First characterization of a Providencia stuartii clinical isolate from a Tunisian intensive care unit coproducing VEB-1-a, OXA-2, qnrA6 and aac(6')-Ib-cr determinants

braz j infect dis.2014;18(2):211–214

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Brief

communication

First

characterization

of

a

Providencia

stuartii

clinical

isolate

from

a

Tunisian

intensive

care

unit

coproducing

VEB-1-a,

OXA-2,

qnrA6

and

aac(6



)-Ib-cr

determinants

Sihem

Mahrouki

_,

_Hela

_Chihi

_,

_Amel

_Bourouis

_,

_Mohamed

_Ben

_Moussa

_,

Omrane

Belhadj

a_{LaboratoryofBiochemistryandBiotechnology,DepartmentofBiology,FacultyofSciencesofTunis,CampusUniversitaire,}

2092El-ManarII,Tunis,Tunisia

b_{LaboratoryofMicrobiology,MilitaryHospitalofTunis,1089Monfleury,Tunis,Tunisia}

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Articlehistory:

Received24December2012 Accepted12October2013 Availableonline27December2013

Keywords: ESBL Providenciastuartii qnrA6 aac(6)-Ib-cr

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AclinicalProvidenciastuartiiisolateSM662wasrecoveredfromapatienthospitalizedinthe intensivecareunitattheMilitaryhospital,Tunisia.Thisisolatewasresistanttopenicillins, cephalosporins,aminoglycosidesandfluoroquinolones.Amarkedinvitrosynergybetween ceftazidimeorcefotaximeandamoxicillin–clavulanicacidonMueller-Hintonagarplates suggestedthepresenceofanextended-spectrum-␤-lactamase.Inaddition,anunusual syn-ergywasfoundbetweencefepimeoraztreonam,andcefoxitinorimipenemonadoubledisk synergytestsuggestingaVEB-typeextended-spectrum-␤-lactamase.Thecharacterization of␤-lactamasesandassociatedresistancegeneswasperformedbyisoelectricfocusing, polymerasechainreactionandnucleotidesequencing.Two␤-lactamasesbandswithpI val-uesof5.4and7.7,whichwerematchedtoTEM-1,VEB-1-aandOXA-2-like␤-lactamases weredetected.TheblaVEB-1-agenewasfoundtobeassociatedwithcomplexgenetic struc-tures,includingReelements.These␤-lactamaseswerenottransferredbyelectroporationor conjugationexperimentstothetransconjugantsandelectroporants.Hybridizationmethods showedthattheextended-spectrum-␤-lactamaseencodinggenemayhaveachromosomal localization.TheisolateSM662producedthequinoloneresistancedeterminantsqnrA6and aac(6)-Ib-crwhichweresuccessfullytransferred.Thedetectionofanassociated chromoso-malquinoloneresistancerevealedthepresenceofagyrAmutationatcodon83(Ser83Ile). ThisisthefirstreportofthelinkageVEB-1-a/OXA-2-likeinP.stuartiiassociatedwiththe descriptionofqnrA6andaac(6)-Ib-crgenesinthisisolate.

©2013 ElsevierEditoraLtda.Allrightsreserved.

Providenciastuartiiisafrequentcauseofurinarytract infec-tions in hospitalized patients.1 _{It plays an important role} asanosocomialpathogeninthedisseminationof plasmid-mediated resistance.2 _{P. stuartii is naturally resistant to}

∗ _{Correspondingauthorat:StreetElHoudaN}◦_{3,OliveCity,1005ElOmrane,Tunis,Tunisia.} E-mailaddress:[email protected](S.Mahrouki).

aminopenicillinsandnarrow-spectrumcephalosporinsdueto achromosomallyexpressedAmblerclassCcephalosporinases (AmpC).1_{However,acquisitionofESBLhasbeenreported.}1,2 blaVEB-1genewasidentifiedinP.stuartiiforthefirsttimein

1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2013.10.004

212

Table1–MICs(␮g/mL)ofvariousantimicrobialagentsobtainedfortheclinicalisolateP.stuartiiSM662,its transconjugantsandelectroporantsandtheE.coliHB101andE.coliDH10Brecipientsstrains.

