Molecular characterization of methicillin-resistant Staphylococcus aureus isolated from a Brazilian university hospital

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

Molecular

characterization

of

methicillin-resistant

Staphylococcus

aureus

isolated

from

a

Brazilian

university

hospital

André

Martins

a,b

_,

_Danilo

_Flávio

_Moraes

_Riboli

a

_,

_Valéria

_Cataneli

_Pereira

a

_,

Maria

de

Lourdes

Ribeiro

de

Souza

da

Cunha

a,∗

a_{DepartmentofMicrobiologyandImmunology,BiosciencesInstitute,UNESP–UnivEstadualPaulista,SãoPaulo,Brazil}

b_{DepartmentofTropicalDiseasesandImagingDiagnosis,BotucatuSchoolofMedicine,UNESP–UnivEstadualPaulista,}

SãoPaulo,Brazil

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Articlehistory:

Received6February2013 Accepted15November2013 Availableonline3January2014

Keywords: Staphylococcusaureus MRSA SCCmec BEC

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TheaimofthisstudywastoperformSCCmectypinginStaphylococcusaureusisolatesand tocharacterizetheclonalprofileoftheseisolates.Forty-sixmecAgene-positivestrains iso-latedbetween2002and2006weresubmittedtoantimicrobialresistancetestingbytheE-test, SCCmectypingbymultiplexPCR,andclonalprofileanalysisbypulsed-fieldgel electrophore-sis.Forty-one(89.1%)isolatesweretypedasSCCmecIIIandfive(10.9%)asSCCmecIV.Four circulatingclonesweredetected,oneofthemcomprisingisolatesrelatedtotheBrazilian epidemicclone.Thisclonewasdetectedthroughoutthestudyperiod.TheSCCmecIII iso-lateswereassociatedwithahighrateofmultidrugresistanceandclonaldisseminationof methicillin-resistantS.aureusinthewardsoftheUniversityHospitaloftheBotucatuSchool ofMedicine,UniversidadeEstadualPaulista.

©2013 ElsevierEditoraLtda.Allrightsreserved.

Staphylococcus aureus is a major human pathogen, which

hasbeenrelated tonumerouspathologicalprocesses, such as food poisoning, pneumonia, bacteremia, impetigo, folli-culitis, and osteomyelitis.1 _{At present, this microorganism} is the main causative agent of nosocomial infections. In Brazil,approximately 20% ofbacteremias are caused by S.

aureus.2_{SimilarrateshavebeenreportedinseveralEuropean}

countries,indicatingthatitisimportanttostudytheroleof thismicroorganisminhealthcare-associatedinfections.3,4

Oxacillinisused asaresistancemarkerfor methicillin-resistantS.aureus(MRSA),sincestrainsresistanttooxacillin

∗ _{Correspondingauthorat:DepartamentodeMicrobiologiaeImunologia,InstitutodeBiociências,UNESP–UniversidadeEstadualPaulista,}

SãoPaulo,Brazil.

E-mailaddresses:cunhamlr@ibb.unesp.br,c.lurdinha@hotmail.com(M.deLourdesRibeirodeSouzadaCunha).

are often multiresistant. Approximately 30–50%ofhospital

S. aureus isolates are oxacillin-resistant, a fact that leaves

fewtreatmentoptions.5–7 ThemecAgeneisthemain mech-anism responsible for methicillin resistance. This gene is carriedbythecassettechromosomemec(SCCmec),amovable geneticelementthatconsistsofincisionandexcisiongenes (ccrcomplex),inadditiontogenesthatencoderesistanceto antimicrobials and heavymetals.8 _{Eleven differentSCCmec} typeshavebeendescribed sofar,9 _{whichdifferintermsof} thenumberofgenesthattheycarryandgenearchitecture.In addition,differentSCCmectypesareassociatedwithhospital

1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2013.11.003

orcommunityisolates,afactmakingSCCmectypingan impor-tantepidemiologicaltool.10_{SomeoftheseSCCmectypescarry} genes encoding multidrug resistance, including resistance to beta-lactams, macrolides, lincosamines, streptogramins, aminoglycosides,and tetracycline.Hence,if abacterialcell acquiressuchSCCmec,itconcomitantlyacquiresamultidrug resistancephenotype.11

