramphenicol,fosfomycin,macrolides,quinolones, sulfonamides,trimethoprimandtetracyclines.PlasmidreplicontypesIncHI2andIncHI2Aweredetected inallmcr-9-positiveisolates.

Conclusion:Theoccurrenceofmcr-9wascommonamongclinicalESBL-producingEnterobacteriaceae isolatesfromhorsesinSwedenandwaslinkedtotheESBL-encodinggeneblaSHV-12andplasmidreplicon

typesIncHI2andIncHI2A.

org/licenses/by-nc-nd/4.0/).

1.Introduction

Colistinwaspreviouslyusedmainlyinfood-producinganimals forthepreventionandtreatmentofinfectionscausedby Enter-obacteriaceae, andglobally usageis still common[1].Recently, colistinhasreceivedrenewedattentionasanimportant antimi-crobialagentforthetreatmentofinfectionscausedby carbape-nem-resistantandmultidrug-resistantGram-negativebacteriain

human medicine [2]. Therefore, the recent description of the plasmid-mediatedcolistinresistancegenemcr-1in Enterobacter-iaceaefromChinaisofconcern[3].Sincethedescriptionofmcr-1, additionalmcrhomologueshavebeendescribedandseveralofthe genesoccurworldwide[1,4].However,theoccurrenceofmcrgenes is rare in Sweden, with onlya handful of cases reportedfrom humans[5].

InMay2019,anovelmcrhomologue,mcr-9,wasdescribedina Salmonella enterica serotype Typhimurium carrying blaSHV-12

isolatedfromahumanpatient[6].Theauthorsalsoshowedthat mcr-9couldbedetectedin335genomesofvariousspeciesinthe National Center for Biotechnology Information(NCBI) database and the that occurrence of mcr-9 could be linked toplasmids

* Correspondingauthorat:Department ofAnimalHealthand Antimicrobial Strategies,NationalVeterinaryInstitute(SVA),Uppsala,Sweden.

E-mailaddress:stefan.borjesson@liu.se(S.Börjesson).

http://dx.doi.org/10.1016/j.jgar.2019.08.007

2213-7165/©2019TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

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belongingtoreplicontypesIncHI2andIncHI2A.Thesamegene andplasmidcombinationwasshortlythereafteralsodescribedin anisolatefromahumanpatientinFrance[7].

Asof2010,theNationalVeterinaryInstitute(SVA)inSweden has encouraged Swedish veterinary laboratories to submit presumptiveextended-spectrum β-lactamase (ESBL)-and plas-mid-encodedAmpCβ-lactamase(pAmpC)-producing Enterobac-teriaceaeforveriﬁcationandgenotypiccharacterisationtoSVA, and from 2017 all identiﬁed ESBL- and pAmpC-producing Enterobacteriaceaearesubjected towhole-genome sequencing (WGS). In a retrospective investigation, a blaSHV-12-positive

Escherichia coli isolated froma horsein 2018 was found tobe positiveforthemcr-9genewhensubjectedtoinsilicodetectionof antimicrobial resistance genes (ARGs) using the ResFinder databaseon6thofMay2019.Detectionofmcr-9markstheﬁrst detectionofmcrgenesfromanimalsinSwedenand,asaresultof thisrandomﬁnding,wedecidedtoinvestigatewhethermcr-9,or othermcr genes,could bedetected in additional ESBL/pAmpC-producing Enterobacteriaceae clinical isolates from horses for whichWGSdatawereavailable.

2.Materialsandmethods 2.1.Isolates

Between 2017–2018, 55 Enterobacteriaceae isolates from horses wereveriﬁed phenotypically as ESBL-producers from submission to the SVA of presumptive ESBL- and pAmpC-producing Enterobacteriaceaeby Swedish veterinary labora-tories[5],andallisolatesweresubjectedtoWGS.Inaddition, oneESBL-isolate from a clinicalsubmission from a horse in 2016hadbeensubjectedtoWGSandwasalsoincludedinthe study. Of the total 56 isolates, 32 harboured blaSHV-12, 22

harbouredblaCTX-M-1,1harbouredblaCTX-M-15and1harbouredonly

blaTEM-1B.

