Molecular characterization of methicillin-resistant Staphylococcus aureus isolated from blood in Rio de Janeiro displaying susceptibility profiles to non-β-lactam antibiotics

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Medical

Microbiology

Molecular

characterization

of

methicillin-resistant

Staphylococcus

aureus

isolated

from

blood

in

Rio

de

Janeiro

displaying

susceptibility

profiles

to

non-

␤-lactam

antibiotics

Alexandra

Vidal

Pedinotti

Zuma

_,

_Danielle

_Ferreira

_Lima

_,

Ana

Paula

D’Alincourt

Carvalho

Assef

_,

_Elizabeth

_Andrade

_Marques

_,

Robson

Souza

Leão

a_{UniversidadedoEstadodoRiodeJaneiro,DepartamentodeMicrobiologia,ImunologiaeParasitologia,RiodeJaneiro,RJ,Brazil} b_{InstitutoOswaldoCruz,LaboratóriodePesquisaemInfecc¸ãoHospitalar(LAPIH),RiodeJaneiro,RJ,Brazil}

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Articlehistory:

Availableonline21December2016 AssociateEditor:AgnesFigueiredo

Keywords: MRSA MLST USA800 USA400 USA1100

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The distinction between healthcare-associated MRSA (HA-MRSA) and community-associatedMRSA(CA-MRSA)infectionshasbecomeincreasinglyblurred.Weassessedthe molecular characterization andantimicrobialresistance profile forMRSAisolatesfrom blood.Mostofall(81.9%)isolatesarerelatedtoknownHA-MRSAandCA-MRSAepidemic lin-eages,suchas,USA300,USA400,USA600,USA800andUSA1100.Thisisthefirstmulticenter studyinRiodeJaneiro.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Overtheyears,thedistinctionbetweenhealthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) has become increasingly blurred, with a growing number of reports indicating that CA-MRSA strains are spread in hospital settings and replacing traditional HA-MRSAstrains,includinginBrazil.1,2_{HA-MRSAstrainstypically}

have a multidrug-resistant phenotype, whereas CA-MRSA strains are often susceptible to a variety of non-␤-lactam antimicrobials.1_{Ontheotherhand,thisresistancepatternis}

∗ _{Correspondingauthorat:DisciplinadeMicrobiologiaeImunologia,FaculdadedeCiênciasMédicas,UniversidadedoEstadodoRiode}

Janeiro,Avenida28deSetembro,87/3◦_{andar20551-030VilaIsabel,RiodeJaneiro,RJ,Brazil.}

E-mail:robson.leao@uerj.br(R.S.Leão).

1 _{Theseauthorshaveequallycontributedforthisstudy.}

changinginthe lastdecade.Thus, someHA-MRSAisolates are susceptible to many non-␤-lactam antibiotics, includ-ing the isolates related USA800 clone, forexample.1,3 The

in vitro antimicrobial susceptibility to clindamycin and/or sulfamethoxazole/trimethoprim is a relevant characteristic describedinstrainswithSCCmecIV.2_{Thegenotypingof}

Brazil-ian MRSA isolates allowed a better understanding of the disseminationpatternsoftheepidemicclones,suchas, typ-icallyHA-MRSAandCA-MRSA.4_{Theknownvirulencefactor}

http://dx.doi.org/10.1016/j.bjm.2016.09.016

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

the exoprotein Panton-Valentine leucocidin (PVL) is tradi-tionally seen in CA-MRSA,5 _{however it has already been}

identifiedinmethicillin-susceptibleS.aureusandsomerare HA-MRSAisolates.1,6_{Theincreasingnumberofreported}

infec-tions causedbyCA-MRSAstrains inBraziljustifies theuse ofsurveillance studiestohelpinunderstandinghow these strainsarriveandcirculateinhealthcaresettings.2

Further-more, recently,Rossi and colleagues7 _{reported a case of a}

BrazilianpatientwithabloodstreaminfectioncausedbyUSA 300-related isolate, designated BR-VRSA, that acquired the

vanAgeneclusterduringantibiotictherapybecoming resis-tanttovancomycin.