P.stuartiiSM662 HB101×SM662 E.coliHB101 DH10B/pSM662 E.coliDH10B

Amoxicillin >512 4 8 4 2 Ticarcillin >512 4 8 8 2 Oxacillin 512 <4 <2 2 2 Cefoxitin 64 8 2 4 4 Cefotaxime >512 <4 <2 2 4 Ceftriaxone 512 <4 <2 2 1 Ceftazidime >512 <4 <2 2 1 Aztreonam 512 4 <2 <4 1 Nalidixicacid >512 >512 2 >512 2 Chloramphenicol 256 8 <2 8 2 Tetracycline 512 8 <2 8 <1 Ciprofloxacin 64 4 2 8 1 Ofloxacin 128 64 2 32 1 Streptomycin 64 256 512 128 1 Tobramycin 128 256 <0.25 128 2 Gentamicin 1 0.5 <0.25 0.5 <0.25 Piperacillin 1 0.5 <0.25 0.5 <0.25 Ertapenem 0.25 <0.25 <2 <0.25 <0.25 Imipenem 2 <2 <2 <2 <1

Alger.1_{VEB-1␤-lactamaseconfershigh-levelresistancetoa} broadspectrum ofcephalosporins;however this activity is inhibitednotonlybyclavulanate,butalsobycefoxitinand imipenem.3_{Untilnow,thebla}

OXA-2genehasnotbeendetected

inProvidencia genusastothebestofourknowledge,but in Tunisia it was described in clinical strains of Pseudomonas aeuroginosa.4_{Adecreasedquinolonesusceptibilityassociated} withqnrA6andaac(6)-Ib-crdeterminantswasalsoreported inTunisiainclinicalstrainsofP.stuartii.5_{Inthecurrentstudy,} wereportforthefirsttimethe co-productionof chromoso-malblaVEB-1-aandblaOXA-2-likegenesinamultidrugresistant

P. stuartii clinical strain isolated at the Militaryhospital in Tunisiaand theirassociation withplasmid-mediatedqnrA6 andaac(6)-Ib-crdeterminants.

OnJuly2008, a46-year-oldmanwas transferredfrom a Tunisian Regional Hospital and hewas hospitalized inthe intensivecareunitattheMilitaryhospitalinTunis,Tunisia foraseverecranialtraumatism.Threemonthsthereafter,the patientnotablydiabeticandepilepticwasfebrileatanytime and subsequentlydeveloped achronicinfection.According to the patient’s medical records,such an infection turned outtobeurinarytractinfectionthatwasdiagnosed follow-ingtheappearanceofaninfectioussyndrome;itwastreated withceftazidimeandofloxacin.AttheendofOctober2008, P.stuartiiSM662isolatewasrecoveredbyatrachealaspirate, althoughsevendayspriortotheisolationofthisstrainhehad receivedacourseofcefotaximeandciprofloxacin.Thepatient wastreatedwithgentamicinandimipenem.Sevendaysafter startingantimicrobialtherapytheclinicaloutcomeindicated treatmentfailureandtheultimatelydied.

TheP.stuartiiSM662strainwasidentifiedusinganAP20Ekit (Biomèrieux,Marcy-l’Etoile,France).E.coliDH10B(Invitrogen, Life Technologies) and streptomycin resistant E. coli HB101 recipientstrainswere usedrespectivelyforthe electropora-tionandconjugationexperiments.␤-Lactamaseswithknown pIswereusedasstandards:TEM-1(pI5.4),TEM-2(pI5.6), TEM-3(pI6.3)andSHV-1(pI7.6).6Antimicrobialsusceptibilitywas determinedbythediskdiffusionmethodonMueller-Hinton

(MH) agar (Bio-Rad, Marnes La Coquette, France) recom-mended bythe Clinical and LaboratoryStandards Institute (CLSI)guidelines.7_{Theisolatewasresistanttomultiple} antibi-otics, including chloramphenicol, kanamycin, tobramycin, sulphonamide,tetracycline,nalidixicacid,ciprofloxacinand ofloxacinwhereasitwassusceptibletoimipenem,ertapenem, gentamicinandpiperacillin.Thedoublediscsynergytestwas positiveshowingamarkedsynergybetweenceftazidimeor cefotaximeandamoxicillin–clavulanicacidonMHagarplates and suggestedthepresenceofaclassAESBL.8 _Inaddition, an unusualsynergy wasfoundbetweencefepimeor aztre-onam,and cefoxitinorimipenemonadoubledisksynergy testsuggestingaVEB-typeESBLproductionaccordingtoNaas etal.3_{Theminimuminhibitoryconcentrations(MICs)(Table1)} weredeterminedbythebrothmicrodilutionmethodand inter-pretedaccordingtotheCLSIcriteria.6 _{Thestrainwasfound} intermediatelyresistanttoimipenemaccordingtothenovel CLSI breakpoints(M100-S23)and thedifference onthe car-bapenem’sactivity(imipenemandertapenem)isduetothe lower activity ofimipenem againstProvidencia spp., Proteus spp.andMorganellamorganii.