2.2.Genomesequencing,bioinformaticsandcolistinsusceptibility testing

DNAwasextractedfrombacterialisolatesusinganEZ11DNA TissueKit (QIAGEN,Hilden, Germany)according tothe manu-facturer’s protocol, and DNA concentrations were determined usingaQubitTM_HS_DNA_Kit_(Life_{Technologies,}_Carlsbad,_CA,_USA).

Sequencing was performed using Illumina-based technology eitherin-houseusinganIlluminaNexteraXTKit(IlluminaInc., SanDiego, CA,USA) and 250-bppaired-end sequencingonan Illumina MiSeq sequencer (Illumina Inc.) or the DNA was submitted to GATC Biotech GmbH (Konstanz, Germany) or SciLifeLab Clinical Genomics (Göteborg, Sweden). To identify potential contamination and bacterial species, reads were checked using Kraken against the MiniKraken 8GB database (19October2017).ReadsweretrimmedwithTrimmomatic0.36 andgenomeassemblywasperformedusingSPAdesv.3.11.1with ‘—careful’ parameter followedby Pilon v.1.22. The presence of ARGsaswellasplasmid replicontypesweredeterminedusing Antimicrobial Resistance Identiﬁcation By Assembly (ARIBA) v.2.13.3withdownloadeddatabasesof ResFinderand Plasmid-Finder (8 May 2019). Seven-loci multilocus sequence typing (MLST)and core genome MLST(cgMLST) wereperformed and visualised in SeqSphere + software (Ridom GmbH, Münster, Germany), whilst plasmid MLST (pMLST) was performed by uploadingassembliestopubmlst.org.Theoccurrenceofthetwo potentialregulatorygenesqseB-likeandqseC-likeformcr-9was checkedonlineusingBLASTn(https://blast.ncbi.nlm.nih.gov/)by aligning qseB and qseC sequences from Kieffer et al. [7] to assemblieswithdefaultsettings.

Sequencereadsfortheincludedisolateshavebeendepositedin the European Nucleotide Archive (ENA) with accession no. PRJEB32700.

Forisolatesveriﬁedtocarrymcr-genes,thecolistinminimum inhibitory concentration (MIC) was determined using micro-dilutionwithMICRONAUTMIC-StripColistin(MerlinDiagnostika, Bornheim-Hersel,Germany)accordingtotherecommendationsof themanufacturer. The results wereinterpreted usingEuropean Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiologicalcut-offvalues(ECOFFs)[http://www.mic.eucast. org;accessed17May2019].

3.Resultsanddiscussion

Of the 56 ESBL-producing Enterobacteriaceae isolates from horsesinSweden,30wereshowntocarrythemcr-9gene(Table1). ExceptforoneisolatethatcarriedonlytheblaTEM-1Bgene,all

mcr-9-positiveisolateswerealsopositive forblaSHV-12andblaTEM-1B.

OnlytwoblaSHV-12-positiveclinicalisolatesfromhorseslackedthe

mcr-9gene.Themcr-9-positiveisolatesoriginatedfromadiverse setofsamples,includinguterus(n=10),wound/surgicalwound (n=7),urine (n=5),abscess(n=2)and otherorigins (n=6) (Table 1). Since mcr-9 was only identiﬁed in isolates carrying blaSHV-12, additional isolates from other sources positive for

blaSHV-12 that had been subjected to WGS at SVA were also

checkedforthepresenceofmcr-9.Atotalof8additionalisolates [broiler production (n = 6),dog (n = 1) and cat (n = 1)] were availableforinsilicotestingbutnoneofthesewerepositivefor mcr-9.Thehighoccurrenceofmcr-9inthis setof isolatesfrom horses was unexpectedas mcr geneshave notpreviously been describedinanimalsinSweden.Furthermore,colistinisonlyused for pigsin Sweden,but polymyxin Bmaybeused topicallyfor companion animals[5].Thecolistin MICs forallmcr-9-positive isolateswerealsobeloworequaltotheEUCASTECOFFsof2mg/L (Table 1).The isolates alsolacked thetwo potentialregulatory genesqseB-likeand qseC-like,whichKiefferet al.[7] suggested could play a role in the inducibility of mcr-9. Thus, both the selectiveeffectofcolistinandtheclinicalimpactofthegeneis thereforeuncertain.Allisolatesalsocarriedaac(6ʹ)-IIcand aph(6)-Id conferring resistance to aminoglycosides, ere(A) conferring resistancemacrolides,andsul1andsul2conferringresistanceto sulfonamides.Inaddition,genesencodingothertypesof amino-glycosideresistancewereidentifiedinallisolates(Table1).Other ARGswerealsoidentifiedamongtheisolates;trimethoprim(87%), chloramphenicol(57%),tetracycline (50%)fosfomycin (43%)and quinolones (17%) (Table 1). In equine medicine in Sweden, benzylpenicillin, gentamicin, streptomycin and trimethoprim/ sulfonamides are the only classes authorised for systemic treatment, leavingfluoroquinolones astheonlyoff-labeloption

