pMEX01, a 70 kb plasmid isolated from Escherichia coli that confers resistance to multiple β-lactam antibiotics

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

pMEX01,

a

70

kb

plasmid

isolated

from

Escherichia

coli

that

confers

resistance

to

multiple

␤-lactam

antibiotics

Jose

Luis

Hernandez-Flores

_,

_Juan

_Caballero

_Pérez

_,

_Carlos

_{Salda ˜na}

_Gutiérrez

_,

Andrés

Cruz

Hernández

_,

_Gerardo

_Soto

_Alonso

_,

_Sergio

_Pacheco

_Hernández

_,

Sergio

Romero

Gómez

_,

_Francisco

_Fernández

_,

_Achim

_M.

_Loske

_,

Juan

Campos

Guillén

a_{UnidadIrapuato,CentrodeInvestigaciónydeEstudiosAvanzadosdelInstitutoPolitécnicoNacional,DepartamentodeIngeniería}

Genética,Irapuato,Guanajuato,Mexico

b_{UniversidadAutónomadeQuerétaro,FacultaddeQuímica,Querétaro,Mexico}

c_{UniversidadAutónomadeQuerétaro,FacultaddeCienciasNaturales,Querétaro,Mexico}

d_{UniversidadNacionalAutónomadeMéxico,CentrodeFísicaAplicadayTecnologíaAvanzada,Querétaro,Mexico}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20February2017 Accepted7November2017 Availableonline3February2018 AssociateEditor:AfonsoBarth

Keywords: Escherichiacoli Plasmid Antibioticresistance blaCTX-M-14gene

a

b

s

t

r

a

c

t

Multidrug-resistantmicroorganismsareofgreatconcerntopublichealth.Geneticmobile elements,suchasplasmids,areamongthemostrelevantmechanismsbywhichbacteria achievethisresistance.WeobtainedanEscherichiacolistrainCM6,isolatedfromcattle pre-sentingseverediarrheicsymptomsintheStateofQuerétaro,Mexico.Itwasfoundtocontain a70kbplasmid(pMEX01)withahighsimilaritytothepHK01-likeplasmidsthatwere pre-viouslyidentifiedanddescribedinHongKong.AnalysisofthepMEX01sequencerevealed thepresenceofablaCTX-M-14gene,whichisresponsibleforconferringresistanceto multi-ple␤-lactamantibiotics.Severalgenesputativelyinvolvedintheconjugativetransferwere alsoidentifiedontheplasmid.ThestrainCM6isofhighepidemiologicalconcernbecause itnotonlydisplaysresistancetomultiple␤-lactamantibioticsbutalsotootherkindsof antibiotics.

PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.

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

Introduction

Plasmids play a crucial role in the dissemination of CTX-Mtype extended-spectrum␤-lactamases (ESBLs),and their

∗ _{Correspondingauthors.}

E-mails:loske@fata.unam.mx(A.M.Loske),juan.campos@uaq.mx(J.C.Guillén).

characterizationmayprovide importantinsightinto under-standingmultidrug-resistantbacterialstrains.␤-Lactamsare among the most widely used antimicrobial agents for the treatmentofbacterialinfections.However,theintensive expo-sure of bacteria to ␤-lactam antibiotics has induced the emergence of several antibiotic resistance strategies, with

https://doi.org/10.1016/j.bjm.2017.11.002

1517-8382/PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

thedevelopmentof␤-lactamasescapableofinactivating ␤-lactams being one ofthe most relevant. In gram-negative bacteria, the production of ESBLs is the principal cause of resistance to ␤-lactam antibiotics.1,2 _{Plasmids encoding}

CTX-M-typecefotaximasewerereportedinGermanyinthe late1980s,3,4 _{and theyare currentlydistributed worldwide,}

including North America,5 _LatinAmerica6 _{and Asia.}7

CTX-M hasbecomeso widespread thatit is now considered to bethemostprevalent␤-lactamaseamongclinicalisolatesof