Whole-cell DNA from P. stuartii SM662 was used as a templateforPCRassays. PresenceofblaTEM-1, blaVEB-1-a and

blaOXA-2-like genes was assessedby PCRand sequencing as

previouslydescribed.6,9,10_{Noampliconswereobtainedwith} blaSHV and blaCTX-M genes.6,11 Furthermore, multiplex PCR amplifications using primersspecific forplasmid-mediated AmpC␤-lactamases(CBLs)12_{werenegative.}

VEBcas-Fand VEBcas-B(Eurogentec,Belgium)locatedat each end ofthe blaVEB-1 cassette wereused toamplify the

entire blaVEB-1 gene.3 Conditions PCR amplification

experi-mentswereperformedusingprimerslocatedintheblaVEB-1a

geneand intheclass1integron variableregion(5CS–3CS) (Eurogentec, Belgium) as described previously.13 Amplifica-tion ofthe class1integron variable region (5CS–3CS) was positive inSM662showingsizeofabout 1200bp. Sequence analysis showed two genes cassettes arrays: aadB+dfrA1. A combination of 5-CS or 3-CS primers and VEBINV1 or

brazj infect dis.2014;18(2):211–214

213

Fig.1–HybridizationpatternswiththeVEB-1andOXA-2 probesafterHindIIIandSmaIdigestionofgenomicDNA. Lanes1and2,SmaIfragmentswithVEB-1andOXA-2 probesrespectively;Lanes3and4,HindIIIfragmentswith VEB-1andOXA-2probesrespectively;M,10-KbDNA marker.

VEBINV2 (Eurogentec, Belgium), respectively, both primers readingoutwardsfromblaVEB-1,wasalsousedforthe

determi-nationofthegeneticcontentofclass1integron.13However, noPCRfragmentswereobtainedsuggestingthattheblaVEB-1a

genecannot bepart ofaclass1 integron.Thishypothesis did not guarantee that this gene was not inserted into a class1integron,sinceVEB-1isusuallydescribedaspartof agenecassetteitselflocatedinaclass1integron.3_Afurther PCRperformedusingprimerspairRe1F(repeatelement)and VEBcas-B,14_{revealedthepresenceofaPCRfragmentofabout} 1.2KbandsuggestedthattheblaVEB-1a genewasassociated

to two Re1 repeated elements in the direct orientation. A previousreportidentifiedthepresenceofRe1repeatelements sequencessurroundingtheblaVEB-1-ageneinP.aeruginosa10.2

clinicalisolatefromIndia[14].

Analyticalisoelectricfocusingofcrude␤-lactamaseextract ofP.stuartiiSM66215_{demonstratedtwobandsof␤-lactamases} activitieswithpIsof5.4and7.7.TEM-1andVEB-1haveboth5.4 whileOXA-2hasthepI7.7.These␤-lactamaseshavebeennot transferredsuggestingthatwerenotmediatedbya conjuga-tiveortransferableplasmid.Thesingleplasmidtransferred (p-SM662)usingaplasmidextractionkitGFXMicroPlasmid Prep(AmershamBiosciences,UK),conferredresistanceonlyto nalidixicacid,ofloxacinandtobramycin(Table1). Hybridiza-tionmethodsafterdigestionrestrictionwithSmaIandHindIII (Biorad®_{, Laboratories, France)}16 _{showed that bla}