Thepresent studyanalyzedthegeneticbackgroundand resistance profile of MRSA isolates recovered from blood betweenMarch2011andMay2012.Theinclusioncriterium for the MRSA isolates studied here was the presence of susceptibilitytothenon-␤-lactamantibiotics trimethoprim-sulfamethoxazole and/or clindamycin.The 61 MRSA blood isolateswerecollectedfrompatientsinfivehospitals(H)inRio deJaneiro,Brazil:H1(n=11;18.1%),H2(n=14;22.9%),H3(n=16; 26.2%),H4(n=7;11.5%)andH5(n=13;21.3%),thelastoneis apediatricunit.Usingdiskdiffusiontest,8_{allMRSAisolates}

selecteddisplayedhighpercentageofsusceptibilitytonon- ␤-lactams.2,8_{Thefollowingresistancerateswasobserved:59.0%}

forclindamycin(CLI),78.7%forerythromycin(ERI)and60.6% forciprofloxacin(CIP).Lowerresistancerateswereobserved forchloramphenicol (CLO),19.7%;gentamicin(GEN),16.4%; rifampicin(RIF),8.2%;tetracycline(TET),11.5%; trimethoprim-sulfamethoxazole(SUT)andlinezolid(LIN),4.9%.Resistance tothreeor morenon-␤-lactam antibioticswasobserved in 55.7%ofthetotalisolatesandonlyinH3wasobservedstrains resistanttoSUT.Noneoftheisolateswereresistantto van-comycin(VAN)byMICdetermination,9_{withaconcentration}

rangingfrom0.25to2␮g/mL.ThemajorityofMRSAisolates (n=45/73.8%)showedMIC of1.0␮g/mL,followingbyMICof 2.0␮g/mL (n=14/22.9%). SCCmec types were carried out by multiplexPCRanalysis thatgenerateda specific amplifica-tionpattern,thatincludesSCCmectypesI,II,III,IVandV,as previouslydescribed.10_{Atotaloftwo(3.3%)MRSAisolates}

con-tainedSCCmecI,58(95.1%)SCCmecIVandone(1.6%)harbored SCCmecIIelement.Two isolatesthat containingSCCmec IV, allSCCmecIandIIexhibitedmultidrugresistance(resistance tothreeormoreclassesofnon-␤-lactamantimicrobialdrugs). Molecularcharacterizationbasedonmultilocussequence typ-ing (MLST)11 _{identified 9 STs previously described among}

MRSAisolates(ST1,ST5,ST8,ST12,ST30,ST45,ST105,ST435, ST1635). Three new STs (ST3163, ST3164 and ST3165) and threenewallelicarrangementswereidentifiedandaddedto themlst.netdatabase.MLSTresultsshowedthatmostofthe isolates(81.9%)belongedtoknownepidemiclineages.The lin-eagesrelatedwiththeclonesUSA800/Pediatric(ST5,SCCmec IV), USA400/MW2/WA-1 (ST1, SCCmec IV), USA1100/OSPC (ST30,SCCmec IV),USA300/USA300-LV(ST8,SCCmecIV),UK EMRSA-3/Cordobes/Chilean (ST5, SCCmec I), USA600/Berlin (ST45, SCCmec IV) and USA100/NYJ (ST5, SCCmec II) were observedinapproximately27.8%,26.2%,18%,3.3%,3.3%,1.6% and1.6%ofisolates,respectively.HA-MRSAandCA-MRSA iso-latestypicallyfoundinBrazil,relatedtoUSA800andUSA400, respectively, were identified in all hospitals (at least two

isolates/hospital).IsolatesrelatedtotheOceaniaSouthwest Pacificclone(OSPC/USA1100)werealsoobservedinall hospi-tals,exceptH4.Beside,oneisolate(newST3165)belongedto thesameclonalcomplexasOSPC(CC30)wasfoundintheH4

(Table1).PCR-amplificationproduct12compatiblewiththePVL

geneslukSandlukFwasdetectedin11(18%)oftheMRSAblood isolates.ThosegeneswererelatedtoMRSAharboringSCCmec IV. Lookingattheepidemiclineage,PVLgenewasfoundin isolatesrelatedtoOSPC(n=8/72.7%).

In recent years, many authors have reported the pres-ence oftypicallyCA-MRSA strainscirculating inhealthcare environments,2 _{including Brazil.}13,14 _{The rising frequency}

of CA-MRSA has several implications innosocomial infec-tions.CA-MRSAisolatesarerecognizedaspresentingahigher spectrum of susceptible to non-␤-lactam antibiotics when compared with HA-MRSA. However, vancomycin remains first-choice antibiotic therapy for serious infections.6 _The

increase use of this late antimicrobial drug is directly relatedtotheemergenceofvancomycin-intermediate(VISA), vancomycin-resistant(VRSA)andvariedmultidrug-resistant MRSA isolates around the world.15,16 In the present study, resistancetovancomycinwasnotdetected;howeveraMIC creep phenomenonissuggested bythehigh percentageof samplesshowingavancomycinMIC≥1.0␮g/mL.15_The

high-est MIC detected was 2␮g/mLand was observed in 22.9% of the isolates. Several authors have reported that treat-mentwithvancomycineventuallyfailsamongpatientswith MRSAbloodstreaminfectionswhichpresentedavancomycin MIC>1␮g/mL.17_{Recently,aVRSAstrainwasisolatedfroma}

bloodcultureinaHospitalinSãoPaulocity,Brazil,inwhich the patienthad previouslybeentreated withvancomycin.7

