Molecular characterization and epidemiology of cefoxitin resistance among Enterobacteriaceae lacking inducible chromosomal ampC genes from hospitalized and non-hospitalized patients in Algeria: description of new sequence type in Klebsiella pneumoniae iso

braz j infect dis.2015;19(2):187–195

ww 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

Original

article

Molecular

characterization

and

epidemiology

of

cefoxitin

resistance

among

Enterobacteriaceae

lacking

inducible

chromosomal

ampC

genes

from

hospitalized

and

non-hospitalized

patients

in

Algeria:

description

of

new

sequence

type

in

Klebsiella

pneumoniae

isolates

Alima

Gharout-Sait

_,

_Abdelaziz

_Touati

_,

_Thomas

_Guillard

_,

_Lucien

_Brasme

_,

Christophe

de

Champs

a_{LaboratoiredeMicrobiologieAppliquée,DépartementdeMicrobiologie,FSNV,UniversitédeBejaia,06000,Algeria} b_{Laboratoired’EcologieMicrobienne,DépartementdeMicrobiologie,FSNV,UniversitédeBejaia,06000,Algeria} c_{LaboratoiredeBactériologie–Virologie-HygièneHospitalière,CHUReims,HôpitalRobertDEBRE,Reims,France} d_{UniversitédeReims-Champagne-Ardenne,Reims,France}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11August2014 Accepted17December2014 Availableonline28January2015

Keywords: Enterobacteriaceae AmpC␤-lactamases Genotyping Algeria

a

b

s

t

r

a

c

t

Inthisstudy,922consecutivenon-duplicateclinicalisolatesofEnterobacteriaceaeobtained from hospitalized and non-hospitalized patients at Bejaia, Algeria were analyzed for AmpC-type ␤-lactamases production. The ampC genes and their genetic environment were characterized using polymerase chain reaction (PCR) and sequencing. Plasmid incompatibilitygroupsweredeterminedbyusingPCR-basedreplicontyping.Phylogenetic grouping andmultilocussequencetyping weredetermined formolecular typingofthe plasmid-mediatedAmpC(pAmpC)isolates.

Of the isolates, 15 (1.6%) were identified as AmpC producers including 14 CMY-4-producingisolatesandone DHA-1-producingKlebsiellapneumoniae.AllAmpC-producing isolates co-expressed the broad-spectrum TEM-1 ␤-lactamase and three of them co-producedCTX-Mand/orSHV-12ESBL.Phylogeneticgroupingandvirulencegenotypingof theE.coliisolatesrevealedthatmostofthembelongedtogroups DandB1.Multilocus sequencetypinganalysisofK.pneumoniaeisolatesidentifiedfourdifferentsequencetypes (STs) with two new sequences:ST1617 and ST1618. Plasmid replicon typing indicates that blaCMY-4 genewas located onbroad host range A/C plasmid,while LVPKreplicon

wasassociatedwithblaDHA-1.AllisolatescarryingblaCMY-4displayedthetransposon-like

structuresISEcp1/ISEcp1-blaCMY-blc-sugE.

OurstudyshowedthatCMY-4wasthemainpAmpCintheEnterobacteriaceaeisolatesin Algeria.

©2015ElsevierEditoraLtda.Allrightsreserved.

∗ _{Correspondingauthor.}

E-mailaddress:ziz1999@yahoo.fr(A.Touati). http://dx.doi.org/10.1016/j.bjid.2014.12.001

188

Introduction

Infection with resistant organisms is a major public healthissue.Enterobacteriaceaeareimportantcausesofboth community-acquired and healthcare-associated infections in adults and children, and production of ␤-lactamases is of greater concern.1 _{Plasmid-mediated AmpC (pAmpC)}