VEB-1a and

blaOXA-2 like genes may have a chromosomal localization

(Fig.1).SeveralstudiesreportedthatblaVEB-1-alikegenesare

mostly plasmid located in Enterobacteriaceae, whereas they arechromosomallylocatedinP.aeruginosaandAcinetobacter

baumannii.1 _{Nonetheless, inourstudy weidentifieda} chro-mosomalVEB-1-atypeESBL.Thisfindingisdescribedforthe firsttimeinTunisiaandsuggeststhatblaVEB-1-acanspread

amongclinicallyrelevantspecies.Here,wedescribea mul-tidrugresistantP.stuartiiSM662co-producedTEM-1andthe narrow-spectrum␤-lactamaseOXA-2-like,togetherwiththe ESBL VEB-1-a. Previous finding reported the simultaneous presenceofblaVEB-1andblaoxa-10 genesinaclinicalstrainof

P. stuartiiV1isolated fromNigeria17_{but ourstudy presents} thefirstreportofthelinkageVEB-1-a/OXA-2-likeinP.stuartii SM662clinicalisolate,tothebestofourknowledge.Blaoxa-2-like

hasbeendetectedinP.aeruginosaisolatesfromTunisia;4_this finding indicated that OXA genes canspread progressively betweenspecies.

Interestingly, a marked association was found between ESBLproductionandmultidrugresistance.Toinvestigatethe coresistance,PCRdetectionandsequencingoftheqnrA,qnrB and qnrS genes18 _{and the} aminoglycoside/fluoroquinolone-modifying enzyme-encoding aac(6)-Ib-cr gene19 _identified the qnrA6 determinantand the variant aac(6)-Ib-cr on the same plasmid.Thetransconjugants andthe electroporants expressednon-susceptibilitytoofloxacin,streptomycinand tobramycin(Table1).Inourstudy,aac(6)-Ib-cr,whichencodes an aminoglycosideacetyltransferase, was foundassociated withVEB-1andOXA-2␤-lactamasesforthefirsttimein clini-calstrainofP.stuartiiinTunisia.PCRdetectionandsequencing ofanadditionalchromosomalquinoloneresistance determi-nantsregions(QRDRs)20_{didnotrevealthepresenceofgyrB,} parC and parE, but wedetect agyrA mutationatcodon 83 (Ser-Ile).Thisobservationmayexplainthehigherlevelof resis-tancetonalidixicacidinourisolate.

In conclusion, our study indicated for the first time in Tunisiathe dissemination ofVEB-1␤-lactamaseassociated withplasmid-mediatedqnrA6andaac(6)-Ib-cr-like determi-nantsinamultidrugresistantP.stuartiiclinicalisolate.The presenceofResequencessurroundingtheblaVEB-1-ageneis

worryingsincetheiroriginandtheirfunctioninthe mobiliza-tionofblaVEB-1-aremainunknownandposeachallengeforthe

treatmentofhospitalinfectionsduetoGram-negative bacte-ria.Therefore,theincidenceofESBL-producingbacterianeeds acontinuousmonitoringofsuchmultidrugresistantstrains andwarrantsfurtherstudyoftheirepidemiologicevolution.

Conflicts

of

interest

Theauthordeclarenoconflictsofinterest.

Acknowledgments

The authors wish to thankPr. Ferjani Mustafa, directorof IntensiveCareUnitWar,Militaryhospital,Tunisforhis help-fulassistancetoobtainclinicaldataoftheisolate.Thiswork wassupportedbytheTunisianMinistryofHigherEducation, ScientificResearchandTechnology.

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1.AubertD,NaasT,LartigueMF,NordmannP.Novelgenetic structureassociatedwithanextended-spectrum␤-lactamase

214

blaVEBgeneinaProvidenciastuartiiclinicalisolatefrom Algeria.AntimicrobAgentsChemother.2005;49:3590–2. 2. FranceschiniN,PerilliM,SegatoreB,etal.Ceftazidimeand

aztreonamresistanceinProvidenciastuartii:characterization ofanaturalTEM-derivedextendedspectrum␤-lactamase, TEM-60.AntimicrobAgentsChemother.1998;42:1459–62. 3. NaasT,BenaoudiaF,MassuardS,NordmannP.Integron

locatedVEB-1extended-spectrum␤-lactamasegeneina ProteusmirabilisclinicalisolatefromVietnam.JAntimicrob Chemother.2000;46:703–11.

4. KtariS,MnifB,ZnazenA,etal.Diversityof␤-lactamasesin Pseudomonasaeruginosaisolatesproducing

metallo-␤-lactamaseintwoTunisianhospitals.MicrobDrug Resist.2011;17:25–30.

