Draft genome sequence of a blaNDM-1- and blaOXA-244-carrying multidrug-resistant Escherichia coli D-ST69 clinical isolate from Egypt

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Genome

Note

Draft

genome

sequence

of

a

bla

NDM-1

-

and

bla

OXA-244

-carrying

multidrug-resistant

Escherichia

coli

D-ST69

clinical

isolate

from

Egypt

Ahmed

M. Soliman

a,b

,

Hazem

Ramadan

c,d

,

Mustafa

Sadek

e

,

Hirofumi

Nariya

f

,

Toshi

Shimamoto

g

,

Lari

M. Hiott

c

,

Jonathan

G. Frye

c

,

Charlene

R. Jackson

c

,

Tadashi

Shimamoto

g,

*

a

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

b

DepartmentofMicrobiologyandImmunology,FacultyofPharmacy,KafrelsheikhUniversity,KafrEl-Sheikh33516,Egypt

c

BacterialEpidemiologyandAntimicrobialResistanceResearchUnit,USNationalPoultryResearchCenter,USDA-ARS,Athens,GA,USA

d

HygieneandZoonosesDepartment,FacultyofVeterinaryMedicine,MansouraUniversity,Mansoura35516,Egypt

e

MedicalandMolecularMicrobiology,FacultyofScienceandMedicine,UniversityofFribourg,Fribourg,Switzerland

f

FacultyofHumanLife,JumonjiUniversity,Niiza352-8510,Japan

g_Laboratory_of_Food_Microbiology_and_Hygiene,_Graduate_School_of_Integrated_Sciences_for_Life,_Hiroshima_University,_1-4-4_Kagamiyama,_{Higashi-Hiroshima}

739-8528,Japan

ARTICLE INFO

Articlehistory: Received10April2020

Receivedinrevisedform3July2020 Accepted14July2020

Availableonline29July2020

Keywords: blaNDM-1 blaOXA-244 Egypt IncI1 Whole-genomesequencing ABSTRACT

Objectives: This study describes the ﬁrst draft genome sequence of a multidrug-resistant (MDR) EscherichiacoliD-ST69clinicalisolatefromEgyptcarryingblaNDM-1andblaOXA-244.

Methods:ThestrainwasisolatedinDecember2014fromawoundpusswabofamalepatientinthecityof KafrEl-SheikhusingMacConkeyagarcontaining2 mg/mLmeropenem.Thestrainwassubjectedto antimicrobialsusceptibilitytesting,conjugationexperiments,andwhole-genomesequencingusingan IlluminaMiSeqplatform.

Results:Thedraftgenomeofthestrain(HR14_AS)was5.08Mbpinsizecontainingatotalof90contigs encoding 4677predictedgenes withanaverageG+Ccontentof 50.7%.StrainHR14_AS belongsto sequencetype69(ST69),phylogroupDandexhibitsanMDRphenotype,withminimuminhibitory concentrations(MICs)of64mg/mLand32mg/mLformeropenemanddoripenem,respectively.Multiple acquiredantimicrobialresistancegenesconferringresistancetomacrolides[mdf(A)],ﬂuoroquinolones [aac(6')-Ib-cr],quinolones(qnrS1), trimethoprim(dfrA14), β-lactams(blaNDM-1,blaOXA-244,blaCTX-M-15,

blaOXA-9andblaTEM-1B)andaminoglycosides[aac(3)-IId,aac(6')-Ib,aadA1andaph(3')-VI]weredetected.

TheblaOXA-244andblaNDM-1geneswerelocatedonthechromosome(Tn6237)andonanIncI1-type

self-conjugativeplasmidof>93kbinsize,respectively.

Conclusions:HerewereporttheﬁrstdraftgenomesequenceofaMDRE.coliD-ST69isolatecarrying blaNDM-1andblaOXA-244.BesidesclonalexpansionoftheE.coliST38pandemicclone,thisstudyfurther

identiﬁedthatthespreadofOXA-244-producingE.colicouldberelatedtomobilisationoftheIS1R-made compositetransposon(Tn6237)carryingblaOXA-244.

©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobial Chemotherapy.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons. org/licenses/by-nc-nd/4.0/).

The Ambler class D OXA-244carbapenemase is a variant of OXA-48 with a single (Arg214Gly) substitution that results in decreased carbapenemase activity [1]. OXA-244 was initially reportedina clinicalKlebsiella pneumoniaeisolaterecovered in Malaga,Spain,in2012[2].Ithassincebeenreportedfrommany

othercountries,includingGermany,Russia,France,Egypt,theUK, theNetherlands,Turkey,Colombia,AlgeriaandLebanon[1,3].The blaOXA-244genehasbeendetectedonaplasmidinK.pneumoniae

and Enterobacter aerogenes as well as integrated into the chromosome of Escherichia coli sequence type 38 (ST38) [1,3]. OXA-244-producingE.colidonotgrowonChromID1CarbaSmart platesbecauseofthereducedcarbapenemaseactivityofOXA-244

[1]. They are therefore difﬁcult to detect, leading to silent dissemination[1].Theaimofthisworkwastodescribetheﬁrst *Correspondingauthor.