␤-lactamaseshaveemergedandarebeingreportedworldwide with varying prevalence rates.2 _{They have been mainly}

detected in Escherichia coli, Klebsiella spp., Salmonella spp., and Proteus mirabilis.3 _{These enzymes confer resistance}

to penicillins, first and third generation cephalosporins, cephamycins,andmonobactamssuchasaztreonam.Theyare poorlyinhibited bythecommercially available␤-lactamase inhibitors such as clavulanic acid, but are inhibited by cloxacillin and phenylboronic acid. Treatment options are severelylimited because pAmpC are oftenassociated with othermultipleresistancegenes,suchasthoseofresistance to quinolones as well as other ␤-lactamase genes.3,4 _The

acquiredampCgeneshaveemergedfollowingmobilizations mediatedbysuchelementsasIS26,ISEcp1,orISCR1.3_Thus,

ISEcp1hasplayedanimportantrole inmobilizing bla CMY-2-likegenes,sinceitisoftenfoundattheir5 flanks.5,6_Ithas

beenidentifiedinplasmidsoftheIncA/CandIncI1groups.5–7

ISCR1 elements have been found adjacent to a number of ampCgenes,includingblaDHA-1,aswellasplasmid-mediated quinoloneresistancedeterminantqnr.8_{Generally,thebla}

DHA-1 gene hasbeen mainly associatedwithInc FIIand Inc L/M plasmids.9

In Algeria, onlyfew reports on plasmid-encoded AmpC (CMY-2andDHA-1)inEnterobacteriaceaestrainsrecoveredfrom hospitalsettingswerepublished.10–12

Theaimofthis study wastoinvestigatethe prevalence and molecular epidemiology ofcefoxitinresistance among Enterobacteriaceae isolates recovered from hospitalized and non-hospitalizedpatientsinBejaialocality(Algeria).The asso-ciationofpAmpCwithextended-spectrum␤-lactamase(ESBL) andplasmid-mediatedquinoloneresistancedeterminantwas alsostudied.

Materials

and

methods

Atotalof922non-duplicateisolates(oneperpatient)of Entero-bacteriaceaewerecollectedfromMarch2005toApril2010from theMicrobiologyLaboratoriesoffivehospitalsandfourprivate laboratoriesinBejaia(Algeria).Theisolateswererecovered fromvariouspathologicalspecimensandwereidentifiedby theAPI20Esystem(bioMérieux,Marcyl’Etoile,France)as fol-lows:E.coli(n=551);Klebsiellapneumoniae(n=221);P.mirabilis (n=125)andSalmonellasp.(n=25).

E.coliJ53AzR_{wasusedasrecipientstrainsforconjugation} experiments. E. coli DH10B (Invitrogen) was used in trans-formationexperimentsandE. coliATCC25922was usedas aqualitycontrolstrainforantimicrobialsusceptibility test-ing.

Antimicrobialsusceptibilitytesting

Antibioticsusceptibility wasdeterminedonMuellerHinton agarbystandarddiskdiffusionprocedureasdescribedbythe EuropeanCommitteeonAntimicrobialSusceptibilityTesting (2014),13_{forthefollowingantibiotics:aztreonam,ticarcillin,}

piperacillin, amoxicillin–clavulanate, ticarcillin–clavulanate, cefoxitin, cefepime, piperacillin–tazobactam, cefuroxime, cefotaxime, ceftazidime, imipenem, tobramycin, amikacin, gentamicin, sulfonamide, trimethoprim, nalidixic acid, ciprofloxacin, norfloxacin, tetracycline, and chlorampheni-col (BioRad, Marnes LaCoquette, France). For tetracycline, the Antibiogram Committee of the French Society for Microbiology recommendations breakpoints were used (http://www.sfm-microbiologie.org).

