Dissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital

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Dissemination

of

multiresistant

Enterobacter

cloacae

isolates

producing

OXA-48

and

CTX-M-15

in

a

Spanish

hospital

Javier

Fernández

a,b

_,

_Ignacio

_Montero

a

_,

_Óscar

_Martínez

b

_,

_Ana

_Fleites

b

_,

_Laurent

_Poirel

c

_,

Patrice

Nordmann

c,d

_,

_M.

_Rosario

_Rodicio

a,∗

a_Department_of_Functional_Biology,_Section_of_{Microbiology,}_University_of_Oviedo,_Oviedo,_Spain b_Service_of_{Microbiology,}_Hospital_{Universitario}_Central_de_Asturias,_Oviedo,_Spain

c_Medical_and_Molecular_{Microbiology,}_‘Emerging_Antibiotic_{Resistance’}_Unit,_Department_of_Medicine,_Faculty_of_Science,_University_of_Fribourg,_Fribourg,

Switzerland

d_HFR_–_Hôpital_Cantonal,_Fribourg,_Switzerland

Twenty-onemultiresistantEnterobactercloacaeisolatesproducingOXA-48(n=10),CTX-M-15(n=7)or both(n=4)␤-lactamasesweredetectedinaSpanishhospitalduringa1-yearperiod(June2013toJune 2014).Theisolateswerealsoresistanttonon-␤-lactamantimicrobials,furthercomplicatingthe ther-apeuticoptions.Genotypingoftheisolatesidentiﬁedtwomajorclones(ST74andST66)thatcaused prolongedoutbreaksindifferentbuildingsofthehospitalaswellassomesporadicisolates(ST78,ST45 andST295).Isolatesbelongingtoclone1(n=7)werecarbapenem-resistantandcarriedtheblaOXA-48gene

onaconjugativeIncL/Mplasmidofca.65kb.Clone2isolates(n=11)wereresistanttocefepimeand har-bouredtheblaCTX-M-15geneonanca.150-kb,non-conjugativeplasmidoftheIncFgroup,co-harbouring

theqnrBandaac(6_)-Ib-cr_genes_encoding_quinolone_resistance._Four_clone₂_isolates_were_also_resistant

tocarbapenemsowingtotheco-productionofOXA-48.Mostoftheisolateswererecoveredfrom criti-callyillpatientsandwereadmittedtointensivecareunits;asinglepatientwastransferredfromanother Spanishhospital.IntrahospitalandinterhospitaldisseminationofmultiresistantE.cloacaeisolatesisof majorclinicalconcernasitcouldleadtoendemicnosocomialsituations.

1. Introduction

Enterobacter cloacae is an opportunistic pathogenfrequently involvedinnosocomialinfections[1].Thisenterobacterialspecies isintrinsicallyresistanttoaminopenicillins,amoxicillin/clavulanic acid(AMC) andcephalosporinsof earlygenerations owingtoa chromosomallyencodedAmpC␤-lactamase.Isolates overproduc-ing AmpCare alsoresistant tobroad-spectrum cephalosporins. Furthermore, E. cloacae has the ability to acquire additional resistance mechanisms, including plasmid-encoded extended-spectrum␤-lactamases(ESBLs)andcarbapenemases[1]. Pheno-typicdetectionofESBL productionmaybedifﬁcultsince itcan bemaskedbyoverexpressionofAmpC.SomeE.cloacaeisolates mayalsoexhibitpermeabilitydefectsduetotheabsenceofporins or porinmutations, furtherincreasing theminimum inhibitory

∗ Correspondingauthorat:DepartamentodeBiologíaFuncional,Áreade Microbi-ología,UniversidaddeOviedo,FacultaddeMedicina,JuliánClavería6,33006Oviedo, Spain.Tel.:+34985103562.

E-mailaddress:[email protected](M.R.Rodicio).

concentrations(MICs)of␤-lactamsandcarbapenems,particularly ertapenem[1].