[8]. It is therefore possible that spread of the mcr-9-carrying isolatesamonghorsesinSwedenhasbeenfacilitatedbyuseofthe few drug classes authorised for horses or off-label use of cephalosporins.Itisimportanttopointoutthatforinfectionsin horsessuchasthosecausedbyisolatesinthisinvestigation,there wouldbenoalternativesauthorisedfortreatmentinSweden.

ThespreadofEnterobacteriaceaecarryingmcr-9,blaSHV-12and

blaTEM-1BamonghorsesinSwedenappearslikelytobebyplasmid

disseminationasthegeneswereidentifiedinisolatesbelongingto thespeciesEnterobactercloacaecomplex(n=15),E.coli(n=10), Klebsiella oxytoca (n = 4) and Citrobacterfreundii (n = 1), with identifiedisolates belongingtoa diverse setof MLSTsequence types(STs)(Table1).Furthermore,occurrenceisnotconnectedto one specific geographic location, animal hospital or studfarm. Although the main spread appears non-clonal, some clonal dissemination was observed. Six E. cloacae isolates from three sitesbelongedtoST116andbasedoncgMLSTtheseisolatescould

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bedividedintotwogroups,butthedifferencebetweenthetwo groupswasonly24alleles(Fig.1).BasedoncgMLST,theremaybe someconnectionbetweenthethreelocations,butinthecurrent investigation additional epidemiological data are lacking to concludethis.TherealsoappearstobecirculationofanE.cloacae belongingtoST113atonespecificanimalhospital,butcgMLSTalso showed that onlythree of the isolateswere similar(Fig.1).In addition,twoidenticalK.oxytocaST37,basedoncomparing3323 allelesusingcgMLST,wereidentifiedfromthesamehospital(data notshown).Furthermore,atonestudfarm,fiveE.coliisolateswere showntobelongtotwoMLSTST1423(n=2)andST1861(n=3) (Table 1), withtherespective STs beingidenticalusingcgMLST (Fig.1).Interestingly,theisolatethatlackedblaSHV-12wasanE.coli

ST9239 that was shown to be essentially identical to another isolatethatcarriedblaSHV-12(Fig.1).Thesetwoisolatesoriginated

fromthesameclinicandfromthesamehorse(Table1).TheE.coli

ST9239 isolatewith blaSHV-12 originated from the ﬁrst sample,

whichcouldindicatethattheisolatelostblaSHV-12butretainedthe

mcr-9andblaTEM-1Bgenes.

Theplasmidinvolvedinthespreadofthemcr-9,andpossibly blaSHV-12andblaTEM-1B,amonghorsesinSwedenprobablybelongs

toreplicontypesIncHI2andIncHI2Aasall30isolateswerepositive forthesetworeplicontypes,andinsomeisolatestheywerethe onlyreplicontypesidentiﬁed.UsingPubMLSTitwasshownthat, with the exception of one isolate, all IncHI2 belonged to the plasmid MLSTpST1. The single isolates that differed in pMLST lackedoneoftheallelesfortheIncHI2-pST,anditwasalsotheE. cloacae ST113 isolate that differed signiﬁcantly from the other threeST113isolates(Fig.1).Theoriginalpublicationofmcr-9also concludedthatthereappearstobeaconnectionbetweenmcr-9, IncHI2andIncHI2A,andthisconnectionwasfurtherestablishedin thestudyby Kiefferetal. [4,7].Furthermore,allisolates inthe

Table1

Genotypiccharacteristicsandcolistinminimuminhibitoryconcentrations(MICs)ofmcr-9-positive,blaSHV-12and/orblaTEM-1B-producingEnterobacteriaceaeclinicalisolates

positiveforplasmidreplicontypesIncHI2andIncHI2AfromhorsesinSweden(n=30).

