Draft genome sequence of an mcr-1/IncI2-carrying multidrug-resistant Escherichia coli B1:ST101 isolated from meat and meat products in Egypt

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Genome

Note

Draft

genome

sequence

of

an

mcr-1/IncI2-carrying

multidrug-resistant

Escherichia

coli

B1:ST101

isolated

from

meat

and

meat

products

in

Egypt

Mustafa

Sadek

a,b

,

Laurent

Poirel

a,c,d,

*,

Patrice

Nordmann

a,c,d,e

,

Hirofumi

Nariya

f

,

Toshi

Shimamoto

f

,

Tadashi

Shimamoto

f

a

MedicalandMolecularMicrobiology,FacultyofScienceandMedicine,UniversityofFribourg,Fribourg,Switzerland

b

DepartmentofFoodHygieneandControl,FacultyofVeterinaryMedicine,SouthValleyUniversity,Qena,Egypt

c_INSERM_European_Unit_(IAME),_University_of_Fribourg,_Fribourg,_Switzerland d

SwissNationalReferenceCenterforEmergingAntibioticResistance(NARA),UniversityofFribourg,Fribourg,Switzerland

e

InstituteforMicrobiology,UniversityofLausanneandUniversityHospitalCentre,Lausanne,Switzerland

f

LaboratoryofFoodMicrobiologyandHygiene,GraduateSchoolofBiosphereScience,HiroshimaUniversity,Higashi-Hiroshima739-8528,Japan

ARTICLE INFO

Articlehistory:

Received11November2019 Accepted22November2019 Availableonline28November2019

Keywords: mcr-1 IncI2 Escherichiacoli Whole-genomesequencing Egypt ABSTRACT

Objectives:Theaimofthisstudywastoinvestigatetheoccurrenceofplasmid-encodedcolistinresistance amongGram-negativebacteriaisolatedfrommeatandmeatproductsinEgyptandtoreportthedraft genomesequenceofanmcr-1/IncI2-carryingmultidrug-resistant(MDR)EscherichiacoliB1:ST101isolate. Methods:Atotalof128colistin-resistantstrainswereisolatedfromvariousmeatandmeatproduct samplesindifferentcitiesinEgypt.MultiplexPCRscreeningforplasmid-mediatedcolistinresistance geneswasperformed.Whole-genomesequencingwasperformedusinganIlluminaNextSeqplatform andthegenomewasassembledusingCLCGenomicsWorkbench7.5.1.

Results:Asinglemcr-1-positiveMDRE.colistrainwasisolatedfrombeefsausages.Thegenomesizeofthe E.colistrainwascalculatedat5044715bp,withatotalof226contigsandaG+Ccontentof50.5%.The strainbelongedtoST101(phylogroupB1).Themcr-1genewaslocatedonanIncI2-typeself-conjugative plasmidof64.6kbinsize.ThestrainshowedaMDRphenotype,withacolistinMICof4mg/L.Alarge numberofacquiredantimicrobialresistancegeneswasidentiﬁed,includinggenesencodingresistanceto colistin(mcr-1),β-lactams(blaTEM-1),phenicols(ﬂoR),trimethoprim(dfrA12),aminoglycosides [aac(3)-IIa,aph(3”)-IbandaadA2],macrolides(mphAandmdfA),tetracyclines(tetA),sulfonamides(sul1andsul2) andquinolones(qnrS1).

Conclusion:Herewereporttheﬁrstdraftgenomesequenceofanmcr-1/IncI2-carryingMDRE.coliB1: ST101 isolatedfrombeef sausageinEgypt.This studyhighlightsthepotential roleplayedby food productsinthespreadofcolistinresistancetohumans.