A reportfrom Rossi andcolleagues7 _{emphasizesthe value}

of effective surveillance on the MIC levels ofvancomycin, aswell asthe importanceofquickresponsebythe clinical microbiologylaboratoryinformingthe medicalteamonthe specificvancomycinMIC.Duetotheinclusioncriteriausedin thisstudy,mostoftheMRSAisolatesexhibitedSCCmectype IV(95.1%). Differently, Caiaffa-Filho18 _{studyingMRSA blood}

isolates from Brazilian patients, showed a high frequency of MRSA carrying SCCmec type II. Thelargest cassettes (I, II and III)enhance the survivalabilities of MRSAwhen in hospitalsettings.However,itisbelievedthatthesmaller cas-settes(particularlytheIV)promoteevolutionaryadvantagesin spreadingbyhorizontaltransferofthiselement,enablingthe increaseofCA-MRSAinhospitals.19_{ThusCA-MRSASCCmec}

IVfrequently showcompetitive advantagescomparedwith multidrug-resistantstrains(HA-MRSA).20_{Severalfactorsseem}

tocontributetotheseadaptiveadvantagesofCA-MRSA iso-lates,suchasthetoxinPVLproduction.5,19 _{Thepresenceof}

PVLgenesamongtheisolatescharacterizedasCA-MRSAhave beenuneven,withpercentagesofpositivityvaryingfrom27 to75%.19,21_{Inthisstudy,11(18%)isolateswerepositivetoPVL}

genesandallcontainedSCCmecIV.

Non multidrug-resistantMRSA isolatescarrying SCCmec IVhave alreadybeen identified inBrazilianhospitals,with patterns similar to USA100, USA300, USA400, USA800 and USA1100clones,4,13,14,22,23_{however,twoisolates,inthisstudy}

relatedtoUSA1100andUSA400,showedmultidrugresistance profiles.

Table1–Characteristicsandidentificationof61MRSAisolatesfromfivehospitalsinRiodeJaneirocity. ST/CC n SCCmec type Clonality Resistance profile(n) Numberisolates positivesofthe lukSF(genes) H1(n) H2(n) H3(n) H4(n) H5(n) 1/1 16 IV USA400 CIP-ERI-CLI-GEN-TET-CLO(1) – – – 1 – CIP-ERI-CLI-GEN-CLO(1) – – – – – 1 CIP-ERI-CLI-CLO-LIN(1) – 1 – – – – CIP-ERI-CLI-GEN(1) – – – – 1 – CIP-ERI-CLI(5) – – 1 2 1 1 CIP-ERI-CLO(1) – – – – – – ERI-TET(1) – 1 – – – 1 TET-CLO(1) – – – 1 – – CIP-ERI(1) – – 1 – – – CIP(1) – – – 1 – – ERI(1) – – – 1 – – Ø(1) – 1 – – – – 5/5 20 I UK-EMRSA-3 SUT-CIP-ERI-CLO(1) – – – 1 – – CIP-ERI-CLI(1) – – – 1 – – II USA100/NYJ CIP-ERI-CLI-GEN(1) – 1 – – – – IV USA800 CIP-ERI-CLI-GEN-TET-CLO(1) – – 1 – – – CIP-ERI-CLI-RIF-CLO(1) – – – – – 1 CIP-ERI-CLI-CLO(1) – – – – – 1 CIP-ERI-CLI-GEN(3) – 1 – – 1 1 CIP-ERI-CLI-RIF(1) – – – – – 1 CIP-ERI-CLO(1) – – – 1 – – CIP-ERI-CLI(3) – 2 1 – – – ERI-CLI(1) – – 1 – – – ERI(5) – 1 1 1 1 1 8/8 2 IV USA300 CIP-ERI-CLI(1) – – – – 1 – CIP-ERI(1) – – 1 – – – 12/12 1 IV – SUT-CIP-ERI-GEN-RIF-LIN(1) – – – 1 – – 30/30 11 IV OSPC CIP-ERI-CLI-CLO(1) 1 – 1 – – – CIP-ERI-CLI(2) 2 – 1 – – 1 ERI-CLI-TET(1) 1 – – 1 – – ERI-CLI(2) 1 1 – – – 1 CIP-ERI(1) – – – – – 1 Ø(4) 3 2 2 – – – 45/45 1 IV USA600/Berlin CIP-ERI-CLI-RIF-TET-LIN(1) – – – – – 1 105/5 2 IV – CIP-ERI-CLI(2) – – 1 1 – – 435/30 1 IV – Ø(1) 1 – 1 – – – 1635/5 3 IV – Ø(3) – – 1 1 1 – 3163a_{/30 1 IV – ERI-CLI(1) 1 – – 1 – –} 3164a_{/30 2 IV – SUT-CIP-ERI-GEN-RIF-TET-LIN(1) – – – 1 – –} Ø(1) – – – – – 1 3165a_{/30 1 IV – Ø(1) 1 – – – 1 –}