Escherichiacoliworldwide.8_{DiversestudiesinHongKong,}9,10

China,11_{and SouthKorea}12 _{havereportedthatCTX-M-14is}

the most frequently found ␤-lactamase enzyme in E. coli, Klebsiella pneumoniae, and Shigella isolates. In Mexico, the presence ofGES and CTX-M type ␤-lactamases have been found inEnterobacteriaceae clinical isolates.13 _{In a previous}

study, we sampled multidrug-resistant E. coli strains from cattlepresentingseverediarrheicsymptomsintheStateof Querétaro,Mexico,andanalyzedtheirdrugresistance spec-tra,plasmidprofiles andconservationusingshockwaves.14

Inthisstudy,weanalyzedthecompletenucleotidesequence ofthe pMEX01 plasmid from E. colistrain CM6, which has a high similarity with the previouslydescribed pHK01-like plasmids.15

Materials

and

methods

Isolationandidentificationofbacteria

Fecalsampleswere obtained from cattlepresentingsevere diarrheic symptoms in the State of Querétaro, Mexico during 2014. Samples from cattle that did not respond to standard antibiotic treatment were selected for further analysis. A total of 10 isolates were identified as E. coli

by analyzing 16S ribosomal deoxyribonucleic acid (rDNA) sequences, which were amplified using the primers fD1

(CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG) and

rD1 (CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC)15 _and

bymatrix-assistedlaserdesorption/ionizationtime-of-flight (MALDI-TOF)massspectrometry,16_{usingaMicroFlexLTmass}

spectrometer(BrukerDaltonics,Bremen,Germany).

Antibioticresistanceprofile

TheplasmidpMEX01 wasextractedfrom E.colistrain CM6 andtransformedbyelectroporation(250V,20and250␮FD) into the E. colistrain BL21 STAR(Invitrogen, Carlsbad,CA, U.S.A.). We then determined the antibiotic resistance pro-files of the E. coli strain CM6 and the E. coli strain BL21 STARcontainingpMEX01.Allantimicrobialtestingwas per-formedonagar-solidifiedLB.The␤-lactamantibioticsassayed included carbenicillin, cephalexin, ceftriaxone, cefotaxime and dicloxacillin. Kanamycin, rifampicin, chloramphenicol, polymyxin,spectinomycinandstreptomycinwerealsotested. Theminimuminhibitoryconcentration(MIC)ofeach E.coli

strainwereevaluatedbythecriteriaoftheClinicaland Lab-oratoryStandards Institute.17 _{E.coliBL21 STARwithoutthe}

pMEX01plasmidwasusedasacontrol.

Sequencingandbioinformatics

E. coli strain CM6 was selected for plasmid isolation and sequencing. Theplasmid was extracted from an overnight cultureofthe selectedstraingrowninLBbroth(BDDIFCO, Mexico) and purified using a Zyppy Plasmid Miniprep Kit (Zymo Research, CA, U.S.A). The plasmid was sequenced with an Illumina HiSeq 2000 sequencing platform at the MacrogenKoreaInstitute(Seoul,RepublicofKorea)usinga whole-genomeshotgunlibrarystrategy.TheplasmidicDNA sequence was reconstructed using multiple bioinformatic tools.Thesequencereadswereassembledusingthegenome assemblerprogramSPAdesv3.1.1.18 _{Theinitialcontigswere}

compared withthe Plasmidsdatabase from NCBIGenBank (accessed April 2016) using Blast+. The best matches indi-cated a high similarity with pHK plasmid family, then we usepHK17aasatemplatetoreorderthecontigsand recon-struct the full plasmid. Open reading frames (ORFs) were deducedandannotatedbyusingNCBIGlimmerv3.02.19–21_ORF

sequenceswereanalyzedagainstproteinfamilydatabasesto obtaininformationabouttheirfunctionality.22,23

Nucleotidesequence

Thenucleotidesequenceforthe pMEX01plasmidhasbeen depositedinGenBankundertheaccessionnumberKU695535.