In members of theEnterobacteriaceae family, CTX-M-15 and OXA-48 are among the most common enzymes involved in resistanceto broad-spectrum cephalosporinsand carbapenems, respectively [2,3]. CTX-M-15 is a class A ESBL encoded by the blaCTX-M-15 gene,whichhasbeenidentifiedinplasmidsof vary-ing size (85–200kb)and structure, and often belonging to the FIIincompatibility(Inc)group[4].OXA-48isanAmblerclassD carbapenemasethatconfersresistancetopenicillinsandreduced susceptibilitytocarbapenems butdoesnot significantly hydrol-yse broad-spectrum cephalosporins [3]. The blaOXA-48 gene has beenalmostalwayslocatedonaconjugativeplasmidofca.62kb assigned to the IncL/M group and has been identified only in enterobacterial species [5,6]. Both CTX-M-15and OXA-48 have beendetectedinseveralenterobacterialspecies,beingparticularly frequentinKlebsiellapneumoniae,whilsttheyarerelatively uncom-monin E.cloacae[2,3].Here wereporttheclinicaland genetic featuresof21E.cloacaeisolatesproducingCTX-M-15,OXA-48or both,whichweremainlyrecoveredfromcriticallyillpatients,ina Spanishhospitalduringa1-yearperiod.

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2. Patientsandmethods

2.1. Setting,patientsandbacterialisolates

‘Hospital Universitario Central de Asturias’ (HUCA) (Oviedo, Spain)isa1100-beduniversityhospitalprovidingcarefora popu-lationofca.342,000inhabitantsintheAsturiasregionofnorthern Spain.Duringthe1-yearperiodfromJune2013toJune2014,all E.cloacaeisolatesrecoveredatthehospital(n=448)wereanalysed phenotypicallyforproductionofcarbapenemasesandESBLs.Atotal of21multiresistantisolates,allrecoveredfromdifferentpatients, producedacarbapenemase,anESBLorboth.MostE.cloacaeisolates wereobtainedfromcriticallyillpatientsadmittedtotwointensive careunits(ICU1andICU2)locatedindifferentbuildingsortoother hospitalwards.Relevantfeaturesregardingthepatientsareshown inTable1.

2.2. Susceptibilitytesting

AllE.cloacae isolates underwent antimicrobial susceptibility testingbythediscdiffusionassay(BectonDickinson,Sparks,MD). The MicroScan® system (Neg Combo Panel Type 53; Siemens Healthcare Diagnostics, Deerfield, IL) was used for determina-tionof MICs and forbacterialidentification,and thelatter was confirmedbymatrix-assistedlaserdesorption/ionisation time-of-flight massspectrometry(MALDI-TOF/MS)(microflexTM_; _Bruker DaltonikGmbH,Bremen,Germany).Resultsofdiscdiffusionassays andMICs wereinterpretedaccordingtoClinicalandLaboratory Standards Institute(CLSI) breakpoints updatedin January 2014 [7].Eteststrips(bioMérieux,Marcy-l’Étoile,France)wereusedfor determiningtheMICsofertapenemandimipeneminthoseisolates suspectedofcarbapenemaseproduction.TheCarbaNPtest recom-mendedfordetectionofcarbapenemases[8]wasalsoperformed forisolateswithelevatedMICs ofertapenemandimipenem.All E.cloacaesuspectedofESBLproduction(double-discsynergytest positiveand/orelevatedMICtocefepime)weretestedwiththe cefepime/cefepime+clavulanicacid(PM/PML)Etest(bioMérieux). 2.3. Identificationofgenesencodingextended-spectrum

ˇ-lactamasesandcarbapenemases,andplasmidanalysis

Genesencodingresistancetobroad-spectrumcephalosporins (blaTEM, blaSHV andblaCTX-M)and carbapenems (blaOXA-48)were identified by PCR amplification [5] followed by sequencing. Transposon Tn1999 (known to carry the blaOXA-48 gene) and plasmid-mediated quinolone resistance genes [qnrA, qnrB, qnrS and aac(6)-Ib-cr]were searchedby PCR amplificationfollowed bysequencingoftheobtainedproducts[3,9].Plasmid DNAwas extractedbytheKadoandLiumethod[5]andwasvisualisedby agarosegelelectrophoresis.