Isolates showinga zone ofinhibition diameter ≤20mm withcefoxitinwereselectedforscreeningforpampCgenes. ESBLproductionwasdetectedbyadouble-disksynergytest (DDST) on Mueller Hinton supplemented with cloxacillin (200mg/L).14 _{Inducibility of the ␤-lactamase was}

deter-mined by the double disk test. The cephalosporins used werecefotaxime,ceftazidime,andcefepime.Clavulanicacid (10␮g) and cefoxitin(30␮g) were used asinducing agents. The plates were examined after overnight incubation at 37◦C.15

Minimuminhibitoryconcentrations(MICs)ofamoxicillin, amoxicillin/clavulanate,piperacillin/tazobactam,cefotaxime, ceftazidime,cefoxitin,imipenem,aztreonam,andcefepime were determined by Etest (AB bioMérieux, Marcy l’Etoile, France).

Molecularcharacterizationofresistancedeterminants Total DNA was extracted byusing a QIAmp DNA MiniKit (QIAGEN)accordingtotheinstructionsofthemanufacturer. A multiplex PCR covering the six families of ampC genes (CMY-2/BIL/LAT,CMY-1/MOX,DHA,FOX,ACC,ACT/MIR)was performed as previously described.16 _{PCR-positive isolates}

were further tested using individual pairs of primers and then sequenced.pAmpC-producingisolatespositiveforthe DDSTwerescreenedforblaCTX-M,blaTEMandblaSHVbyPCRas describedpreviously.17

ScreeningofqnrA,qnrB,qnrS,qnrC, qnrDandqepAgenes was carried out with a multiplex real-time PCR assay using SYBR Green I and Roche LightCycler1 as described previously.18_{Pyrosequencingmethodwasusedforthe}

detec-tionofaac(6)-Ib-crandaac(6)-Ibgenes.19

All PCR products were sequenced and the sequencing resultswerecomparedtoreportedsequencesavailablein Gen-Bank.

Transferofresistance

ConjugationwasperformedonMuellerHintonagar supple-mentedwithsodiumazide(100mg/L)andcefotaxime(1mg/L). Transconjugantsgrowing onthe selectionplates were sub-jectedtoantimicrobialssusceptibility,DDSTandPCRanalysis toconfirmthepresenceoftheAmpCphenotype.

brazj infect dis.2015;19(2):187–195

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Moleculartyping

PossiblegenomicrelatednessofstrainswasanalyzedbyRAPD using genomic DNA as previously described.20 _Multilocus

sequence typing (MLST) was performed on the K. pneumo-niaeisolatesusingsevenconservedhousekeepinggenes(gapA, infB,mdh,pgi,phoE,rpoBandtonB).21_{A detailedprotocol of}

theMLSTprocedure,includingallelictypeandSTassignment methods, is availablein MLST databases from the Pasteur Institute,Paris,France,atthewebsitehttp://www.pasteur.fr/ recherche/genopole/PF8/mlst/Kpneumoniae.html.

PhylogeneticgroupsandvirulencegenotypingofE.coli PCRswereperformedtodeterminethephylogeneticgroups (A,B1,B2,C,D,E,F,andcladeI)oftheE.coliisolates,using thenewlyrevisedClermontmethod.22_{Allisolatesbelonging}

togroupB2wereanalyzedbytwomultiplexPCRasdescribed previously.23 _{Thepresenceofeightvirulence factors found}

in ExPEC was investigated by PCR. These factors included sfa/foc(Sand F1Cfimbriae), papGalleles (Gadhesinclasses ofPfimbriae), papC(C adhesinclasses ofPfimbriae), hlyA (alpha-haemolysin A),cnf (cytotoxicnecrotizating factor 1), fyuA(genesofyersiniabactin),iutA(aerobactinreceptor),and ibeA(invasionproteinIbeA).

Plasmidreplicontyping

Plasmidsincompatibility(Inc)groupsweredeterminedusing PCR-basedreplicontyping(PBRT).24_{FourmultiplexPCRwere}

usedforthedetectionofA/C,T,FIIAs,W,N,FIB,L/M,I1-I␥,X, HI2,FIA,andYreplicons.RepliconsP,R,U,F,FIC,HI1,B/Oand KweredetectedbysimplexPCR.24,25 _{RepliconsFII1K,FII2K,}

NewXXXalsonamedZK,LVPK,andAmetweredetectedusing PCRmethoddescribedbyD.DecréandG.Arlet.