Results

Sequenceassemblyandcomparativeanalysisofthe pMEX01plasmid

The plasmid (pMEX01) from E. coli strain CM6 was iso-lated and sequenced.Thesequencing ofpMEX01 produced 26,133,370short-sequencesas2×100 paired-endreads.The initialassemblyofthesequencereadsproduced1247contigs, with an average size of 4879bp, an expected 2119× aver-agecoverageandacalculatedN50valueof20,000.Thefine assemblyusingpHK17asequenceasareferenceshowedthe presenceofacircularrepliconconsistingof70,093nucleotides, withanaverageGCcontentof52.27%(Fig.1).

ThepMEX01plasmidiscomparableinsizeandishighly similartootherplasmidsisolatedandsequencedfromE.coli

inAsia,includingpHK17a (99%),pHK01(99%),pHK08(99%), andpHK09(99%);pKF3-70(98%)fromK.pneumoniae;pEG0356 (99%)(exceptfortheblaCTX-M-14allele)fromS.sonnei;and

pO26-L(98%)aplasmidfromaCanadianE.coliisolatethatcarries atetracyclineresistancegenecluster(Fig.2).Ourassembly covers 88.71%ofthe reference sequence (pHK17a) withan averagecoverage of7547×.AsequenceanalysisofpMEX01 showed that it belongs tothe incompatibilitygroup IncFII. Analyses using severalalgorithms and approaches showed thatthepMEX01plasmidhad98potentialORFs,withonly8 showingsimilaritiestounknownproteinsfromtheNCBI non-redundantmicrobialproteindatabase(SupplementaryTable S1). pMEX01 contains a number of primary structural fea-turesthatare highlysimilartopHK01-likeplasmids.These includeaDNAtransferregion(tragenes),requiredforefficient

63000 56000 49000 42000 35000 28000 21000 14000

∗

∗

∗

∗

Fig.1–CircularmapofpMEX01(innermap)comparedtopHK17a(outermap).ThelengthofpMEX01is70,093bp.Dark green,ORFrelatedtoplasmidreplication;lightgreen,ORFrelatedtoplasmidstabilization;red,CTX-M-14␤-lactamase;gray, ORFrelatedtotransposases,insertionsequences;yellow,ORFrelatedABCtransportsystem;lightblue,ORFrelatedto plasmidconjugation,transfer;darkblue,miscellaneousORF.InnercircleindicatesaGCplot,withpurpleandbrown indicatingbelowandaboveaverageGCcontents,respectively.AsterisksindicateORFwithsignificantdifferences.

S. sonnei strain EG0430 plasmid pEG0430

S. sonnei plasmid pEG0356 E. coli plasmid pHK17a

E. coli plasmid pEC13

E. coli strain CM6 plasmid pMEX01

E. coli strain G1 plasmid pHK08 E. coli strain Combat2D2 plasmid pHK01

E. coli strain C017e-caz-1 plasmid pHK09

0.004

E. coli strain H30 plasmid pO26-L K. pneumoniae plasmid pKF3-70

Fig.2–PhylogeneticdendrogramforpMEX01.TheassembledsequencewascomparedwiththeNCBIPlasmiddatabase (accessedApril2016)withBlastn(megablast)toidentifyhighlysimilarfullplasmidsequenceswithE-value<10−10.Plasmid sequenceswerealignedandadendrogramwasgeneratedusingtheNJalgorithminClustalWv2.1withthedefault

conjugativetransfer;thetrbgenes,whichareinvolvedinpilus assembly,theputativeplasmidmobilitygene(mob);the con-jugationgenetrwB; andthetraTgene,whichappearstobe responsibleformaintainingtheregulationoftransfer efficien-cies.ThefinOgeneisinvolvedininhibitingtheexpressionof theregulatorygenetraJinaprocessknownasfertility inhi-bition.In addition, pMEX01 carriesgenetic informationfor the control ofreplication, including a structuralgene (rep) thatencodesaninitiationprotein(Rep),aswellasaprotein involvedinthetoxin–antitoxinsystemmechanismthat par-ticipatesinplasmidmaintenance(relE/parE).Genesencoding an ironABC transport system (eit genes) are also present, including a permease, a substrate-binding protein, and an export-associatedprotein.Theplasmidharborsawell-defined potentialtransposonconsistingofIS903,ablaCTX-M-14gene,

andanISEcp1 element (tnpAgene), whichaccordingtothe sequenceanalysisanddistancethatseparatesthesegenetic elements,correspondstoatypeIItransposon(Fig.1).