PlasmidIncgroupsweredeterminedbyPCR-basedreplicon typ-ing[5].Conjugationexperimentswereperformedusingasdonor isolatesrepresentativesofthreeantibioticresistancephenotypes, namelyresistancetocarbapenemsbutsusceptibilitytocefepime (Ecl10),resistancetocefepimebutsusceptibilitytocarbapenems (Ecl8andEcl9),orresistancetoboth(Ecl15).Arifampicin-resistant Escherichiacoli J53strain wasusedasrecipient forconjugation assays.Transconjugantswereselectedoneosin–methyleneblue agar(Oxoid,Madrid,Spain)containingrifampicin(100mg/L)plus ertapenem (0.5mg/L) or rifampicin plus ceftriaxone (10mg/L). Plasmid DNA extracted from each of the donor E.cloacae and theirtransconjugantswastransferredtoanylonmembraneand wasthenhybridisedwithprobesspeciﬁcfortheblaCTX-M-15and blaOXA-48 genes.Probes werelabelled usingthe PCRDIG Label-ingMix(RocheAppliedScience,Penzberg,Germany),followedby

gelextractionwiththeGFXTM_DNA_and_Gel_Band_{Puriﬁcation}_Kit (AmershamBiosciences,LittleChalfont,UK).

2.4. Pulsed-ﬁeldgelelectrophoresis(PFGE)analysis

TotalDNA fromthe21E.cloacae isolateswasdigestedwith XbaI (Takara Bio Europe, Saint-Germain-en-Laye, France; 30U, 4hat 37◦C). Thegenerated fragments wereseparatedby PFGE usingaCHEF-DRIIISystem(Bio-RadLaboratories,Hercules,CA) under the conditions recommended by PulseNet (http://www. pulsenetinternational.org).Similarity betweenXbaIproﬁles was evaluatedbytheJaccard’scoefﬁcient(S),andclusteranalysiswas performedbytheunweightedpair-groupmethodwitharithmetic averages(UPGMA)usingthesoftwareprogramMVSP(Multivariate StatisticalPackageforPCs;RockWareInc.,Golden,CO).

2.5. Multilocussequencetyping(MLST)

MLST wasperformed for representativeisolates with differ-entPFGEproﬁles(Ecl10,Ecl3,Ecl9,Ecl8,Ecl21,Ecl5andEcl7)as describedpreviously[10].Thedatabaseavailableathttp://pubmlst. org/ecloacaewasusedforassigningsequencetypes(STs).

3. Results

3.1. Antimicrobialsusceptibilityandgeneticbasisforresistance tobroad-spectrumcephalosporinsandcarbapenems

AsshowninTable2,20ofthe21E.cloacaeisolateswereresistant tocefotaximeand11werealsoresistanttocefepime.Moreover,14 isolatesdisplayedintermediatesusceptibilityorresistanceto car-bapenems.Inadditionto␤-lactamantibiotics,allE.cloacaeisolates were variably resistant to other antimicrobial groups, includ-ingquinolones(nalidixicacidandciproﬂoxacin),aminoglycosides (gentamicinandtobramycin)andtrimethoprim/sulfamethoxazole. The blaCTX-M-15 gene was detected in all isolates resistant to cefepime and positive for the PM/PML test (n=11), whilst theblaOXA-48 genewasfoundinallisolates withelevatedMICs of imipenem and positive for the Carba NP test (n=14). Both blaCTX-M-15 and blaOXA-48 wereidentiﬁed in fourisolates. Over-all,isolatesharbouringblaCTX-M-15,blaOXA-48orbothaccountedfor 1.6%,2.2%and0.9%ofthetotalnumberofE.cloacaerecoveredin HUCAoverthe1-yearstudyperiod(n=448).