In Brazil, oxacillin resistance rates are high, with an incidence of30–50% depending on the region studied.12–14 However, little isknown about the dissemination of these clonalisolates,althoughstudiesontheclonalprofileofMRSA infectionshavebeenconductedinothercountries.Fivelarge pandemic clonesof MRSAisolateshave been identified by epidemiologicaltechniques.Recentfindingspermittedusto confirm important changes in the epidemiology of MRSA infectionsinBrazilianhospitals.Atthebeginningofthepast decade,theBrazilianepidemicclone(BEC)/typeIII/ST239was themainlineageinBrazilianhospitals.15_{Overrecentyears,} typeIVisolateshaveemergedinhospitalsettingsandhave becomeacommontypeofMRSAfoundininpatients world-wide.Studies conductedinRiode Janeiro,Brazil,identified USA400/type IV/ST1 followed by USA800/ST5 as the main lineages.16–19 Antimicrobialresistanceand virulencefactors areuniquetoeachclone.

Sinceoxacillinresistancehasbecomeamajorproblemin hospitalswhichleavesfewtherapeuticoptionsandsincethe presenceofpandemicclonesindicatesclonaldissemination ofMRSAisolates, theaimofthe presentstudy wasto per-formmolecularcharacterizationofMRSAstrainsisolatedfrom theUniversity Hospitalofthe Botucatu SchoolofMedicine (HCFMB),UNESP.

Forty-sixmecAgene-positiveS.aureusstrains(MRSA) iso-latedfrombloodculturesofpatientshospitalizedindifferent wardsofHCFMB-UNESPbetween2002and 2006and stored intheculturecollectionofthe DepartmentofMicrobiology andImmunology,UNESP,werestudied.Thestrainswere iso-latedusingthestandardmicrobiologicaltechniquesdescribed by Koneman et al.20 _{In this study, the isolates of the 30} HCFMB-UNESP wards were divided into complexes accord-ingto specialties:InternalMedicineWards, SurgeryWards, PediatricWards,GynecologyandObstetrics,EmergencyRoom, IntensiveCareUnits,andotherwards.

The in vitro susceptibility of the isolates to the

follow-ing drugs was tested: oxacillin, netilmicin, erythromycin, sulfamethoxazole-trimethoprim, and vancomycin. For this purpose,theminimalinhibitoryconcentration(MIC)ofthese drugswasdeterminedbytheE-test.Theisolateswere clas-sifiedassensitiveorresistantaccordingtothecut-offlevels (␮g/mL)recommendedbytheCLSI.21

SCCmec typing was performed by multiplex PCR as described byMilheiric¸o et al.22 _{TheS.aureus isolateswere} typedbyPFGEaccordingtothemodifiedprotocolofMcDougal etal.23 _{Amongthe46 mecAgene-positiveS.aureusisolates,} 23 were selected based on a similar profile of MIC deter-minedwiththe E-teststrip.Theisolateswere divided into groups accordingto the MIC obtainedfor all antimicrobial agentstested andone isolaterepresentative ofeachgroup wasselected. All S. aureusstrains carryingSCCmec typeIV were submittedto typing byPFGE. SmaI (Fast DigestSmaI, FermentasLifeScience,Canada)wasusedforgenomicDNA

restriction. Electrophoresis was performed on 1% agarose gelpreparedwith0.5MTBE (PulsedFieldCertifiedAgarose, BioRad Laboratories, USA) using the CHEF-DR III System (BioRad Laboratories) underthe followingconditions:pulse time interval of 5–40s for 21h on a linear ramp; 6V/cm; 120◦ angle; 14◦ C; 0.5M TBE as running buffer. Lambda Ladder PFG Marker (New England BioLabs, UK) was used as amolecular weightmarker. The gelswere stained with GelRed (10,000× in water, Biotium, USA) for 1h and pho-tographedunderUVtransillumination.Forsimilarityanalysis, theDicecorrelationcoefficientwasestimatedanda dendro-gram wascreated bytheUPGMA method(unweightedpair group methodusing arithmeticaverages)using the BioNu-merics6.1software(AppliedMaths,Belgium).Bandposition toleranceand optimizationwereset at1.5and 1%, respec-tively.Asimilaritycoefficientof80%waschosenforcluster definition.