Since strain CM6 was isolated from the feces of cattle thatdidnotrespondtothestandardtreatment,weassessed theantibioticresistanceprofileofstrainCM6togaininsight intoitspotentialmultidrug-resistance.Wefoundthatithas a resistance not only to high concentrations of ␤-lactam antibiotics (Table 1), but also to several other antibiotics, includingkanamycin,streptomycinandtospectinomycin,but notrifampicinandpolymyxin,towhichCM6issusceptible. Tocorrelatethesephenotypictraitswiththepresenceofthe pMEX01plasmid,the E.coliBL21 STARtransformant carry-ingtheplasmidwasevaluated.Theresultsshowedthatthe presenceofpMEX01providestheE.coliBL21STARstrainwith resistancetoall␤-lactamantibioticsanalyzed(Table1).Taken together,ourfindingssuggestthatpMEX01ishighlysimilar tootherpHK01-likeplasmidsdisseminatedworldwidewith anextended-spectrum␤-lactamase.

Discussion

ThisstudydemonstratedthepresenceofapHK01-like plas-mid(pMEX01)inE.colistrainCM6thatwasisolatedfromfecal samplesofcattleintheStateofQuerétaro,Mexico.Although pMEX01ishighsimilartopHK17a(99%),severalopen read-ingframessignificantlydifferbetweenthetwoplasmids.For

Table1–Minimuminhibitoryconcentration(␮g/mL)of

E.colistrainCM6andE.coliBL21STARtransformants carryingpMEX01plasmid.

Antibiotic CM6 BL21STARpMEX01

Carbenicillin >400 >400 Dicloxaillin >400 >400 Cephalexin >400 >400 Ceftriaxone >400 >400 Cefotaxime >400 >400 Kanamycin >30 30 Rifampicin 10 10 Chloramphenicol >30 30 Polymyxin 2 2 Spectinomycin >100 100 Streptomycin >50 50

instance, CDS10 and 17, which are involved in transposi-tion,haveidentitiesof40.4%and81.5%,respectively,when comparedwiththeircorrespondingopenreading framesin pHK17a.CDS37,whichcodesforaputativeydaA,has62.2% identityandhasaprematurestopcodon.Interestingly,CDS88, whichencodestheTypeIVconjugativetransfersystem cou-plingproteinTraD,is85.5%identicaltothecorrespondingTraD inpHK17aandhasaprematurestopcodon.Thesechanges inthisopenreadingframemayhaveasignificantimpacton thefitnessofpMEX01sincethelast37aminoacidresidues are involvedinplasmidspecificityand transferefficiency,24

althoughthishastobeverifiedexperimentally.

InthepMEX01plasmid,blaCTX-M-14wastheonly␤-lactam

resistant determinant found. We compared the antibiotic resistanceprofileofCM6againsttheE.coliBL21STAR trans-formant carrying thepMEX01 plasmid.AlthoughbothCM6 andtheE.coliBL21STARtransformantexhibitedresistanceto awiderangeof␤-lactams,bothstrainsdidnotexhibit sim-ilar resistance to kanamycin, spectinomycin, streptomycin or chloramphenicol, indicating that these resistance traits mightbeencodedonadifferentextra-chromosomalelement presentintheCM6isolate.Indeed,pMEX01wasnottheonly plasmiddetectedwhentotalCM6DNAwasanalyzedbyPFGE (datanotshown).Theaminoglycosideandchloramphenicol resistancesdisplayedbyCM6,alongwiththeresistanceto dif-ferentgenerationsof␤-lactams,mayexplainwhytheafflicted cattlewerenotresponsivetostandardtreatment.