Species Year Strain Colistin

MIC MLST Additional plasmid replicon types(Inc) ARGsa Origin/samplesite

Citrobacterfreundii 2018 3553 2 ST233 aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,blaCMY-82,catA2,

dfrA19,qnrB10,tetD

AnimalhospitalA/surgical wound

Escherichiacoli 2017 3243 0.5 ST1861 FIB,FII aadA1,aadA2,aph(3')-Ia,dfrA1,dfrA19 StudfarmA/uterus 3283 0.25 ST1861 FIB,FIC aadA1,aadA2,aph(3')-Ia,dfrA1,dfrA19 StudfarmA/uterus 3294 0.5 ST1861 FIB,FIC aadA1,aadA2,aph(3')-Ia,dfrA1,dfrA19,mdfA StudfarmA/uterus 3271 0.25 ST2557 aac(6')-Ib,ant(3”)-Ia,aph(3')-Ia,mdfA,tetD AnimalhospitalB/surgical

wound 3479b

0.5 ST9329 FIB,FIC,FII aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,catA2,dfrA19,mdfA, qnrB2,tetD

HorseclinicA/urine 2018 3486b _0.25 _ST9329 _FIA,_FIB,_FI _aac(6')-Ib,_aadA2,_aph(3')-Ia,_{aph(3”)-Ib,}_dfrA19,_mdfA,_qnrB2 _Horse_clinic_A/urine

3632 0.5 ST1252 Col

(MG828)

aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,catA2,dfrA19,mdfA, qnrB2

AnimalclinicX/wound secretion

3662 0.25 ST1423 aadA2,aph(3')-Ia,aph(3”)-Ib,dfrA19,mdfA StudfarmA/uterus

3663 0.5 ST1423 aadA2,aph(3')-Ia,aph(3”)-Ib,catA2,dfrA19,mdfA StudfarmA/uterus

3666 0.25 ST4398 Col

(MG828), FI

aac(6')-Ib,aph(3')-Ia,aph(3”)-Ib,catA2,dfrA19,mdfA Privatepractitioner/uterus

Enterobactercloacae complex

2017 3100 0.25 ST113 FIB,FII aac(6')-Id,aadA1,aadA2,ant(3”)-Ia,aph(3')-Ia,aph(3”)-Ib, blaACT,catA2,dfrA19,fosA,tetD

Animalhospital B/post-operativefuniculitis 3114 0.25 ST116 FIB aac(6')-Id,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,dfrA19,fosA LabAc/abscess

3229 0.25 ST116 FIB,P aac(6')-Id,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,dfrA19,fosA AnimalhospitalA/blood

3290 0.5 ST116 Col

(MG828)

aac(6')-Id,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,fosA LabAc/sinus

3401 0.5 ST116 FIB aac(6')-Id,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,dfrA19,fosA AnimalhospitalA/urine

3203 0.25 ST1021 A/C aac(3)-IIa,aadA1,aph(3”)-Ib,blaACT,blaSCO-1,catA2,dfrA19,

dfrA8,fosA,qnrA1,tetD

AnimalhospitalA/uterus 3424 0.5 ST51 aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,catA2,dfrA19 AnimalhospitalB/skinbiopsy

3458 0.25 ST102 aac(6')-Ib,ant(3”)-Ia,aph(3')-Ia,blaACT,catA2,fosA,tetD AnimalhospitalB/surgical

wound 2018 3546 0.5 ST113 aac(6')-Ib,aadA1,aadA2,aph(3”)-Ib,blaACT,catA2,dfrA19,

fosA,tetD

AnimalhospitalB/surgical wound

3581 1 ST113 FIB,FII aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,catA2,dfrA19,

fosA,tetD

AnimalhospitalB/abscess 3661 1 ST113 FIB,FII aac(6')-Ib,aadA1,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,catA2,

dfrA19,fosA,tetD

AnimalhospitalB/tracheal 3558 1 ST116 FIB aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,dfrA19,fosA AnimalhospitalB/urine