GeneticorganizationofblaampCgenes

For the analysis of genetic arrangement of the resistance genes,overlappingPCRamplificationofinternalregionsofthe transposon-likeelementthatcarriedblaCMY-4wasperformed basedonknownsequences.

Genetic structures surrounding the blaDHA-1 gene were studied by PCR mapping, cloning and sequencing method using a large variety of primers based on the previously reportedstructures.8

The nucleotide sequence and the deduced protein sequence were analyzed using the Basic Local Alignment Search Tool(BLAST)throughthe Internet(http://www.ncbi. nlm.nih.gov/BLAST/). Multiple sequence alignment of deducedpeptidesequenceswascarriedoutwiththeVector NTIprogram(Invitrogen).

Results

Bacterialisolatesandantibioticsusceptibility

Amongthe922Enterobacteriaceaeisolates,15isolatesshowed decreasedsusceptibility tocefoxitin: nine isolatesofE. coli

(9/551),fiveisolates(5/221)ofK.pneumoniaeandoneisolate (1/125)ofP.mirabilis.Tenisolateswererecoveredfromurine. SixE.coliisolatesandoneK.pneumoniaeisolatewerefrom com-munitypatients(7/712),andtheremainingeightisolateswere collectedfromhospitalizedpatients(8/210).

Allisolatesexhibitedresistancetoticarcillin,piperacillin, ticarcillin–clavulanic acid, amoxicillin–clavulanic acid, cefuroxime, cefotaxime, ceftazidime, aztreonam, and cefoxitin. Isolates exhibited intermediate resistance to piperacillin–tazobactam(60%)andcefepime(40%).Resistance of the isolates to non-␤-lactam antibiotics was high for sulfonamide (80%), tobramycin (71.4%),gentamicin (71.4%), tetracycline(71.4%), chloramphenicol (64.3%)and trimetho-prim(57.1%),andlowfornalidixicacid(28.6%)andamikacin (6.6%). The isolates remain susceptible to imipenem and fluoroquinolones.TheMICsrangesarelistedinTable1.

TheESBLphenotypic screeningbydoublediskdiffusion synergytestshowedthatoneisolateofK.pneumoniaeandtwo isolatesofE.coliwereESBLproducers(Table1).

Inducibility of ␤-lactamases was recognized by the disk antagonism test, which demonstrated blunting of the cephalosporindisksadjacenttothecefoxitinandclavulanic aciddisksinonlyoneisolateofK.pneumoniae413.This phe-notype suggestedthe presenceofan inducible AmpC-type ␤-lactamase.

Genotypicanalysisofantibioticresistancegenes

BymultiplexPCR,weobtainedampliconsin15isolates:nine E.coliisolates,fiveK.pneumoniaeisolates,andoneP.mirabilis isolate(Table1).

PCR and sequencing analysis revealed the presence of blaCMY-4inallisolatesexceptoneisolateofK.pneumonia,which producedblaDHA-1.

In addition, two E. coli (CMY-4) co-produced CTX-M-15 ESBLsandoneisolateofK.pneumoniae(DHA-1)co-produced CTX-M-3andSHV-12ESBL.AllisolatescarriedblaTEM-1.

PCRamplificationofPMQRyieldedamplificationinoneK. pneumoniaeisolateonly.ThisstrainexpressedboththeqnrB4, aac(6)-Ib, blaDHA-1, blaCTX-M-3, blaSHV-12 and blaTEM-1 genes (Table1).

NoampliconswereobtainedforqnrA,qnrS,qnrD,qnrCand qepAinallisolates.