The CTX-M enzymes are a subfamily of the extended-spectrum ␤-lactamases (ESBLs) that confer resistance to ␤-lactam antibiotics.Morethan onehundred typesof CTX-M enzymes havebeen reported andare classified into five classes(CTX-M-1,CTX-M-2,CTX-M-8,CTX-M-9and CTX-M-25);theseenzymesareassociatedwithISEcp1-likeinsertion sequences(IS)formobilizationandarethemostbroadly dis-tributedenzymesamongawiderangeofbacteria,particularly theEnterobacteriaceaefamily.25,26_{TheCTX-M-14enzyme}

iden-tified in the E. coli strain CM6 pMEX01 plasmid has been sub-classifiedintotheCTX-M-9group.27_{AlthoughE.coliis}

rec-ognizedasthemajorsourceofESBLs,28_{itisacceptedthatthe}

CTX-Mgrouphasachromosomalorigin fromKluyvera geor-gianaandisthelikelysourceofCTX-M-9family,ofwhichthe pMEX01 CTX-M-14enzymeisamember.29 _{Members ofthe}

CTX-M-5familyhavebeenreportedinNorthAmerica,25_but

tothebestofourknowledge,thisisthefirstreportofa mem-beroftheCTX-M-9familyinthisregion.Thisfindingsuggests that multiple geneticprocesses are involved in the evolu-tion and persistenceofthe pHK01-likeplasmids indiverse countries.12_{The␤-lactamresistancegenepresentinpMEX01}

likelyemergedfromabacteriallineagewithmultidrug resis-tanceandmaintenanceofapHK01-likeplasmid.ThepMEX01 plasmidhasapotentialtransposoncomprisedofanIS903,the

blaCTX-M-14geneandanISEcp1element,whichhasimportant

epidemiologicalimplicationsforthedisseminationofthis ␤-lactamase.30 _{Infact,diversestudieshavedemonstratedthe}

widedisseminationofpHK01plasmidamongE.coliisolates obtainedfrompatientswithurinarytractinfectionsinHong Kong.31 _{PlasmidvariantscloselyrelatedtopHK01havealso}

been isolatedamongE.coliisolatesfrompigs(pHK17a)and humanfecalsamples(pHK09)inHongKong.32_The

K. pneumonia isolates from human-fecal samples(pKF3-70) inChina26 _{and among S.sonnei isolatesfrom humanfecal}

samples(pEG356)circulatinginsouthernVietnam.33_Previous

studieshaveshownthatShigatoxin-producingE.coli(STEC) aremedicallyimportantfoodbornepathogensresponsiblefor diverseoutbreaksofhemorrhagiccolitisandhemolytic ure-micsyndrome.Thesebacteriaoftencarryavarietyofplasmids thatencodevirulencefactorsandantibiotic-resistancegenes, suchasthepO26-Lplasmid,acloselyrelatedvariantofpHK01 thathasatetracyclineresistanceoperon;thisplasmidwas iso-latedfromE.coli026:H11strainH30,whichwasisolatedfrom achildpresentingdiarrheicsymptomsinCanada.34

Consid-ering thesestudies and the fact that the pMEX01 plasmid has a similar structure as several other plasmids isolated fromdiversepartsoftheworld,wecaninferthatnotonly arecattleanimportantreservoir,butalsothatfarmworkers couldbeimportantforthedisseminationofE.colistrainswith multidrugresistanceand forthe persistence ofpHK01-like plasmids.

TheeconomicimportanceofcattleintheStateof Queré-taro,Mexico,aswellasthediversefederalregulationsfortheir transporthasforcedpublichealthauthoritiestocreatediverse strategiestoreducethetransferofthesemulti-drug-resistant bacteriainhumanandanimalpopulations,andunderstand theroleoftheseE.colistrainsanditsplasmidsin pathogen-esis.Therefore,adetailedmolecularcharacterizationofthis plasmidfromdiversefarmsandgeographicaloriginsinthe StateofQuerétaro,Mexicoshouldbeinvestigatedtoidentify theepidemiologicpatternofpMEX01plasmidsequencesand changesovertime.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors would like to thank Paula Bernardino, René PrezaandGuillermo Vázquezfortechnicalassistance.This study waspartiallyfinanced bytheUniversidadAutónoma deQuerétaro,México(PFCE2016,projectsFOFIUAQ2016and FOPER2017toCSG).