E-mailaddress:tadashis@hiroshima-u.ac.jp(T.Shimamoto).

http://dx.doi.org/10.1016/j.jgar.2020.07.015

2213-7165/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

JournalofGlobalAntimicrobialResistance22(2020)832–834

ContentslistsavailableatScienceDirect

Journal

of

Global

Antimicrobial

Resistance

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draft genome sequence of a blaNDM-1- and blaOXA-244-carrying

multidrug-resistant (MDR) E. coli D-ST69 clinical isolate from Egypt.

IsolateHR14_ASwasrecoveredfromawoundpusswabofa malepatientadmittedtotheorthopaedicdepartmentofahospital inthe cityof KafrEl-Sheikh,Egypt, on10December2014. The isolatewasselectedonMacConkeyagarsupplementedwith2

m

g/ mL meropenem and was identiﬁed using an API 20E system (bioMérieux,Marcy-l’Étoile,France)followedbyPCRsequencingof thesmallsubunitribosomal RNA(16SrRNA)geneaspreviously described[4].Antimicrobialsusceptibilitytestingwasperformed bythediskdiffusionassayandbrothmicrodilutionmethodandthe resultswereinterpretedinaccordancewiththe2018guidelinesof the Clinical and Laboratory Standards Institute (CLSI). Isolate HR14_ASshowedresistancetomeropenem[minimuminhibitory concentration(MIC)=64

m

g/mL],doripenem(MIC=32

m

g/mL), aztreonam, ampicillin, amikacin, amoxicillin/clavulanic acid, cefotaxime,ceftazidime, ceftriaxone,cefoxitin,gentamicin, nali-dixic acid, ciproﬂoxacin, kanamycin and sulfamethoxazole/tri-methoprimbut remainedsusceptibletofosfomycin,tigecycline, chloramphenicolandcolistin(MIC<0.5

m

g/mL).Theisolatewas also positive for carbapenemase production by the modiﬁed carbapeneminactivationmethod[4].

Subsequently,PCRandDNAsequencingwereusedtoscreenfor carbapenemase-encodinggenes,extended-spectrumβ-lactamase (ESBL) genes, plasmid-mediated quinolone resistance genes, integrons, mobile colistin resistance genes (mcr-1 and mcr-8) and16SrRNAmethylasegenesaswellasforE.coliphylogrouping

aspreviouslyreported[4].TheresultsdemonstratedthatHR14_AS carriedblaNDM-1,blaOXA-244,blaCTX-M-15,blaTEM-1BandqnrSandwas

assignedtophylogroupD.

TotalgenomicDNAwasextractedfromanovernightcultureof theisolateusingaGenEluteTMBacterialGenomicDNAKit(Sigma– Aldrich,StLouis,MO,USA).Sequencinglibrarieswereconstructed using a Nextera XT Library Preparation Kit, and paired-end sequencing was performed with an Illumina MiSeq system (Illumina Inc.) using a 500-cycle MiSeq Reagent Kit. Genomes wereassembledusingtheA5-miseqpipeline.Thedraftgenomeof HR14_ASwas 5.08 Mbpin sizecontaininga totalof 90 contigs encoding 4677predictedgeneswithanaverageG+Ccontentof 50.7%andamediumgenomedepthcoverageof70,withanN50of

250881bp.Multilocussequencetyping(MLST)usingMLST2.0 software(https://cge.cbs.dtu.dk/services/MLST/)assignedthe iso-latetoST69.Toourknowledge,thisistheﬁrstreportofablaNDM-1

-and blaOXA-244-carryingMDR E. coli D-ST69 globally. ST38 was

previously identiﬁed as thecommon sequence type associated withclonalexpansionofOXA-244-producingE.coliinEuropean countries [3,5,6]. However, other sequence types of OXA-244-producingE.coliweredetectedinEgyptandAlgeria(ST361and ST3541),respectively[1,3].

SerotypeFinder 2.0 ( https://cge.cbs.dtu.dk/services/Serotype-Finder/) and FimTyper 1.0 ( https://cge.cbs.dtu.dk/services/Fim-Typer/) were used to analyse the serotype and fimH subtype, respectively,andidentifiedtheO17:H18-fimH27profile. Further-more, VirulenceFinder 2.0 (

https://cge.cbs.dtu.dk/services/Viru-lenceFinder/) identiﬁed virulence genes including air

Fig.1.(A)SchematicrepresentationofthegeneticenvironmentofblaNDM-1identiﬁedfromthedraftgenomesequenceofEscherichiacolistrainHR14_ASanalysedinthis

study,includinglinearcomparisonwithplasmidtig00000217(accessionno.CP021941)inKlebsiellapneumoniaestrainAR_0145fromtheUSAandplasmidpK66-45-1 (accessionno.CP020902)inclinicalK.pneumoniaestrainK66-45fromNorway.(B)LinearcomparisonofthegeneticenvironmentofblaOXA-244identiﬁedfromE.colistrain

HR14_ASanalysedinthisstudyandthechromosomeofaclinicalE.coliST38strain28Eco12(accessionno.CP038505)isolatedinColombia.Thegeneticenvironmentof blaNDM-1andblaOXA-244wasaph(3')-VI–ISAba125(IS30family)–ISSpu2(IS630family)–blaNDM-1–bleMBL(A)andIS1R(IS1family)–blaOXA-244–lysR–IS10A(IS4family)(Tn6237)

(B),respectively.ThefigurewasdrawnusingEasyfig(http://mjsull.github.io/Easyfig/).