ThegeneticlocationofblaCTX-M-15andblaOXA-48wasestablished byplasmidanalysis,hybridisationandconjugationexperiments.As showninFig.1AandTable2,allESBL-producerscarriedaplasmid ofca.150kbhybridisingwiththeblaCTX-M-15 probeand belong-ingtotheIncFgroup.Furthermore,allimipenem-resistantisolates harboured a commonplasmidof ca. 65kbthat gave a positive hybridisationsignalwiththeblaOXA-48probeandbelongedtothe IncL/Mgroup.Noteworthy,theblaOXA-48genewasalways identi-ﬁedintransposonTn1999.1.

ToinvestigatethepotentialoftransferoftheIncFandIncL/M plasmids carrying the blaCTX-M-15 and blaOXA-48 genes, conjuga-tionexperimentswereperformedusingEcl8andEcl9(blaCTX-M-15), Ecl10(blaOXA-48)andEcl15(blaCTX-M-15 andblaOXA-48)asdonors. Whenselectionwasperformedwithertapenem,E.coli transcon-jugantswerereadilyobtainedboth fromEcl10and Ecl15donor strains.ThreeindependentE.colitransconjugantsanalysedfrom each matingexperiment showed resistanceto ampicillin, AMC andertapenem,werepositivebyPCRfortheblaOXA-48geneand carried an ca. 65kb plasmid (Fig. 1A;Table 2).By contrast,no transconjugantswereobtainedafterthreeindependent conjuga-tionsusingEcl8and Ecl9asdonorsandceftriaxoneasselective agent, andonly two transconjugantsafter thesame numberof

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Table1

Featuresofthe21patientsinfectedorcolonisedbymultiresistantEnterobactercloacaeisolates. Patient Sex/age(years) Hospitalunit Admission

diagnosis/colonisationor infectiontype

Sampleorigin Dateof isolation

Treatment Outcome

1 M/46 ICU1 Pancreatitis/surgical

woundinfection

Woundexudate 25/06/2013 Tobramycin Discharged

2 F/56 ICU1 Lungcancer/pneumonia Lungnecropsy 26/06/2013 TZP Died

3 F/77 ICU2 Cardiacsurgery/respiratory

colonisation

Tracheobronchial aspirate

5/07/2013 Untreated Discharged

4 M/19 ICU1 Severeheadinjury/urinary

tractinfection

Urine 12/08/2013 Ciproﬂoxacin Died

5 M/81 Reanimation Vascular surgery/respiratory infection Tracheobronchial aspirate 23/12/2013 Ciproﬂoxacin/ gentamicin/meropenem Died 6 M/81 ICU2 Vascular surgery/pneumonia Tracheobronchial aspirate 26/12/2013 Imipenem Discharged

7 M/76 Generalsurgery Abdominal

surgery/surgicalwound infection

Woundexudate 27/12/2013 TZP Discharged

8a _F/57 _Pneumology_unit _Lung_{transplantation}

follow-up/respiratory infection

Sputum 17/01/2014 Amikacin/meropenem Discharged

9 M/41 ICU2 Septicshock/pneumonia Tracheobronchial

aspirate 21/01/2014 Meropenem Discharged 10 F/33 ICU1 H1N1ﬂu pneumonia/respiratory colonisation Tracheobronchial aspirate 23/01/2014 Untreatedb _Discharged 11 F/53 ICU1 H1N1ﬂu pneumonia/respiratory colonisation Tracheobronchial aspirate 27/01/2014 Untreatedb _Discharged