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2017.11.002.

r

e

f

e

r

e

n

c

e

s

1.MedeirosAA.Evolutionanddisseminationof

beta-lactamasesacceleratedbygenerationsofbeta-lactam

antibiotics.ClinInfectDis.1997;24(suppl1):S19–S45.

2.MulecJ,StarcicM,Zgur-BertokD.F-likeplasmidsequences

inentericbacteriaofdiverseorigin,withimplicationof

horizontaltransferandplasmidhostrange.CurrMicrobiol.

2002;44(4):231–235.

3.BradfordPA.Extended-spectrumbeta-lactamasesinthe21st

century:characterization,epidemiology,anddetectionof

thisimportantresistancethreat.ClinMicrobiolRev.

2001;14(4):933–951[tableofcontents].

4.BauernfeindA,GrimmH,SchweighartS.Anewplasmidic

cefotaximaseinaclinicalisolateofEscherichiacoli.Infection.

1990;18(5):294–298.

5.MolandES,BlackJA,HossainA,etal.Discoveryof

CTX-M-likeextended-spectrumbeta-lactamasesin

EscherichiacoliisolatesfromfiveUSStates.AntimicrobAgents Chemother.2003;47(7):2382–2383.

6.BonnetR,SampaioJL,ChanalC,etal.AnovelclassA

extended-spectrumbeta-lactamase(BES-1)inSerratia

marcescensisolatedinBrazil.AntimicrobAgentsChemother.

2000;44(11): 3061–3068.

7.ChanawongA,M’ZaliFH,HeritageJ,etal.Characterisationof

extended-spectrumbeta-lactamasesoftheSHVfamilyusing

acombinationofPCR-singlestrandconformational

polymorphism(PCR-SSCP)andPCR-restrictionfragment

lengthpolymorphism(PCR-RFLP).FEMSMicrobiolLett.

2000;184(1):85–89.

8.LivermoreDM,CantonR,GniadkowskiM,etal.CTX-M:

changingthefaceofESBLsinEurope.JAntimicrobChemother.

2007;59(2):165–174.

9.HoPL,ChowKH,LaiEL,etal.Extensivedisseminationof

CTX-M-producingEscherichiacoliwithmultidrugresistance

to‘criticallyimportant’antibioticsamongfoodanimalsin

HongKong,2008–10.JAntimicrobChemother.

2011;66(4):765–768.

10.DuanRS,SitTH,WongSS,etal.Escherichiacoliproducing

CTX-Mbeta-lactamasesinfoodanimalsinHongKong.

MicrobDrugResist.2006;12(2):145–148.

11.TianGB,WangHN,ZouLK,etal.DetectionofCTX-M-15

CTX-M-22,andSHV-2extended-spectrumbeta-lactamases

(ESBLs)inEscherichiacolifecal-sampleisolatesfrompigfarms

inChina.FoodbornePathogDis.2009;6(3):297–304.

12.TamangMD,NamHM,KimSR,etal.Prevalenceand

molecularcharacterizationofCTX-M

beta-lactamase-producingEscherichiacoliisolatedfrom

healthyswineandcattle.FoodbornePathogDis.

2013;10(1):13–20.

13.BarriosH,Garza-RamosU,Ochoa-SanchezLE,etal.A

plasmid-encodedclass1integroncontainsGES-type

extended-spectrumbeta-lactamasesinEnterobacteriaceae

clinicalisolatesinMexico.AntimicrobAgentsChemother.

2012;56(7):4032–4034.

14.LoskeAM,Campos-GuillenJ,FernandezF,etal.Enhanced

shockwave-assistedtransformationofEscherichiacoli.

UltrasoundMedBiol.2011;37(3):502–510.

15.WeisburgWG,BarnsSM,PelletierDA,etal.16Sribosomal

DNAamplificationforphylogeneticstudy.JBacteriol.

1991;173(2):697–703.

16.LartigueMF.Matrix-assistedlaserdesorptionionization

time-of-flightmassspectrometryforbacterialstrain

characterization.InfectGenetEvol.2013;13:230–235.

17.ClinicalandLaboratoryStandardsInstitute[CLSI].