The continuing spread of multidrug-resistant (MDR) Gram-negativebacteria,especially carbapenem-resistantbacteria,is a globalhealthconcernandhasledtothere-useofoldantibiotics, particularlycolistin,fortreatingcriticalinfections[1,2].Resistance tocolistinwas initially linkedtomutational changes identiﬁed within chromosomal genes that contribute tothe biosynthesis of lipopolysaccharide. Recently, a plasmid-mediated colistin resistancegene(mcr-1)wasreportedfromhumans,animals,food

and the environment in China [3]. This gene encodes a phosphoethanolaminetransferaseresponsibleforacquired resis-tancetocolistinandhasnowbeenreportedworldwide.

Theaimofthisstudywastoevaluatetheoccurrenceof plasmid-mediated colistin resistance among Gram-negative bacteria isolated frommeat andmeat productsin Egypt. Atotal of 128 colistin-resistant strains [minimum inhibitory concentration (MIC)>2mg/L]wereisolatedfromvariousmeatandmeatproduct samples (fresh beef,frozenbeef, mutton, mincedmeat, burger, sausage,luncheon,koftaandpastırma)randomlycollectedfrom differentsupermarkets, slaughterhousesand butcher’s shops in different cities in Egypt. Isolates were recovered by direct * Corresponding authorat: Medical andMolecular Microbiology, Facultyof

ScienceandMedicine,UniversityofFribourg,Fribourg,Switzerland. E-mailaddress:laurent.poirel@unifr.ch(L.Poirel).

http://dx.doi.org/10.1016/j.jgar.2019.11.015

JournalofGlobalAntimicrobialResistance20(2020)41–42

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

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spreadingonMacConkeyagarsupplementedwith2mg/Lcolistin. PCR screening for plasmid-mediated colistin resistance genes (mcr-1to-8)wasperformedbutresultednegative[4],exceptfora singleEscherichiacoliisolateinwhichthemcr-1genewasdetected. Thisstrain(MC13)wasrecoveredfromabeefsausagesample.

AntimicrobialsusceptibilitytestingofE.colistrainMC13was performedbythediskdiffusionmethodaccordingtotheClinical and LaboratoryStandardsInstitute(CLSI)guidelines, except for colistin.ThecolistinMICwasdeterminedbybrothmicrodilutionin cation-adjusted Mueller–Hinton broth (Bio-Rad, Marnes-la-Co-quette,France)and theresultwas interpretedaccording tothe European Committee on Antimicrobial Susceptibility Testing (EUCAST)/CLSIjointguidelines(www.eucast.org).Escherichiacoli strainMC13 was resistantto ampicillin, ciproﬂoxacin, nalidixic acid, kanamycin, gentamicin, chloramphenicol and tetracycline but remained susceptible to broad-spectrum cephalosporins, carbapenemsandaztreonam.ThecolistinMICwas4mg/L,being intheresistantrange.

WholegenomicDNAwasobtainedfromanovernightcultureof strainMC13usingaPureLink1GenomeDNAMiniKit(Invitrogen, Darmstadt,Germany)andagenomiclibrarywaspreparedusingan IlluminaNexteraXTDNALibrarywith2300-bppaired-endreads onanIllumina MiSeqinstrument(Illumina Inc.,SanDiego,CA, USA). De novo genome assembly was performed using CLC GenomicsWorkbench7.5.1.Theassembleddraftgenomesequence ofE.coliMC13consistedof226contigscomprising5044715bp withaG+Ccontentof50.5%.Multilocussequencetyping(MLST) analysis (https://cge.cbs.dtu.dk/services/MLST/) showed that strain MC13 belonged to ST101, pathogenic phylogroup B1, a backgroundthathaspreviouslybeenassociatedwiththe produc-tionofMCR-1inapatientwithaurinarytractinfectioninSouth Africa[5].

Onlyasinglemcr-1-positiveE.colihumanclinicalisolatehas beenidentiﬁedinEgyptsofar,fromapatientwithbacteraemia[6]. Regardinganimals, the mcr-1 gene was identiﬁed in an E. coli isolate from a cow suffering from subclinical mastitis [4]. Furthermore,a single mcr-1-positive E. coli strain was isolated fromrawmilkcheesein2019[7].