12 M/79 Cardiacsurgery Cardiacsurgery/urinary tractinfection

Urinec _29/01/2014 _SXT _Discharged

13 M/36 ICU2 H1N1ﬂu

pneumonia/pneumonia

Pleuralﬂuid 11/02/2014 TZP/colistinb _Died

14 M/62 Cardiacsurgery Cardiac

surgery/asymptomatic bacteriuria

Urine 17/02/2014 Untreated Discharged

15 F/30 Cardiology Heart

transplantation/surgical woundinfection

Woundexudate 19/02/2014 Colistin/tigecycline Discharged

16 F/61 ICU2 Cardiacsurgery/surgical

woundinfection

Woundexudate 17/03/2014 Imipenem Discharged

17 F/66 Internalmedicine Respiratory failure/asymptomatic bacteriuria

18 M/69 Neurology Epilepticattack/urinary cathetercolonisation

19 F/92 Maxillofacial

surgery

Maxillofacial surgery/urinarytract infection

Urine 29/04/2014 Meropenem Discharged

20 M/66 Cardiacsurgery Cardiacsurgery/surgical woundinfection

Woundexudate 13/05/2014 Unknownd _Discharged

21 M/79 ICU2 Vascularsurgery/catheter

sepsis

Cathetertip 14/05/2014 Imipenem Discharged

ICU,intensivecareunit;TZP,piperacillin/tazobactam;SXT,trimethoprim/sulfamethoxazole.

a_Patient_transferred_from_another_hospital_in_a_nearby_region,_where_the_E._cloacae_infection_had_already_been_diagnosed. b_Patients_10,₁₁_and₁₃_were_treated_with_oseltamivir.

c _Another_E._cloacae_isolate_with_a_different_resistance_pattern_and_not_producing_{extended-spectrum}_{␤-lactamase}_(ESBL)_or_OXA-48_was_detected_in_the_same_sample. d _The_sample_from_this_patient_was_collected_in_a_primary_care_centre_a_few_days_after_have_being_discharged_of_the_Cardiac_Surgery_Unit_of_the_hospital._There_are_no

treatmentdataforthispatient.

matings and ceftriaxone selection using Ecl15 as donor. Both Ecl15transconjugantswereresistanttoampicillin, AMC, broad-spectrum cephalosporinsand aminoglycosides,had an elevated MICofciproﬂoxacinandcarriedtwoplasmids,oneofca.150kb andtheotherslightlylargerthan65kb,whichhybridisedwiththe blaCTX-M-15andblaOXA-48probes,respectively(Fig.1A;Table2).The lattercouldhavederivedfromtheca.65kbplasmidthrough acqui-sition ofadditional DNA. Takentogether, theseresultsstrongly suggestthattheca.150kbIncFplasmidcarryingblaCTX-M-15isnot self-transferable,butitcouldbemobilisedbytheca.65kb plas-midthoughataratherlowfrequency.TheqnrBandaac(6)-Ib-cr genesweredetectedin theEcl15transconjugantsas wellasin

theE.cloacaeisolatescarryingtheIncFplasmid,allwithdecreased susceptibilitytociproﬂoxacin.

3.2. GenotypingoftheCTX-M-15-and/orOXA-48-producing Enterobactercloacaeisolates

Typingofthe21E.cloacaeisolatesbyPFGEyieldedseven pro-files(X1–X7)(Fig.1B).Adendrogramofsimilaritybasedonthese profiles revealedtwo mainclustersof E.cloacae(clusters Iand II) that were strongly associated with thetwo separate build-ingsofHUCA(Fig.1C).ClusterIcomprisedtwoprofiles(X1and X5)withsevenandoneisolates,respectively.ArepresentativeX1

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Table2

MicrobiologicalcharacteristicsofclinicalEnterobactercloacaeisolatesandtheirtransconjugants. Isolatea,b _{Antimicrobial}_resistance

phenotype ESBL/CARB gene ERT/IPMMIC (mg/L) Corresponding plasmids PFGEproﬁle/ cluster/buildingc MLST Ecl1,Ecl2,Ecl4,Ecl10,