PerformanceStandardsforAntimicrobialSusceptibilityTesting; Twenty-ThirdInformationalSupplement.CLSIDocument M100-S23.Wayne,PA:ClinicalandLaboratoryStandards

Institute;2013.

18.BankevichA,NurkS,AntipovD,etal.SPAdes:anewgenome

assemblyalgorithmanditsapplicationstosingle-cell

sequencing.JComputBiol.2012;19(5):455–477.

19.SalzbergSL,DelcherAL,KasifS,etal.Microbialgene

identificationusinginterpolatedMarkovmodels.Nucleic

20.DelcherAL,HarmonD,KasifS,etal.Improvedmicrobial

geneidentificationwithGLIMMER.NucleicAcidsRes.

1999;27(23):4636–4641.

21.WheelerDL,ChurchDM,FederhenS,etal.Database

resourcesoftheNationalCenterforBiotechnology.Nucleic

AcidsRes.2003;31(1):28–33.

22.deCastroE,SigristCJ,GattikerA,etal.ScanProsite:detection

ofPROSITEsignaturematchesandProRule-associated

functionalandstructuralresiduesinproteins.NucleicAcids

Res.2006;34(WebServerissue):W362–W365.

23.FinnRD,BatemanA,ClementsJ,etal.Pfam:theprotein

familiesdatabase.NucleicAcidsRes.2014;42(Database

issue):D222–D230.

24.SastreJI,CabezonE,delaCruzF.Thecarboxylterminusof

proteinTraDaddsspecificityandefficiencytoF-plasmid

conjugativetransfer.JBacteriol.1998;180(22):6039–6042.

25.CantonR,CoqueTM.TheCTX-Mbeta-lactamasepandemic.

CurrOpinMicrobiol.2006;9(5):466–475.

26.YiH,XiY,LiuJ,etal.SequenceanalysisofpKF3-70in

Klebsiellapneumoniae:probableoriginfromR100-likeplasmid ofEscherichiacoli.PLoSOne.2010;5(1):e8601.

27.BonnetR.Growinggroupofextended-spectrum

beta-lactamases:theCTX-Menzymes.AntimicrobAgents

Chemother.2004;48(1):1–14.

28.ValverdeA,CoqueTM,Sánchez-MorenoMP,etal.Dramatic

increaseinprevalenceoffecalcarriageof

extended-spectrum␤-lactamase-producing

EnterobacteriaceaeduringnonoutbreaksituationsinSpain.Jf ClinMicrobiol.2004;42(10):4769–4775.

29.OlsonAB,SilvermanM,BoydDA,etal.Identificationofa

progenitoroftheCTX-M-9groupofextended-spectrum

beta-lactamasesfromKluyverageorgianaisolatedinGuyana.

AntimicrobAgentsChemother.2005;49(5):2112–2115.

30.PoirelL,LartigueMF,DecousserJW,etal.ISEcp1B-mediated

transpositionofblaCTX-MinEscherichiacoli.Antimicrob

AgentsChemother.2005;49(1):447–450.

31.HoPL,LoWU,WongRC,etal.CompletesequencingoftheFII

plasmidpHK01,encodingCTX-M-14,andmolecularanalysis

ofitsvariantsamongEscherichiacolifromHongKong.J

AntimicrobChemother.2011;66(4):752–756.

32.HoPL,LoWU,YeungMK,etal.DisseminationofpHK01-like

incompatibilitygroupIncFIIplasmidsencodingCTX-M-14in

Escherichiacolifromhumanandanimalsources.VetMicrobiol.

2012;158(1–2):172–179.

33.NguyenNT,HaV,TranNV,etal.Thesuddendominanceof

blaCTX-MharbouringplasmidsinShigellaspp.circulatingin

SouthernVietnam.PLoSNeglTropDis.2010;4(6):e702.

34.FratamicoPM,YanX,CaprioliA,etal.ThecompleteDNA

sequenceandanalysisofthevirulenceplasmidandoffive

additionalplasmidscarriedbyShigatoxin-producing

EscherichiacoliO26:H11strainH30.IntJMedMicrobiol.

2011;301(3): 192–203.