Conjugationandreplicontyping

Bymatingassay,theampCgenesweretransferredfromthree ofthefiveK.pneumoniae,fiveofthenineE.coliisolatesandfrom theP.mirabilisisolate.Susceptibilityresultsofthe transconju-gantsareshowninTable1.

PBRToftheplasmidIncgroupsshowedthattheplasmids carryingblaCMY-4belongedtotheIncA/Cgroupandthe plas-midcarryingblaDHA-1belongedtothegroupIncLVPK(Table1). Moleculartyping

RAPD-typingrevealedthepresenceofdiversebacterial pop-ulation and no predominant clone was identified in our collection.

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Table1–MicrobiologicalfeaturesofpAmpCproducingEnterobacteriaceae.

Isolatesand transconjugants Dateof isolation SpecimenHospital/ private labora-tory Ward Plasmid-mediated blaAmpC Other ␤-lactamases genes PMQRs genes MICs(mg/L) RAPD clone MLST(K. pneumoniae) Replicon typing

AMX AMC TZP CTX CAZ ATM FEP IMP FOX

K.pneumoniae47 04/04/2007 Urine AWH Surgery CMY-4 TEM-1 – >256 48 >256 >32 >256 32 <04 0.38 64 K.A. 17 IncA/C, LVPK TC47 CMY-4 – >256 16 128 >32 128 32 <04 0.19 32 IncA/C

K.pneumoniae123 04/05/2005 Urine AWH Pediatrics CMY-4 TEM-1 – >256 32 >256 >32 >256 32 >64 0.38 64 K.B. 1617 IncA/C, FIB,F,P TC123 CMY-4 – >256 16 16 12 12 <04 <04 0.25 32 IncA/C

K.pneumoniae613 05/04/2010 Feces AZH Medicine CMY-4 – >256 16 08 >32 192 32 <04 0.25 64 K.D. 1618 ND

K.pneumoniae615 03/05/2010 Feces AZH Medicine CMY-4 – >256 16 08 >32 192 32 <04 0.25 64 K.D. 1618 ND

K.pneumoniae413 21/04/2008 Feces AZH Surgery DHA-1 TEM-1; CTX-M-3; SHV-12 QnrB4 >256 16 02 >32 48 32 16 0.19 16 K.C. 834 IncL/M, LVPK,HI2 TC413 DHA-1 TEM-1 QnrB4, acc(6)-Ib >256 02 0.38 0.19 03 <04 <04 0.032 16 IncLVPK

E.coli234 15/04/2007 Urine AZH Surgery CMY-4 TEM-1; CTX-M-15

– >256 32 >256 >32 >256 32 >64 0.5 64 E.A. IncA/C,F, P,FII2K TC234 CMY-4 – >256 16 12 >32 96 32 <04 0.25 64 IncA/C

E.coli412 22/04/2008 Feces AZH Pediatrics CMY-4 TEM-1 – >256 32 >256 >32 >256 32 <04 0.5 32 E.B. IncA/C, FIA,A/C, FIB,F,B/O, U TC412 CMY-4 – >256 16 >256 >32 48 32 <04 0.25 32 IncA/C,

E.coli535 14/04/2009 Urine LPL CommunityCMY-4 TEM-1 – >256 16 16 >32 >256 32 <04 0.25 64 E.C. IncA/C, FIB,HI1

E.coli538 14/02/2009 Urine LPL CommunityCMY-4 TEM-1 – >256 16 08 >32 128 32 <04 0.19 64 E.D. IncA/C, FIB,FIA, FII1K,U TC538 LPL CMY-4 – >256 16 04 >32 64 32 <04 0.19 32 IncA/C

E.coli539 05/04/2009 Urine LPL CommunityCMY-4 TEM-1 – >256 16 16 >32 >256 32 <04 0.25 64 E.C. IncA/C, FIB,HI1

E.coli545 24/02/2009 Urine LPL CommunityCMY-4 TEM-1 – >256 16 08 >32 128 32 <04 0.19 64 E.D. IncA/C, FIB,FIA, FII1K,U