Ecl11,Ecl17,Ecl18

AMX-AMC-FOX-CTX-ERT-IPM-NAL-CIP-SXT

blaOXA-48 4/2 65kbd X1/I/1 ST74

Tc-Ecl10-ERT(1,2and3) AMX-AMC-ERT blaOXA-48 2/0.5 65kbd N/A N/A

Ecl3

AMX-AMC-FOX-CTX-GEN-ERT-IPM-NAL-CIP-SXT

blaOXA-48 3/2 65kbd X5/I/2 ST78

Ecl6,Ecl12,Ecl14,Ecl16, Ecl20 AMX-AMC-FOX-CTX-FEP-GEN-TOB-NAL-CIPe blaCTX-M-15 ≤0.5/≤1 150kbf X2/II/2 ST66 Ecl9 X3/II/2 Ecl8 AMX-AMC-FOX-CTX-FEP-GEN-TOB-NAL-CIPe_-SXT blaCTX-M-15 ≤0.5/≤1 150kbf X4/II/1 ST66

Ecl19,Ecl21 AMX-AMC-FOX-CTX-FEP-ERT-IPM-GEN-TOB-NAL-CIPe

blaCTX-M-15,

blaOXA-48

4–6/2–4 150kbf_,₆₅_kbd _X2/II/2 _ST66

Ecl13,Ecl15 X3/II/2

Tc-Ecl15-ERT(1,2and3) AMX-AMC-ERT blaOXA-48 2/0.5 65kbd N/A N/A

Tc-Ecl15-CRO(1and2) AMX-AMC-CTX-FEP-ERT-GEN-TOB-CIPe blaCTX-M-15, blaOXA-48 1/0.38 150kbf_, >65kbd N/A N/A Ecl5 AMX-AMC-FOX-ERT-IPM-NAL-CIP-SXT blaOXA-48 4/4 65kbd X6/–/1 ST45 Ecl7 AMX-AMC-FOX-CTX-ERT-IPM-GEN-TOB-NAL-CIP-SXT blaOXA-48 8/4 65kbd X7/–/1 ST295

ESBL,extended-spectrum␤-lactamase;CARB,carbapenemase;MIC,minimuminhibitoryconcentration;PFGE,pulsed-ﬁeldgelelectrophoresis;MLST,multilocussequence type;AMX,amoxicillin;AMC,amoxicillin/clavulanicacid;FOX,cefoxitin;CTX,cefotaxime;ERT,ertapenem;IPM,imipenem;NAL,nalidixicacid;CIP,ciproﬂoxacin;SXT, trimethoprim/sulfamethoxazole;GEN,gentamicin;FEP,cefepime;TOB,tobramycin;CRO,ceftriaxone;N/A,notapplicable.

a_Enterobacter_cloacae_(Ecl)_isolates_with_numbers_{corresponding}_to_patient_numbers_in_Table₁_._Ecl_isolates_used_as_donors_in_conjugation_experiments_are_shown_in_bold.

EclisolatesforwhichtheMLSTwasdeterminedareunderlined.

b_Escherichia_coli_{transconjugants}_designated_with_Tc_followed_by_the_E._cloacae_donor_isolate_and_the_{antimicrobial}_used_for_selection:_ERT_(ertapenem)_or_CRO_{(ceftriaxone);}

thenumberoftransconjugantstestedisshowninparenthesis.NotransconjugantswereobtainedforEcl9andEcl8afterselectionwithCRO.

c _See_Fig.₁_C_and_text_for_details. d_IncL/M_plasmids.

e_Isolates_positive_for_the_qnrB_and_aac(6_)-Ib-cr_genes. f _IncF_plasmids.