Amongthe46mecAgene-positiveS.aureusstrainsisolated frombloodculturesbetween2002and2006,41(89.1%)were typedasSCCmecIIIandfive(10.9%)asSCCmecIV.Most(n=14) ofthe41 SCCmecIIIisolateswere identifiedintheInternal Medicinecomplex,followedbynineisolatesinotherwards, eightisolatesintheSurgerycomplex,sevenisolatesinthe Emergencycomplex,twoisolatesintheIntensiveCareUnit complex,andoneisolateintheNurserycomplex.SCCmectype IVwasdetectedintwoisolatesfromtheEmergencycomplex andinoneisolateeachfromtheSurgery,InternalMedicine andIntensiveCareUnitcomplexes.

Resistance to the drugs tested varied according to SCCmec type. A higher percentage of isolates resis-tant to erythromycin (n=40), netilmicin (n=38) and trimethoprim–sulfamethoxazole(n=39)wasobservedamong SCCmec III MRSA. An association was observed between multidrugresistanceandpresenceoftheSCCmecIIIcassette, with38 (92.7%)isolatesbeingresistant tothreeofthe four drugstestedandonlyoneisolatebeingresistanttotwo,one andnoneofthedrugs,respectively.FourofthefiveSCCmecIV isolatesweresusceptibletothedrugstestedandonestrain was resistant to erythromycin. SCCmec III MRSA predomi-natedinallyearsstudied;however,SCCmecIVisolateswere notdetectedin2005.

SusceptibilitytovancomycinvariedaccordingtoSCCmec cassettetype.TheMIC50andMIC90ofvancomycinforSCCmec IVisolateswere2and3␮g/mL,respectively.Thesevalueswere higher than thoseobtainedforSCCmec III isolates(1.5 and 2␮g/mL,respectively).Nodecrease invancomycin suscepti-bilitywasobservedinSCCmecIIIandIVisolatesoverthestudy period.AmongSCCmecIIIisolates,theMIC50ofvancomycin showedaslightdecreasefrom2002(2␮g/mL)to2003(1␮g/mL), followedbyaslightincreasein2004(1.5␮g/mL),andremained stablein2005and2006(2␮g/mL).ForSCCmecIVisolates,MIC valuesdecreasedfrom2002to2003and remainedstablein subsequentyears.

AnalysisofSCCmecIIIisolatesbyPFGErevealedthe pres-ence offour clones.These clonesweredivided based ona similaritycoefficient≥80%andidentifiedwithcapitalletters. There wasapredominanceofcloneA,comprisingfive iso-lates. When all isolateswere analyzed,16 S.aureusstrains isolatedfromtheInternalMedicine,Surgery,Emergencyand other wardscomplexeswereincludedinthisclone(Table1

Table1–DistributionofclonesofMRSAisolatescarryingSCCmectypeIIIaccordingtowardcomplex.

Wardcomplex

Surgery Internalmedicine Otherwards Emergency ICU

N % N % N % N % N % CloneA 5 100 3 42.9 6 75 1 25 1 50 CloneB 0 0 1 14.3 0 0 1 25 0 0 CloneC 0 0 2 28.6 1 12.5 2 50 1 50 CloneD 0 0 1 14.3 1 12.5 0 0 0 0 Total 5 100 7 100 8 100 4 100 2 100

Clone:identifiedinthedendrogramofSCCmectypeIIIStaphylococcusaureusgeneratedbyDice/UPGMAanalysis(Bionumerics,AppliedMaths) ofPFGEprofiles(SmaI)(similarity≥80%).

N:numberofMRSAisolatesbelongingtotherespectiveclone.

Surgerycomplex:surgery,cardiacsurgery,thoracicsurgery,gastricsurgery,orthopedicsurgery,andurologicsurgerywards.

Internalmedicine:dermatology,gastrointestinalclinic,internalmedicine,intensivecareunit,cardiology,cardiologyclinic,hematologyand pulmonologywards.

Otherwards:chemotherapy,oncology,infectiousandparasiticdiseases,neurology,anddialysiswards. Emergency:emergencyroomandemergencyroom-intensivecareunit.

ICU:intensivecareunitincludingintensivecareunit,centralintensivecareunitandcoronaryintensivecareunit.

andFig.1).ThisclonepredominatedintheSurgeryandother wardscomplexes.