E.coli560 01/12/2009 Urine LPL CommunityCMY-4 TEM-1 – >256 16 08 >32 128 32 <04 0.19 64 E.D. IncA/C, FIB,FIA, FII1K,U

b r a z j i n f e c t d i s . 2 0 1 5; 1 9(2) :187–195

191

E.coli611 28/05/2010 Feces AZH Surgery CMY-4 TEM-1, CTX-M-15

– >256 16 04 >32 64 32 16 0.25 64 E.F. IncFIA,I1, F,U,FII1K TC611 CMY-4 – >256 16 02 >32 08 32 <04 0.25 32 IncA/C

P.mirabilis128 07/04/2005 Urine AZH Surgery CMY-4 TEM-1 – >256 48 12 >32 48 <04 <04 0.25 32 – IncA/C

TC128 CMY-4 – >256 16 0.75 >32 24 <04 <04 0.25 32 IncA/C LPL,Lalaouiprivatelaboratory;DPL,Djamaprivatelaboratory;AZH,Amizourhospital;AWH,Amriwhospital;TZP,piepracillin–tazobactam;AMX,amoxicillin;AMC,amoxicillin–clavulanicacid;FOX, cefoxitin;ATM,aztreonam;CTX,cefotaxime;CAZ,ceftazidime;FEP,cefepime;IMP,imipenem;ND,notdetermined.

192

MLSTanalysisofthefiveAmpC-producingK.pneumoniae identifiedfourdifferentSTs,includingST17,ST834andtwo newsequencetypes:ST1617(Kp123)andST1618(Kp613and 615)(Table1).InST1618,wedescribedanewallele’smdhand rpoBdesignatedrespectively145and108.Thetypingresults generatedbyRAPDanalysisamongtheisolateswere compat-iblewiththoseobtainedbyMLST.

E.coliphylogeneticgroupsandvirulencefactors

OfthenineE.coliisolates,threebelongedtogroupD,threeto groupB1(recoveredfromurine),twotogroupB2,andthelast onetogroupF(Table2).

Fiveisolatesharboredgenesencodingsiderophores(fyuA, iutA).

TheE.coli412isolatewasassignedtotheB2sub-groupVII andSTc14.Thisstrainwasisolatedfromthefecesofa hos-pitalizedpatient.ThisisolatecontainedablaTEM-1geneand, ablaCMY-4gene,whichwastransferredwithanIncA/C plas-mid.Thefollowingvirulencegenes(papC,papG II,sfa, hlyA, cnf1,fyuAandiutA)weredetectedinthisisolate(Table2).

Byusingallele-specificPCRmethodfordetectingthemain E.coliB2STc,E.coli611isolatewasunassigned;itdidnotgive anyPCRproductsexceptfortheinternalcontrol.

CharacterizationofthegeneticcontextsofblaAmpCgenes

AnalysisofthegeneticstructureoftheblaCMY-4geneinour collectionshowed that it waslocatedon atransposon-like DNAelementconsistingofaspecificISEcp1/ISEcp1-bla CMY-4-blc-sugEstructure.Thisstructurewassimilartothatfoundin plasmidpCC416(GenBankAJ875405).

Aregiontypicalofacomplexsul1-typeintegron,fromthe intgenetoCR1wasamplifiedusingPCRmappingandthen sequenced. By cloning the region encompassingampC and ampR,arecombinantplasmid(p413C)withaninsertthat con-ferredinducibleresistancetoceftazidimewasselected.The insertwasfoundtocontainblaDHA-1andtheregulatorygene ampR,whichwasdownstreamofblaDHA-1.Thisinsertshared alsopartofpRBDHA’sbackbonecarryingacomplexintegron (GenBank AJ971343).PCR andDNA sequencingresults con-firmedthattheplasmidencodedatleastthree␤-lactamase

genes:blaTEM-1,blaSHV-12,andblaDHA-1,andaplasmid medi-atedresistancetoquinolone(QnrB4).