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(enteroaggregativeimmunoglobulinrepeatprotein),eilA (Salmo-nellaHilA homologue), gad (glutamatedecarboxylase) and lpfA (long polar fimbriae). ResFinder 3.2 ( https://cge.cbs.dtu.dk/ser-vices/ResFinder/)identifiedadiversityofacquiredantimicrobial resistance genes conferring resistance to macrolides [mdf(A)], fluoroquinolones[aac(6')-Ib-cr],quinolones(qnrS1),trimethoprim (dfrA14),β-lactams(blaNDM-1,blaOXA-244,blaCTX-M-15,blaOXA-9and

blaTEM-1B)andaminoglycosides[aac(3)-IId,aac(6')-Ib,aadA1and

aph(3')-VI].

PlasmidFinder 2.0 ( https://cge.cbs.dtu.dk/services/Plasmid-Finder/)identiﬁedfourdifferentincompatibilitygroups,including IncI1-I

g

,IncFII(K),IncRandIncY.Conjugationexperimentswere performedusingazide-resistantE.coliJ53asrecipientasdescribed previously[4]. Transconjugants wereselected on Luria–Bertani agarsupplementedwithsodiumazide(100

m

g/mL)andampicillin (100

m

g/mL).SeveraltransconjugantswereselectedforcolonyPCR and plasmid analysis as described previously [4]. The NDM-positiveE.colitransconjugantwithonlyoneplasmidwasselected. Itwasresistanttomeropenem(MIC=32

m

g/mL),doripenem(MIC =8

m

g/mL),amoxicillin/clavulanicacid,aztreonam,ceftazidime, ceftriaxone, cefotaxime, cefoxitin and gentamicin. The blaNDM-1

genewaslocatedonanIncI1-I

g

-typeself-conjugativeplasmidof >93kb insizeasdeterminedbyPCR-basedreplicontyping[4]. Furthermore,blaNDM-1,blaCTX-M-15,blaTEM-1BandqnrS1geneswere

co-transferred, indicating that they were located on the same conjugative IncI1 plasmid, which belonged to ST37-CC3 as detectedbypMLST(https://cge.cbs.dtu.dk/services/pMLST/). How-ever, the blaOXA-244 gene was not successfully transferred by

conjugation, transformation or electroporation, suggesting its chromosomallocation.

A BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) search in combination with identiﬁcation of insertion sequence (IS) elements using ISFinder (https://www-is.biotoul.fr/blast.php) illustrated the geneticenvironment of blaNDM-1 and blaOXA-244,

which was aph(3')-VI–ISAba125 (IS30 family)–ISSpu2 (IS630 family)–blaNDM-1–bleMBL (Fig. 1A) and IS1R (IS1 family)–bla OXA-244–lysR–IS10A (IS4 family) (Tn6237) (Fig.1B) for blaNDM-1 and

blaOXA-244,respectively.

A BLASTn search using thewholeblaNDM-1 contig identiﬁed

similarsequencesfromplasmidtig00000217(GenBankaccession no.CP021941)inK.pneumoniaestrainAR_0145fromtheUSAand plasmidpK66-45-1(GenBankaccessionno.CP020902)inclinical K.pneumoniaestrainK66-45fromNorway(100%querycoverage and99.99%sequenceidentity)(Fig. 1A).Moreover,aBLASTnsearch usingthewholeblaOXA-244contigidentiﬁedanidenticalsequence

from the chromosome of clinical E. coli ST38 strain 28Eco12 (GenBank accession no. CP038505) isolated in Colombia (100% querycoverageand100%sequenceidentity)(Fig.1B)[7].These

resultssuggesttheprobableactivityofTn6237transposonbyits movement to different E. coli clones or different sites in the chromosomeofE.coli.

Inconclusion,herewereporttheﬁrstdraftgenomesequenceof a blaNDM-1-and blaOXA-244-carryingMDRE.coliD-ST69globally.

BesidesclonalexpansionoftheE.coliST38pandemicclone,this studyfurtheridentiﬁedthatthespreadofOXA-244-producingE. colicouldberelatedtomobilisationoftheIS1R-madecomposite transposon(Tn6237)carryingblaOXA-244.

ThedraftgenomesequenceofE.colistrainHR14_AShasbeen deposited at DDBJ/ENA/GenBank under BioProject no. PRJNA612707andtheaccessionno.isJAAQFO000000000. Funding

AMS was supported by a fellowship from the Ministry of Education, Culture, Sports, Science and Technology of Japan [Fellowshipno.153532].Thisworkwasfundedbythe USDepart-mentofAgriculture(USDA)[project6040-32000-009-00]. Conﬂictofinterest

Nonedeclared. Ethicalapproval

Notrequired. References

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