ThereferencestrainHU25CEB,whichbelongstothe Brazil-iancloneofpandemiccirculation(BEC),wasalsoincludedin cloneA,suggestingarelationshipbetweentheseisolatesand theBrazilianepidemicclone.Strains isolatedbetween2002 and2006wereincludedinthis clone(Fig.1).CloneB com-prisedtwostrainsisolated from the InternalMedicineand Emergencycomplexes between2002and2004(Table1and

Fig.1).CloneCcomprisedthreeisolates(Fig.1).However, anal-ysisofMICbytheE-testresultedintheinclusionofsixstrains isolatedin2002,2004,2005and2006fromtheIntensiveCare Unit,EmergencyandotherwardscomplexesincloneC,with ahigherprevalenceintheInternalMedicineandEmergency complexes(Table1andFig.1).CloneDcomprisedonlytwo strainsisolatedin2006fromtheInternalMedicineandother wardscomplexes(Table1andFig.1).Twentyisolatesexhibited apolyclonalprofile.

Key Smal

Smal

50 60 70 80 90 100 SCCmec Complex of wards

Internal medicine Internal medicine Internal medicine Internal medicine Internal medicine Internal medicine Internal medicine Internal medicine Emergency Emergency Other wards

A

B

C

D

Other wards Other wards Pediatric wards Emergency Internal medicine Surgery wards Surgery wards Surgery wards 30091/05 III III III IIIa III III III III III III III III III III III III III III III III III III 31597/04 33597/02 HU 25 847/02 30195/04 3373-86/05 32956/04 396/02 31431/04 538/02 30139/04 1369/02 30151/05 85/2082 ANS 46 932/06 1308/06 1594/06 326/06 358/02 30052/05 89.8 88.0 86.6 82.1 78.5 76.9 90.9 69.3 61.4 59.9 53.1 12.1 60.0 92.3 47.7 66.1 81.0 87.0 90.0

Fig.1–DendrogramofSCCmectypeIIIStaphylococcusaureusisolatesgeneratedbyDice/UPGMAanalysis(Bionumerics, AppliedMaths)ofPFGEprofiles(SmaI)(similarity≥80%)andcomparisonwithinternationalMRSAclonesharboringSCCmec typeIII.TheinternationalcloneswerekindlyprovidedbyDr.AntonioCarlosCamposPignatari,LaboratórioEspecialde MicrobiologiaClínica,DisciplinadeInfectologia,UniversidadeFederaldeSãoPaulo-EscolaPaulistadeMedicina,andDr. AgnesMarieSáFigueiredo,InstitutodeMicrobiologiaProf.PaulodeGóes,UniversidadeFederaldoRiodeJaneiro,Brazil.

TheSCCmecIVisolatesshowedlowclonalityandonlytwo strainsisolatedin2002and2004fromtheInternalMedicine andIntensiveCareUnitcomplexescouldbeclustered.

ThepresentstudyshowedapredominanceoftheSCCmec

IIIcassetteinS.aureus.Thisfactemphasizestheimportance ofappropriateantibiotictherapysincenosocomial

Staphylo-coccusspp.isolatesareknowntobeassociatedwithmultidrug

resistance.TherewasastrongassociationbetweenSCCmec IIIisolatesandmultidrugresistance,whichcanbeexplained bythenosocomialoriginoftheseisolates.Thedrugofchoice forthetreatmentofinfectionswithmultiresistantMRSAand

S.aureusisvancomycin;however,vancomycin-resistant

iso-latesorisolateswithintermediate susceptibilityhavebeen identified.There arealsostudies describingdecreased sus-ceptibilityofhospitalisolatestovancomycin.24,25 _Decreased vancomycin susceptibilitywas notobservedinthe present study,withtheMIC50andMIC90valuesofSCCmecIIIMRSA isolatesbeingstableoverthestudyperiod.However,an asso-ciationbetweenthepresenceofSCCmecIIIandisolateswith intermediatevancomycinresistancehasbeenreported.26

Analysis of the prevalence ofSCCmec cassette types in MRSAisolatesoverthestudy periodshowedanincreasein thenumber ofSCCmecIII from2002to2005,followed bya decreasein2006.TheprevalenceofSCCmecIVwaslowand stablebetween2002and2004andthistypewasdetectedagain onlyin2006.