ST,sequencetypeobtainedbyMLST;CC,clonalcomplex;SCCmec,Staphylococcalcassettechromosomemec;H,hospital;–,notfound;a_,

STdescribedinthisstudy;CLI,clindamycin;ERI,erythromycin;CIP,ciprofloxacin;CLO,chloramphenicol;GEN,gentamicin;RIF,rifampicin; TET,tetracycline;SUT,trimethoprim-sulfamethoxazole;LIN,linezolid;Ø,isolatedwithresistanceonlyforcefoxitin;lukSFgenesencoding Panton-ValentineleucocidinweredetectedbyaPCR-basedmethod.

Theemergence of MRSA clones related to USA400 and USA800was previouslydescribed in Riode Janeiroin only twohospitals,22_{andmorerecently,MRSArelatedtoUSA400,}

isolatedfromblood,wasdescribedassociatedwithinfective endocarditisinRiodeJaneiro.24_{Thismulticenterdataindicate}

thattheseMRSAlineagesarestillcirculatingintheBrazilian healthcaresettings.

In Brazil the most common lineage of CA-MRSA is related to OSPC, especially causing community associated infections.13,23,25 _{In this study, eleven isolates (18%) were}

relatedtothisclone.ThepresenceofisolatesrelatedtoOSPC clone has already been described in hospitalized patients intwohospitalsinRiodeJaneiro,14,22 _{however, fewstudies}

whileintheothercasetheOSPCrelatedisolatewascollected fromwound.22 _{Here,OSPCrelatedisolateswereobservedin}

mostofthe studiedhospitals,though,if oneconsidersthe clonalcomplex(CC30),then,wecaninferthespreadofthese MRSAgeneticallyrelated toOSPC cloneinall hospitals.In Uruguay,aBrazilianneighboringcountry,replacementof HA-MRSAbyCA-MRSAinhospitalsettingshavebeendescribed, wherethemostcommonCA-MRSAisolatesrecoveredfrom invasiveandsuperficialinfectiondiseasesarerelatedtothe OSPCclone.20

In the present study, isolates related to USA300, UK EMRSA-3 and USA100 were rarely observed. However, a USA300-relatedisolatehasalsobeenidentifiedinonehospital inRiodeJaneirofromapneumoniacase.13

MRSAbelongingtothelineageUSA100/ST5-MRSA-IIwas observed just in one hospital. This lineage has also been observedinbloodsamplesinBrazil,highlightingthatoneof thesereportsdescribestwoisolatesofS.aureusresistantto daptomycin.27 _{InBrazil,tothebestofourknowledge,there}

arefewreportsofUSA600/ST45-MRSA-IVisolates,whichwere collectedfromskininfectioncaseandfromcatheter.23,28

Theepidemic cloneUK MRSA-3 (ST5-MRSA-I) tradition-allyclassifiedasHA-MRSAisdistributedinEuropeandLatin America,including Brazil.3,29,30 _{Using thephenotypic}

crite-riaestablishedinthisscreening,wecouldidentifyanisolate relatedtothiscloneinourcollectionofMRSAblood.Recently our group described the presence ofthis clonein respira-torysecretionsofcysticfibrosis patientsinRiodeJaneiro.1

Untilnow,thereisnoreportofUK-MRSA-3inbloodstream infections in Brazil. In conclusion, a number of different internationalclones,displayinglower levelofantimicrobial resistancetonon-␤ lactams,are circulatingindifferentRio deJaneirohospitalscausingbloodstreaminfections;including MRSAlineagesthatarenotfrequentlyobservedinourcountry. Thus,werecommendasurveillanceincludingalargernumber ofMRSAbloodisolates,fromdifferentRiodeJaneiro hospi-tals,inordertoexposetherealpictureoftheantimicrobial susceptibilityprofilesfoundamongMRSAisolates,whichcan significantlyvarydependingonthedominantlineagespresent inthestudiedhospitals.

Conflicts

of

interest

Theauthorsdeclarethatthereisnoconflictofinterest.

Acknowledgments

Wethankthepersonnelatthemicrobiologylaboratoriesof theparticipanthospitalsfortheirgenerouscollaborationin sendingustheMRSAbloodisolates.Thisworkwassupported bytheFundac¸ãodeAmparoàPesquisadoEstadodoRiode Janeiro.

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