3649 0.25 ST116 aac(6')-Ib,aph(3')-Ia,ant(3”)-Ia,aph(3”)-Ib,blaACT,fosA LabAc/uterus

3523 0.5 ST88 aac(6')-Ib,aph(3')-Ia,aph(3”)-Ib,blaACT,dfrA19,fosA AnimalhospitalC/wound

3545 0.25 ST254 FIB aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,blaACT,dfrA19,tetA LabBc/uterus

Klebsiellaoxytoca 2016 2798 0.5 ST238 aac(6')-Ib,aadA2,ant(3”)-Ia,aph(3')-Ia,blaOXY,catA2,dfrA19,

qnrB2,tetD

AnimalhospitalA/wound 2017 3163 0.25 ST37 aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,blaOXY,catA2,dfrA19,

tetD

AnimalhospitalB/ thrombophlebitis 3280 0.25 ST37 aac(6')-Ib,aadA2,aph(3')-Ia,aph(3”)-Ib,blaOXY,catA2,dfrA19,

tetD

AnimalhospitalB/urine 3320 0.5 ST2 aadA2,aph(3')-Ia,aph(3”)-Ib,blaOXY,catA2dfrA19,tetD StudfarmB/uterus

MSLT,multilocussequencetyping;ARG,antimicrobialresistancegene.

a

Allisolatescarriedaac(6ʹ)-IIc,aph(6)-Id,ere(A),sul1andsul2.

b _Two_isolates_collected_from_the_same_horse_from_samples_collected₁₀_days_apart,_with_the_later_isolate₃₈₄₆_being_negative_for_bla SHV-12. c_Private_laboratories_not_connected_to_a_speciﬁc_animal_hospital_or_stud_farm.

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originalstudyandthecurrentonealsocarriedaac(6ʹ)-IIc, aph(6)-Id,ere(A),sul1andsul2.Thisraisesthequestionofwhetheritis globalspreadofspeci_ﬁcIncHI2andIncHI2Aplasmids,oraplasmid having both replicons, which may also carry multiple genes encoding antimicrobial resistance. The next step would be to establishthefull sequence of thedetected IncHI2and IncHI2A plasmidstoconﬁrm that theyin factcarry mcr-9,blaSHV-12and

blaTEM-1B,andifsohowmcr-9islocatedcomparedwiththeother

ARGs.

ItappearsthattheepidemiologyofblaSHV-12-carryingisolates

amonghorsesinSwedenmightdifferfromtherestofEurope.A previousFrenchstudyshowedthatblaSHV-12inisolatesfromhorses

was mainly connected to IncX plasmids, and previously IncX3 plasmidshavebeenshowntopredominantlycarryblaSHV-12[9,10].

Inthecurrentstudy,theIncXrepliconwasnotdetectedinisolates carryingmcr-9andblaSHV-12,althoughthetwoSwedishblaSHV-12

isolatesfromhorsesthatlackedmcr-9werepositivefortheIncX3 replicon.

Inconclusion,wewereabletoshowthatmcr-9iscommon(30/ 56; 54%) among clinical ESBL-producing Enterobacteriaceae isolates from Swedish horses, with the occurrence linked to

multidrug-resistantisolatescarryingblaSHV-12andblaTEM-1Baswell

asplasmidsbelongingtoreplicontypesIncHI2andIncHI2A. Funding

ThisinvestigationreceivedfundingfromtheSwedishBoardof Agriculture[Dnr6.2.18-15925/2017]. Competinginterests Nonedeclared. Ethicalapproval Notrequired. Acknowledgments

The authors would liketo thank all of the laboratories and veterinarianswhosubmittedsuspectedESBL-producingisolatesto theNationalVeterinaryInstitute(SVA).Theauthorsarealsograteful