Inviewoftheincreaseinthenumberofoxacillin-resistant

Staphylococcusspp.andtheemergenceofheteroresistantand

vancomycin-resistantisolates,studiesinvestigatingthe dis-seminationoftheseisolatesinhospitalsareneeded.PFGEhas beenusedsuccessfullyforthispurpose.Inthepresentstudy,

23 S. aureusisolates were typed bythis method. Although

a high degree of clonality has been reported for Brazilian nosocomialMRSA,27 _{onlyfour cloneswere detectedinthe} present study, with the circulation of isolates in various wardsandprevalenceduringthestudiedyears.CloneA com-prisedthelargestnumberofSCCmecIIIisolates,whichwere isolatedbetween2002and2006fromthefollowingward com-plexes:Surgery,InternalMedicine,otherwards,Emergency, andIntensiveCareUnit.TheHU25strain,whichbelongsto BEC,showedasimilarityofmorethan80%tocloneA.Hence, allisolatesbelongingtocloneAarerelatedtoBEC.The Brazil-ianclonepredominatesinhospitalsinallregionsofBrazil,in SouthAmericaandinsomeEuropeancountriessuchas Por-tugal,accountingfor70%ofMRSAisolatedfromhospitals.15_A highprevalenceofBEC-likeisolateswasdetectedinhospitals inthenortheasternandsoutheasternregionsofBrazil.28,29_In thepresentstudy,isolatesbelongingtocloneAwere identi-fiedinallyearsstudied, afinding thatcanbeexplainedby thefactthatcloneAhasbeenthepredominantclonein vari-ousBrazilianhospitalssincethe1990s.29,30_{Althoughtheclone} relatedtoBEChasbeenthemostpredominant,itsprevalence wasnothomogeneous,sinceothercloneswerefoundinthis study,whichcanmeanareplacementinthefuturecirculation oftheBECclonebyotherclones.

TheisolatescharacterizedasSCCmec IVshoweda poly-clonalprofile.However,PFGEpermittedtheidentificationofa clonethatdisseminatedin2002and2004.Isolatesofthisclone wererecoveredfromtheCardiologyandIntensiveCareUnit wards.ThisclonedidnotshowsimilaritytostrainsUSA800or

HAR24,whichbelongtothe pediatricandEMRSA-15clone, respectively, and are characterized by the presence of the SCCmecIVcassette.AlthoughSCCmecIVisolatesdidnot pre-dominate inthis study,replacementofBECwithEMRSA-15 hasbeenreportedinPortugal.31_{AhighpercentageofSCCmec} IVMRSAisolates(46%)was describedinaSwissuniversity hospital,withthepredominanceoffourmajorclones.This findingcanbeexplainedbythereplacementofhospitalclones withpediatricclonesinhealthcare-associatedinfections or byamassiveprevalenceofisolatesofcommunityorigin.32_In Brazil,threestrainsgeneticallyrelatedtocloneUSA800were isolatedinthenortheasternregion,afindingsuggestingthe introductionofthiscommunity-acquiredcloneintohospital settings.28_{StudiesconductedinRiodeJaneirosuggestthat,in} Brazil,USA400/typeIVisolatesofcommunityoriginhave grad-uallyreplacedBEC/typeIIIinthehospitalenvironment.16–19 However,onlyoneSCCmecIVS.aureusstrainisolatedfromthe Emergencywardshowedasimilarityof60.9%withthisclone, indicatingthattheyaredifferentclones.Thehighprevalence ofBECisolatesinthepresentstudymightbeexplainedbythe collectionofS.aureusstrainsincludedinthestudy(2002–2006). Furtherstudiesneedtobeconductedatthishospitalto deter-mine whetherachangeisoccurringinthe clonalprofileof isolates.

The present results confirm the importance ofthe epi-demiological characterizationof MRSAisolatesconsidering thehighratesofmultidrugresistanceamongnosocomialS.

aureusisolatesandclonaldisseminationofMRSAisolatesin

thewardsofHCFMB-UNESP.

Funding

ThisstudywassupportedbyFAPESP(Fundac¸ãodeAmparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho NacionaldeDesenvolvimentoCientíficoeTecnológico).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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1.KloosWE,BannermanTL.StaphylococcusandMicrococcus.In: MurrayPR,BaronEj,PfallerMA,TenoverFC,YolkenRH, editors.Manualofclinicalmicrobiology.Washington,DC: ASMPress;1995.p.282–98.