isolateandtheX5isolatebelongedtoST74andST78,respectively, withinthesameclonalcomplex(Fig.1C;Table2).AllX1isolates wererecoveredinbuilding1,whichhousesthemainICUofthe hospital(ICU1),wherefiveofthemweredetected.Theremaining twocamefromtheinternalmedicineandneurologyservices.All isolateswithinclusterIwereresistanttocarbapenemsowingto productionofOXA-48andallwerealsoresistanttobroad-spectrum cephalosporins, consistentwith overexpression of the chromo-somalblaampCgene.ClusterIIwasassociatedwithasecondbuilding ofthehospitalencompassingthecardiacandmaxillofacialsurgery unitsandanotherICU(ICU2).Itincluded11isolateswithidentical (X2;7isolates)orcloselyrelated(X3andX4,with3and1isolates, respectively)profilesassignedtoST66,andallexceptone(Ecl8;X4 profile)wererecoveredinbuilding2(Fig.1C;Table2).Interestingly, Ecl8wasidentifiedfromasinglepatientwhohadbeentransferred fromahospitalinthenearbyregionofSantander,wheretheE. cloacaeinfectionhadalreadybeendiagnosed.Isolateswithin clus-terIIwereallresistanttobroad-spectrumcephalosporinsowing totheproductionofCTX-M-15,and4ofthe11isolatesalso har-bouredblaOXA-48andwerethereforeresistanttocarbapenems.Two distantly related profiles (X6 and X7)were shown by OXA-48-producersbelongingtoST45andST295,respectively,andfound inbuilding1(Fig.1C;Table2).

4. Discussion

EnterobactercloacaeisolatesproducingOXA-48,CTX-M-15or both ␤-lactamases emerged as important pathogens in HUCA, a general university hospital in northern Spain. In fact, they

accountedfor4.7%(21/448)ofthetotalE.cloacaeisolates recov-ered over a 1-year period, although they were not previously reported in this region. Together with some sporadic isolates, two majorclones (ST74 andST66,falling intwo different clus-tersaccordingtoPFGEanalysis)arecausingprolongedoutbreaks inseparatebuildingsofthehospital.TheST74cloneconsistedof carbapenem-resistantisolatescarryingtheblaOXA-48gene.Thisis theﬁrstoutbreakcausedbyOXA-48-producingE.cloacaereported inEurope,butapreviousoutbreakhasbeennoticedina neona-tal ICU in Jerusalem [11]. In contrast to that observed here, isolates fromIsrael carried a derivativeof theOXA-48 plasmid thathadacquiredtheblaCTX-M-14gene.Thesecondclone identi-ﬁed(ST66)correspondedtoblaCTX-M-15-positiveisolateswiththe ESBLgenelocatedonanca.150-kb,non-conjugativeplasmidof theIncFgroup.Someofthemco-harbouredtheIncL/Mplasmid and co-produced OXA-48.Interestingly, oneof theclone 2 iso-lates was apparently transferred fromanother hospital located in a nearbyregionofSpain,soin additiontointrahospital dis-semination,interhospitalspreadiscontributingtotheemergence ofCTX-M-15-producingE.cloacaeinHUCA.Additionaloutbreaks of E. cloacae producing CTX-M-15 have occurred in a new-born ICUin Spainaswellas in achildren’s hospitalin Finland [12,13].

Noticeably,someisolatesbelongingtoclone2alsocarriedthe blaOXA-48plasmid,whichcouldhavebeenacquiredfromother OXA-48-producingEnterobacteriaceaecirculatinginthehospital[5;our unpublishedresults].Thismightbeinaccordancewiththehigh frequency ofdisseminationof theblaOXA-48-carryingplasmidas previouslydemonstrated[14].

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Fig.1. MoleculartypingandgeneticrelationshipsofEnterobactercloacaeisolatesproducingOXA-48andCTX-M-15␤-lactamasesinaSpanishhospital.(A)Plasmidproﬁles ofrepresentativeE.cloacaeisolatesandtheirtransconjugants.Lanesiandii,plasmidsobtainedfromEscherichiacoli39R861(NCTC50192)andplasmidRP4usedasmolecular sizestandardsforuncutDNA;lane1,Ecl9;lane2,Ecl15;lane3,Tc-Ecl15-CRO(1);lane4,Tc-Ecl15-ERT(1);lane5,Tc-Ecl15-ERT(2);lane6,Tc-Ecl15-ERT(3);lanes7, Ecl10;andlane8,Tc-Ecl10-ERT(1).PlasmidshybridisingwithblaCTX-M-15andblaOXA-48probesaremarkedwitharrowheadsandasterisks,respectively.(B)Pulsed-ﬁeldgel