5. ArpinC,ThabetL,YassineH,etal.Evolutionofan

incompatibilitygroupincA/CplasmidharboringblaCMY-16and

qnrA6genesanditstransferthroughthreeclonesof Providenciastuartiiduringatwo-yearoutbreakinatunisian burnunit.AntimicrobAgentsChemother.2012;56:1342–9. 6. MahroukiS,ChihiH,BourouisA,Ben-MoussaM,BarguellilF,

BelhadjO.FirstcharacterisationinTunisiaofplasmid mediatedAmpCbêta-lactamaseDHA-1coexpressedTEM-24 andqnrA6inamultidrugresistantProteusmirabilisclinical strain.AfrJMicrobRes.2011;5:3913–8.

7. ClinicalandLaboratoryStandardsInstitute.Performance standardsforantimicrobialsusceptibilitytesting.In:20th InformationalSupplementM100-S20.Wayne,Penn:Clinical andLaboratoryStandardsInstitute;2007.

8. LivermoreDM,BrownDFJ.Detectionof␤-lactamase-mediated resistance.JAntimicrobChemother.2001;48:59–64.

9. KimJY,ParkYJ,KimSI,KangMY,LeeSO,LeeKY.Nosocomial outbreakbyProteusmirabilisproducingextendedspectrum ␤-lactamaseVEB-1inaKoreanUniversityHospital.J AntimicrobChemother.2004;54:1144–7.

10.DeChampsC,PoirelL,BonnetR,etal.Prospectivesurveyof ␤-lactamasesproducedbyceftazidime-resistantPseudomonas aeruginosaisolatedinaFrenchHospitalin2000.Antimicrob AgentsChemother.2002;46:3031–4.

11.BourouisA,DuboisV,CoulangeL,etal.Firstreportof CTX-M-9inaclinicalisolateofEnterobactercloacaeina TunisianHospital.PatholBiol.2011;59:187–91.

12.Pérez-PérezFJ,HansonND.Detectionofplasmid-mediated AmpC␤-lactamasesgenesinclinicalisolatesbyusing multiplexPCR.JClinMicrobiol.2002;40:2153–62. 13.NaasT,PoirelL,KarimA,NordmannP.Molecular

characterizationofIn50,aclass1integronencodingthegene fortheextended-spectrum␤-lactamaseVEB-1inPseudomonas aeruginosa.FEMSMicrobiolLett.1999;176:411–9.

14.AubertD,GirlichD,NaasT,NagarajanS,NordmannP. Functionalandstructuralcharacterizationofthegenetic environmentofanextended-spectrum␤-lactamaseblaVEB genefromaPseudomonasaeruginosaisolateobtainedinIndia. AntimicrobAgentsChemother.2004;48:3284–90.

15.MahroukiS,BenAchourN,ChouchaniC,BenMoussaM, BelhadjO.Identificationofplasmid-encodedextended spectrum␤-lactamasesproducedbyaclinicalstrainof Proteusmirabilis.PatholBiol.2009;57:e55–9.

16.SambrookJ,MacCallumP,RussellD.Molecularcloning:a laboratorymanual.3rded.ColdSpringHarbor,N.Y:Cold SpringHarborLaboratoryPress;2001.

17.AibinuIE,PfeiferY,OgunsolaF,OdugbemiT,KoenigW, GhebremedhinB.Emergenceof␤-lactamasesOXA-10,VEB-1 andCMYinProvidenciaspp.fromNigeria.JAntimicrob Chemother.2011:1931–2,http://dx.doi.org/10.1093/jac/dkr197. 18.CattoirV,PoirelL,RotimiV,SoussyCJ,NordmannP.Multiplex PCRfordetectionofplasmid-mediatedquinoloneresistance qnrgenesinESBL-producingenterobacterialisolates.J AntimicrobChemother.2007;60:394–7.

19.PerilliM,ForcellaC,CelenzaG,etal.EvidenceforqnrB1and aac(6)-Ib-crinCTX-M-15-producinguropathogenic EnterobacteriaceaeinanItalianteachinghospital.Diagn MicrobiolInfectDis.2009;64:90–3.

20.MammeriH,VanDeLooM,PoirelL,Martinez-MartinezL, NordmannP.Emergenceofplasmid-mediatedquinolone resistanceinEscherichiacoliinEurope.AntimicrobAgents Chemother.2005;49:71–6.