2.GalesAC,SaderHS,RibeiroJ,ZoccoliC,BarthA,PignatariAC. AntimicrobialsusceptibilityofGram-positivebacteria isolatedinBrazilianhospitalsparticipatingintheSENTRY program(2005–2008).ClinInfectDis.2009;13:90–8. 3.JohnsonAP.Methicillin-resistantStaphylococcusaureus:the

Europeanlandscape.JAntimicrobChemother.2011;66Suppl. 4:iv43–8.

4.GoosensH.Europeanstatusofresistanceinnosocomial infections.Chemotherapy.2005;51:177–81.

5.VindelA,CuevasO,CercenadoE,etal.Staphylococcusaureusin Spain:molecularepidemiologyandutilityofdifferenttyping methods.JClinMicrobiol.2009;47:1620–7.

6.MartinsA,PereiraVC,CunhaM,MariadeLourdesdeL. OxacillinresistanceofStaphylococcusaureusisolatedfromthe

universityhospitalofBotucatuMedicalSchoolinBrazil. Chemotherapy.2010;56:112–9.

7. ZhanelGG,AdamHJ,LowDE,etal.CanadianAntimicrobial ResistanceAlliance(CARA).Antimicrobialsusceptibilityof 15,644pathogensfromCanadianhospitals:resultsofthe CANWARD2007–2009study.DiagnMicrobiolInfectDis. 2011;69:291–306.

8. MartinsA,deLourdesRibeirodeSouzaDaCunhaM. MethicillinresistanceinStaphylococcusaureusand coagulase-negativestaphylococci:epidemiologicaland molecularaspects.MicrobiolImmunol.2007;51:787–95. 9. Internationalworkinggroupontheclassificationof

staphylococcalcassettechromosomeelements(IWG-SCC); 2011.Availablefrom:http://www.sccmec.org/Pages/SCC TypesEN.html[accessed07.10.11].

10.MombachPinheiroMachadoAB,ReiterKC,PaivaRM,Barth AL.Distributionofstaphylococcalcassettechromosomemec (SCCmec)typesI,II,IIIandIVincoagulase-negative

staphylococcifrompatientsattendingatertiaryhospitalin southernBrazil.JMedMicrobiol.2007;56:1328–33.

11.ItoT,OkumaK,MaXX,YuzawaH,HiramatsuK.Insightson antibioticresistanceofStaphylococcusaureusfromitswhole genome:genomicislandSCC.DrugResistUpdate. 2003;6:41–52.

12.SaderHS,MoetGJ,JonesRN.Antimicrobialresistanceamong Gram-positivebacteriaisolatedinLatinAmericanhospitals.J Chemother.2009;21:611–20.

13.GrotheC,daSilvaBelascoAG,deCássiaBittencourtAR, ViannaLA,deCastroCintraSessoR,BarbosaDA.Incidenceof bloodstreaminfectionamongpatientsonhemodialysisby centralvenouscatheter.RevLatAmEnfermagem. 2010;18:73–80.

14.CamposGB,SouzaSG,LobOTN,etal.Isolation,molecular characteristicsanddisinfectionofmethicillin-resistant StaphylococcusaureusfromICUunitsinBrazil.NewMicrobiol. 2012;35:183–90.

15.OliveiraDC,TomaszA,deLencastreH.Secretsofsuccessofa humanpathogen:molecularevolutionofpandemicclonesof methicillin-resistantStaphylococcusaureus.LancetInfectDis. 2002;2:180–9.

16.VivoniAM,DiepBA,MagalhãesACG,etal.Clonal compositionofStaphylococcusaureusisolatesataBrazilian UniversityHospital:identificationofinternationalcirculating lineages.JClinMicrobiol.2006;44:1686–91.

17.SchuenckRP,NouérSA,deOliveiraWinterC,etal.Polyclonal presenceofnon-multiresistantmethicillin-resistant StaphylococcusaureusisolatescarryingSCCmecIVinhealth care-associatedinfectionsinahospitalinRiodeJaneiro, Brazil.DiagnMicrobiolInfectDis.2009;64:434–41.