electrophoresis(PFGE)profilesgeneratedfromE.cloacaeisolatesbyXbaIdigestion.Lane␭,LambdaLadderPFGMarker(NewEnglandBiolabs,Beverly,MA)andlaneB,DNA fromSalmonellaentericaserovarBraenderupH9812digestedwithXbaI,bothusedassizestandards.LaneX1,Ecl10;laneX2,Ecl6;laneX3,Ecl9;laneX4,Ecl8;laneX5,Ecl3; laneX6,Ecl5;andlaneX7,Ecl7.(C)DendrogramofsimilarityshowingtherelatednessbetweentheXbaIprofiles.Isolatesassignedtoeachprofileaswellasthehospital wardandbuildinginwhichtheywerecollectedareshown.Relevantgenesandplasmidscarriedbyeachisolatearealsoindicated.ClustersIandIIcompriseST74(clone 1)/ST78(positiveforblaOXA-48)andST66(clone2,positiveforblaCTX-M-15±blaOXA-48)isolates,respectively.X,XbaIprofile;ST,sequencetype;ICU,intensivecareunit;Inc,

incompatibilitygroup;S,Jaccard’scoefﬁcientofsimilarity.

OccurrenceoftheqnrBand aac(6)-Ib-cr genesin enterobac-terial isolates producing OXA-48 or ESBLs, including some of the CTX-M-15-producing E. cloacae isolates causing the out-break in a neonatal unit in Spain, was previously noticed [12].

MostE.cloacaereportedinthisstudywereassignedtoSTs pre-viouslyassociatedwithESBL-andcarbapenemase-producersand consideredashigh-riskinternationalclones(ST45,ST66,ST74and ST78),withthelattertwobelongingtothesameclonalcomplex [10,15].Noteworthy, noneof these clones had beenpreviously

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associatedwithOXA-48production.Sincephylogeneticstudieson multiresistantE.cloacaehavebeeninitiatedonlyrecently,further studiesarerequiredtoexpandtheknowledgeofthese multiresis-tantbacteria.

AlthoughthenumberofE.cloacaeproducingcarbapenemases orESBLshasincreasedinrecentyears,theirrealprevalencecould be underestimated because of difficultiesin detecting both ␤-lactamases.Inthepresentstudy,theCarbaNPtestprovedtobevery efficientforaccurateidentificationofcarbapenemase-producers.

5. Conclusions

Intrahospital and interhospital dissemination of multiresis-tant E. cloacae isolates producing CTX-M-15, OXA-48 or both ␤-lactamases is a cause of concerndue tothe limited options remaining for the treatment of affected patients, most with critical underlying conditions. Interestingly, as observed for K. pneumoniae,emergenceofbothbroad-spectrumresistancetraits insingleE.cloacae isolatescorrespondstothetransferof genes havingdisseminatedfromcommunitypathogensintonosocomial pathogens. Considering that the community reservoir of some oftheseresistancetraitsis signiﬁcantlygrowingnowadays,the likelihood of witnessing an increased number of nosocomial outbreaksduetomultiresistantE.cloacaeisveryhigh.

Funding:This workhasbeensupported byproject FIS-PI11-00808(FondodeInvestigaciónSanitaria,InstitutodeSaludCarlos III,MinisteriodeEconomíayCompetitividad,Spain),co-fundedby theEuropeanRegionalDevelopmentFundoftheEuropeanUnion: awaytoMakingEurope,andbytheUniversityofFribourg (Fri-bourg,Switzerland).AshortstayofJFattheUniversityofFribourg wassupportedbya grantfromtheSociedadEspa ˜nolade Enfer-medadesInfecciosasyMicrobiologíaClínica(SEIMC).IMwasthe recipientofapredoctoralgrantfromtheFundaciónparaelFomento enAsturiasdelaInvestigaciónCientíﬁcaAplicadaylaTecnología [FICYTBP09-069].

Competinginterests:Nonedeclared. Ethicalapproval:Notrequired.

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