Fig.1.Minimumspanningtree(MST)ofthecoregenomemultilocussequencetyping(cgMLST)relationshipof(A)Escherichiacoliand(B)Enterobactercloacaecomplex isolatescarryingtheblaSHV-12andmcr-9genesfromhorsesinSweden,basedongenespresentinallcomparedisolates.Numbersonthelinesbetweenisolatesindicate

differencesinallelesbetweenisolates;thelengthsoflinesarebasedonthenumberofgenedifferencesonalogscale;andthediameterofcirclesisbasedonthenumberof isolatesonalogscale.(A)MSTbasedon2483allelespresentinsevenE.coliclinicalisolatescarryingblaSHV-12andmcr-9fromhorsesinSweden.cgMLSTisbasedonthe

EnteroBasecgMLSTdatabaseavailablethroughSeqSphere+software;theSTdenotestheMLSTnumberasdeﬁnedbythescheme(https://enterobase.warwick.ac.uk/).* IndicatestheisolatelackingtheblaSHV-12gene.(B)MSTbasedon3873allelespresentintenE.cloacaecomplexclinicalisolatescarryingblaSHV-12andmcr-9fromhorsesin

Sweden.cgMLSTisbasedontargetgenesextractedfromtheEnterobacterhormaechei34998genome(GenBankaccessionno.CP012167.1)usingSeqSphere+software,with theresultingcgMLSTschemeconsistingof4273targetgenesforphylogeneticanalyses;theSTdenotestheMLSTnumberasdeﬁnedbytheschemeathttps://pubmlst.org/.

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toProf.LaurentPoirelforprovidingthesequencesoftheqseB-like andqseC-likegenesidentiﬁedinthestudybyKiefferetal.[7]. References

[1]KempfI,JouyE,ChauvinC.Colistinuseandcolistinresistanceinbacteriafrom animals.IntJAntimicrobAgents2016;48:598–606.

[2]FalagasME, Karageorgopoulos DE, Nordmann P. Therapeuticoptions for infections with Enterobacteriaceae producing carbapenem-hydrolyzing enzymes.FutureMicrobiol2011;6:653–66.

[3]LiuYY,WangY,WalshTR,YiLX,ZhangR,SpencerJ,etal.Emergenceof plasmid-mediated colistinresistance mechanism MCR-1 in animals and humanbeingsinChina:amicrobiologicalandmolecularbiologicalstudy. LancetInfectDis2016;16:161–8.

[4]LimaT,DominguesS,DaSilvaGJ.Plasmid-mediatedcolistinresistancein Salmonellaenterica:areview.Microorganisms2019;7:,doi:http://dx.doi.org/ 10.3390/microorganisms7020055pii:E55.

[5]NationalVeterinaryInstitute(SVA).Swedres-Svarm summary2018. Con-sumptionof antibiotics and occurrence of resistancein Sweden. Solna/ Uppsala,Sweden:SVA;2019.

[6]CarrollLM,GaballaA,GuldimannC,SullivanG,HendersonLO,WiedmannM. Identiﬁcationofnovelmobilizedcolistinresistancegenemcr-9ina multidrug-resistant, colistin-susceptible Salmonella enterica serotype Typhimurium isolate. mBio 2019;10:, doi:http://dx.doi.org/10.1128/mBio.00853-19 pii: e00853-19.

[7]KiefferN,RoyerG,DecousserJW,BourrelAS,PalmieriM,OrtizDeLaRosaJM, etal.mcr-9,aninduciblegeneencodinganacquiredphosphoethanolamine transferaseinEscherichiacoli,anditsorigin.AntimicrobAgentsChemother 2019;63:,doi:http://dx.doi.org/10.1128/AAC.00965-19pii:e00965-19. [8]SwedishMedicalProductsAgency.Dosageofantibioticsforhorses–new

recommendation [inSwedish]. Informationfrom the Swedish Medicines Agency2015;26(Supplement)4–16...[Accessed13January2020]https:// lakemedelsverket.se/antibiotikatillhast.

[9]SadikalayS,ReynaudY,Guyomard-RabenirinaS,FalordM,DucatC,FabreL, etal.Highgeneticdiversityofextended-spectrumβ-lactamasesproducing Escherichiacoliinfecesofhorses.VetMicrobiol2018;219:117–22. [10]DobiasovaH,DolejskaM.PrevalenceanddiversityofIncXplasmidscarrying

ﬂuoroquinoloneandβ-lactamresistancegenesinEscherichiacolioriginating from diverse sources and geographical areas. J Antimicrob Chemother 2016;71:2118–24.