18.Silva-CarvalhoMC,BonelliRR,SouzaRR,etal.Emergenceof multiresistantvariantsofthecommunityacquired

methicillin-resistantStaphylococcusaureuslineage

ST1-SCCmecIVin2hospitalsinRiodeJaneiro,Brazil.Diagn MicrobiolInfectDis.2009;65:300–5.

19.CabocloRMF,CavalcanteFS,IorioNLP,etal.Staphylococcus aureusinRiodeJaneirohospitals:disseminationofthe USA400/ST1andUSA800/ST5SCCmectypeIVandUSA100/ST5

SCCmectypeIIlineagesinapublicinstitutionandpolyclonal presenceinaprivateone.AmJInfectControl.2013;41:e21–6. 20.KonemanEW,WinnWC.DiagnósticoMicrobiológico:Textoe

AtlasColorido.6thed.RiodeJaneiro:Guanabara-Koogan; 2008.p.1760.

21.CLSI.Performancestandardsforantimicrobialsusceptibility testing.CLSIapprovedstandardM100-S15.Wayne,PA,USA: ClinicalandLaboratoryStandardsInstitute;2011.

22.Milheiric¸oC,OliveiraDC,deLencastreH.Updatetothe multiplexPCRstrategyforassignmentofmecelementtypes inStaphylococcusaureus.AntimicrobAgentsChemother. 2007;51:3374–7.

23.McdougalLK,StewardCD,KillgoreGE,ChaitramJM, McAllisterSK,TenoverFC.Pulsed-fieldgelelectrophoresis typingofoxacillin-resistantStaphylococcusaureusisolates fromtheUnitedStates:establishinganationaldatabase.J ClinMicrobiol.2003;41:5113–20.

24.SteinkrausG,WhiteR,FriedrichL.VancomycinMICcreepin non-vancomycin-intermediateStaphylococcusaureus(VISA), vancomycin-susceptibleclinicalmethicillin-resistantS. aureus(MRSA)bloodisolatesfrom2001–05.JAntimicrob Chemother.2007;60:788–94.

25.DhawanB,GadepalliR,RaoC,KapilA,SreenivasV.Decreased susceptibilitytovancomycininmethicillin-resistant

Staphylococcusaureus:a5yearstudyinanIndiantertiary hospital.JMedMicrobiol.2010;59:375–6.

26.HsuehPR,LeeSY,PerngCL,ChangTY,LuJJ.Clonal disseminationofmethicillin-resistantand vancomycin-intermediateStaphylococcusaureusina Taiwanesehospital.IntJAntimicrobAgents.2010;36:307–12. 27.CuryGG,MobilonC,StehlingEG,etal.Moleculartypingof

methicillin-resistantStaphylococcusaureus(MRSA)strains isolatedintwometropolitanareasofSãoPauloState, southeastBrazil.BrazJInfectDis.2009;13:165–9. 28.Sousa-JuniorFC,Silva-CarvalhoMC,FernandesMJ,etal.

Genotypingofmethicillin-resistantStaphylococcusaureus isolatesobtainedintheNortheastregionofBrazil.BrazJMed BiolRes.2009;42:877–81.

29.TeixeiraMM,AraújoMC,Silva-CarvalhoMC,etal.Emergence ofclonalcomplex5(CC5)methicillin-resistantStaphylococcus aureus(MRSA)isolatessusceptibleto

trimethoprim–sulfamethoxazoleinaBrazilianhospital.BrazJ MedBiolRes.2012;45:637–43.

30.ReinertC,McCullochJA,WatanabeS,ItoT,HiramatsuK, MamizukaEM.TypeIVSCCmecfoundindecadeoldBrazilian MRSAisolates.EmergInfectDis.2008;12:1995–8.

31.AmorimML,FariaNA,OliveiraDC,etal.Changesinthe clonalnatureandantibioticresistanceprofilesof

methicillin-resistantStaphylococcusaureusisolatesassociated withspreadoftheEMRSA-15cloneinatertiarycare Portuguesehospital.JClinMicrobiol.2007;45:2881–8. 32.ValsesiaG,RossiM,BertschyS,PfyfferGE.Emergenceof

SCCmectypeIVandSCCmectypeVmethicillin-resistant StaphylococcusaureuscontainingthePanton-Valentine leukocidingenesinalargeacademicteachinghospitalin centralSwitzerland:externalinvadersorpersisting circulators.JClinMicrobiol.2010;48:720–7.