Discussion

pAmpChavebeenfoundworldwidebutarelesscommonthan ESBLs.3 _{Theyare emergingworldwideinvarious speciesof}

Enterobacteriaceaeasamechanism ofacquiredresistanceto cefoxitin. In ourstudy 1.6%(n=15) ofthe screened Entero-bacteriaceae isolates were cefoxitin-resistant and produced plasmid-mediatedAmpC␤-lactamases.PrevalenceofpAmpC in Algeria is not known, due to the limited number of epidemiological surveys.In Algeria,Iabaden et al.reported a prevalence of plasmid mediated AmpC ␤-lactamases of 2.18%.11 _{Mata etal. reportedasignificant increasein}

over-all prevalenceofEnterobacteriaceae carrying acquiredAmpC in a Spanish hospital which was 0.43%, rising from 0.06% (1999)to1.3%(2007).26_{Aprevalenceof12.5%wasreportedby}

Mohamudhaetal.inIndia.27

OurstudydemonstratedthatpAmpC-producing Enterobac-teriaceae mightbethecauseofnosocomialandcommunity infections in Algeria. Of note, we found that 40% of the caseswererecoveredfromnon-hospitalizedpatients. Isola-tionofpAmpC-producingEnterobacteriaceaefromcommunity was reported by many authors.28,29 _{Nursing homes and}

community-based sourcesofpAmpC-producerscanpose a serious riskof transmission tohospitalized patients when infectedorcolonizedpatientsareadmitted.Gudeetal.have foundthisresistancemechanismonisolatesfrom commu-nitypatientsinahighrate,underscoringtheneedforclose surveillanceoftheseisolates.30 _{Severalstudiesreportedthe}

isolationofpAmpC-producingEnterobacteriaceaeisolatesfrom foodproducts,suchasretailchickenmeat,retailmeat,and cheese.31–33 _{Thus,foodchainmightbearelevantvehiclefor}

transmissionoftheseenzymesinthecommunity.Theyhave also been detected in drinking water and river beaches.34

Thesesourcescouldcontributetothespreadofglobal pAmpC-producers inaddition toapossibletransmission ofmobile geneticelementscarryingresistancegenesamongstrains.30

InAlgeria,CMY-2andDHA-1werepreviouslyreportedby Messaietal.(2006)10_{,Iabadeneetal.(2009)}11_{andNedjaietal.}

(2012).12_{ThisisthefirstisolationofCMY-4inclinicalisolates}

Table2–Distributionandcombinationpatternsofvirulencegenesandphylogeneticgroupsdetectedin pAmpC-producingE.coli.

Strain Adhesin Toxin Ironsystem Invasin Phylogeneticgroup

papC papGII sfa hlyA cnf1 fyuA iutA ibeA

234 − − − − − − − − F 412 + + + + + + + − B2 535 − − − − − + + − D 538 − − − − − − − − B1 539 − − − − − + + − D 545 − − − − − − − − B1 560 − − − − − − − − B1 606 − − − − − + + − D 611 − − − − − + + − B2

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(nosocomialandcommunityinfections)inAlgeria.Thus,the firststrain (K.pneumoniae 123)was isolatedin 2005from a patienthospitalizedatBejaiahospital(Algeria).The predomi-nanceofCMY-4wasconsistentwithworldwideobservations. DHA-1hasbeenmostlyreportedinAsia.5,35

Inour study,E. coliisolateswere mainly groupsB2 and D strains which are commonly extra-intestinal pathogenic strains, while phylogenetic groups A and B1 strains, usu-allycommensal,werelessfrequent.36_{CMY-2productionwas}

reportedinphylogeneticgroupDE.coliinhumansandstray dogs.5,37

InthestudyofMnifetal.,thenon-ST131-groupB2isolates, whichwereassociatedtoCTX-M-15ESBLs,hadahigher fre-quencyofseveralgenesencodingkeyvirulencefactorssuch asadhesinshra,sfa/foc,papCandpapGII,andthetoxinshylA andcnf1thanhadtheST131isolates.38_{Inourstudy,asingle}

isolateharboredseveralvirulencegenesiutA,papCandsfa/foc andbelongedtophylogeneticgroupB2.