SerotypingandfimHsubtypingwereperformedusing Seroty-peFinder2.0(https://cge.cbs.dtu.dk/services/SerotypeFinder/)and FimTyper1.0(https://cge.cbs.dtu.dk/services/FimTyper/), respec-tively,andrevealedtheO15:H10-fimH86profile.Virulencegenes, including cma (colicin M), ireA (siderophore receptor), gad (glutamate decarboxylase), lpfA (long polar fimbriae) and iss (increasedserumsurvival),weredetectedbyVirulenceFinder2.0 (https://cge.cbs.dtu.dk/services/VirulenceFinder/).Various antimi-crobial resistance genes were predicted in the genome by ResFinder3.2(https://cge.cbs.dtu.dk/services/ResFinder/), includ-inggenesencoding resistancetoampicillin(blaTEM-1),phenicols

(ﬂoR),trimethoprim(dfrA12),aminoglycosides[aac(3)-IIa,aph(3 ”)-IbandaadA2],macrolides(mphAandmdfA),tetracyclines(tetA), sulfonamides(sul1andsul2)andquinolones(qnrS1).

The plasmid harbouring mcr-1 (designatedpEGYMCR-1) was foundtobe64600bpinsizeandbelongedtotheIncI2group(broad hostrange).Ofnote,thefirstreportedmcr-1genefoundinanimal andhumanisolatesinChinawasalsoidentifiedonanIncI2-type plasmid[3].Thesequenceofthisplasmidwasidenticaltothatofthe mcr-1-positive plasmid pMCR-GN775 (accession no.KY471307) identifiedfromanE.coliST624isolaterecoveredfromaCanadian patientwhopreviouslyreceivedhealthcareinEgypt[8].

Generally,themcr-1geneisidentiﬁedasbeinginassociation withinsertionsequenceISApl1,whichmayplayamajorroleinits mobilisation[2].Ofnote,thepEGYMCR-1plasmiddidnotdisplay anISApl1elementinthevicinityofthemcr-1gene.However,the

mcr-1–pap2elementwasidentiﬁedinasimilarcontext,between thetop(encoding a DNAtopoisomeraseIII) and nikB(relaxase) genes,likeinpMCR-GN775andotherIncI2plasmidsaspreviously reported[8].

Transferofthemcr-1-harbouringIncI2plasmid(pEGYMCR-1) wasdemonstratedbyaﬁlter-matingassayusingazide-resistantE. colistrainJ53asrecipient.TransconjugantswereselectedonLB agar supplemented with sodium azide (150mg/L) and colistin (2mg/L).Thetransconjugantwasresistantonlytocolistin(MIC= 4mg/L),andnootherresistancedeterminantwasco-transferred, indicatingthatonlythemcr-1genewaslocatedontheconjugative plasmid.

In conclusion, here we report the occurrence of a colistin-resistantmcr-1geneinaMDRE.colistrainrecoveredfromameat productinEgypt.Withtheemergenceofmcr-1-bearingbacteriain meatandmeatproducts,thishighlightsthepotentialreservoirfor colistin resistance, thereby leading to a higher possibility of transmission of resistant bacteria frommeat tohumans. Strict monitoringandsurveillanceofresistantbacteriaamongfoodsof animalorigin,particularlymeatandmeatproducts,arestrongly neededtopreventtheirdisseminationtohumans.

ThedraftgenomesequencingprojectofE.colistrainMC13had beendepositedatGenBankunderaccessionno.PRJNA587479. Funding

This work was funded by the Egyptian Government, the University of Fribourg (Fribourg, Switzerland) and the Swiss NationalScienceFoundation[projectFNS-407240_177381]. Competinginterests

Nonedeclared. Ethicalapproval

Notrequired. References

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