Ourresultsshowed that AmpC-producingK.pneumoniae isolatesbelongedtodifferentsequencetypes.ST17hasbeen previously found in Cadiz, associated with CTX-M-15, in Freiburg and in Seoul,in Barcelona, associated with DHA-1.21,39–41_{ST17belongstotheST17complex,whichcontains}

foursingle-locusvariantsandsixdouble-locusvariants.41K. pneumoniaeST834strainswerepreviouslyinvolvedinblaKPC disseminationinNewJersey.42_{Besidesthelownumberof}

iso-lates,wehavedetectedtwonewssequencestypes:ST1617and ST1618.

Inthisstudy,allisolatesproducingblaCMY-4andblaDHA-1 co-expressed the broad-spectrum TEM-1 ␤-lactamase and three of them co-produced CTX-Mand/or SHV ESBL. This enzymecombinationcomplicatestheirdetectionand treat-ment.blaCMY-4 gene was locatedon broad host range A/C conjugativeplasmidwhichwasamongthemostcommonly reported worldwide.In the last decades,Inc A/C plasmids have been associated with the spread of the AmpC beta lactamase CMY-2, in strains isolated from human, beef, chicken,turkey,andpork,revealingthatthiscommonplasmid backboneisbroadlydisseminatedamongresistantzoonotic pathogens.9,43

In our study, the genetic organization of blaCMY-4 and its variants was highly conserved. All the isolates carried the transposon-like element ISEcp1 (ISEcp1/ISEcp1-bla CMY-blc-sugE),asdocumentedpreviously.44

TheblaDHA-1genewaspreviouslyfoundondifferent plas-mids of Inc groups A/C, FIA, FII, L/M, N, R and HI2 or of unknownIncgroups.9,11,26,41,45_{Nevertheless,itisworthnoting}

that blaDHA-1 gene was located on LVPK conjugative plas-mid.LinkageofblaDHA-1andqnrB4genesofsimilarstructures hasbeendescribed inisolatesofK. pneumoniae.8 _The

asso-ciationamongblaDHA-1,qnrB4,andaac(6)-Ib-crwasreported before.46_{TheK.pneumoniae413straininourstudyharboreda}

combinationof␤-lactamasegenes(blaCTX-3,blaDHA-1,blaSHV-12 andblaTEM-1),PMQRdeterminants(qnrB4gene)and amino-glycoside acetyltransferasegene (aac(6)-Ib).Despite several investigations, we could not determine the origin of this multiresistant strain. To our knowledge, this is the first descriptionofthisassociationofgenesincludingblaCTX-M-3in thesamestrain.Identificationofthesequencessurrounding theblaDHA-1genefoundanampRgeneincludedinacomplex

sul-1-typeintegronthatwaslikelysimilartothosepreviously reported.8

Useofantibioticsinbothhumansandanimals,theglobal mobility of populations, and food products perpetuate the spreadofmultiresistantbacterialclonesandresistancegenes. Early identification ofthese organisms isnecessary as the appropriate treatment might reduce the spread of these resistantstrainsandconsequentlymortalityofhospitalized patientscanbereduced.Thisemphasizestheneedforsuch enzymes detection for preventingthis emerging resistance intohospitalsandforcontrollingitsspreadwithinthe com-munity.Thatwillavoidtherapeuticfailuresandnosocomial outbreaks.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WearegratefultoJ.Madouxforhercontributiontothiswork. WethanktheteamofcuratorsoftheInstitutPasteurMLST databases for curatingthe data and making them publicly availableathttp://www.pasteur.fr/mlst.

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