Phenotypic and molecular characterization of resistance to macrolides, lincosamides and type B streptogramin of clinical isolates of Staphylococcus spp. of a university hospital in Recife, Pernambuco, Brazil

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

Article

Phenotypic

and

molecular

characterization

of

resistance

to

macrolides,

lincosamides

and

type

B

streptogramin

of

clinical

isolates

of

Staphylococcus

spp.

of

a

university

hospital

in

Recife,

Pernambuco,

Brazil

Jussyêgles

Niedja

da

Paz

Pereira

,

Marcelle

Aquino

Rabelo,

Jailton

Lobo

da

Costa

Lima,

Armando

Monteiro

Bezerra

Neto,

Ana

Catarina

de

Souza

Lopes,

Maria

Amélia

Vieira

Maciel

DepartmentofTropicalMedicine,UniversidadeFederaldePernambuco,Recife,PE,Brasil

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t

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o

Articlehistory:

Received30January2016 Accepted11March2016 Availableonline16April2016

Keywords: Staphylococcus Clindamycin Inducibleresistance ermgenes

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b

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Introduction:ThereisamechanismofmacrolideresistanceinStaphylococcusspp.whichalso affectsthelincosamidesandtypeBstreptograminscharacterizingtheso-calledMLSB resis-tance,whoseexpressioncanbeconstitutive(cMLSB)orinducible(iMLSB)andisencoded mainlybyermAandermCgenes.ThecMLSBresistanceiseasilydetectedbysusceptibility testingusedinthelaboratoryroutine,butiMLSBresistanceisnot.Therapywithclindamycin incasesofinfectionwithisolatediMLSBresistancemayfail.

Objective:Tocharacterizethephenotypic(occurrenceofcMLSBandiMLSBphenotypes)and molecular(occurrenceofermAandermCgenes)profilesofMLSBresistanceofclinicalisolates ofsusceptibleandmethicillin-resistantStaphylococcusaureusandCNS(coagulase-negative Staphylococcus)frompatientsofauniversityhospital,inPernambuco.

Methods:Theantimicrobialsusceptibilityof103isolateswasdeterminedbythedisk diffu-siontechniqueinMueller–Hintonagarfollowedbyoxacillinscreening.TheiMLSBphenotype wasdetectedbyDtest.IsolateswithcMLSBandiMLSBphenotypesweresubjectedto poly-merasechainreaction(PCR)forthedetectionofermAandermCgenes.

Results:ThecMLSBandiMLSBphenotypeswererespectivelyidentifiedin39(37.9%)andfive (4.9%)isolates.TheiMLSBphenotypewasfoundonlyinfour(10.8%)methicillin-susceptible S.aureusandone(4.5%)methicillin-resistantS.aureus.Inthe44isolatessubjectedtoPCR, four(9.1%)onlyermAgenewasdetected,alowerfrequencywhencomparedtoonlyermC17 (38.6%)geneandtoone(2.3%)isolatepresentingbothgenes.

Conclusion:IntheStaphylococcusspp.analyzed,theermCgenewasfoundmoreoftenthan theermA,althoughtheiMLSBphenotypehadbeenlessfrequentthanthecMLSB.Itwas importanttoperformtheDtestforitsdetectiontoguidetherapeuticapproaches.

©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-ND license.(http://creativecommons.org/licenses/by-nc-nd/4.0/) ∗ _{Correspondingauthor.}

E-mailaddress:ju-biomed@hotmail.com(J.N.d.P.Pereira). http://dx.doi.org/10.1016/j.bjid.2016.03.003

1413-8670/©2016ElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense.(http://creativecommons.org/ licenses/by-nc-nd/4.0/)

Introduction

The increasing prevalence of methicillin resistance in Staphylococcusspp. isa growingproblem.Thishasrenewed interest regarding the use ofmacrolide, lincosamides, and type B streptogramin antimicrobials for the treatment of staphylococcalinfections.Clindamycin,a lincosamide, rep-resents a common choice for some of these infections, particularlyforinfections ofthe skinand soft tissues,and analternativeincaseofintolerancetopenicillinor methi-cillinresistance.1–3_{InStaphylococcusspp.,oneoftheresistance}

mechanisms consists of ribosomal target modification, affectingmacrolides,lincosamides,andtypeBstreptogramins characterizes the so-called MLSB resistance. Its expression

can be constitutive (cMLSB) or inducible (iMLSB) and is

encoded by ermA (erythromycin ribosome methylase) and ermCgenes,whicharethemaindeterminantsofresistancein staphylococci.2,4,5

It is important to know the type of MLSB resistance

forestablishing adequate therapy, sinceStaphylococcus spp. with constitutive resistance present in vitro resistance to all macrolides, lincosamides, and type B streptogramins. In addition, in vivo therapy with clindamycin for Staphy-lococcus spp. infection with inducible resistance can select constitutiveermmutants,resultingintreatmentfailure.3,4,6,7

It is noteworthy that cMLSB resistance is easily detected

by susceptibility testing used in the laboratory routine, while iMLSB resistance is not. Using these detection

methods, Staphylococcus spp. with inducible resistance has in vitro resistance to erythromycin and susceptibility to clindamycin.5,6,8,9

Todetecttheinducibleclindamycinresistancein Staphy-lococcus spp., one of the tests recommended by the CLSI (Clinicaland Laboratory StandardsInstitute) isthe double-diskdiffusiontest(DTest)andwhentheisolatedpresentsuch resistance,theCLSIrecommendsreportingthemasresistant toclindamycin.10_{Then,dataontheantimicrobial}

susceptibil-ityareimportantinthechoiceoftherapyagainstinfections, butfalsesusceptibilityresultscanbeobtainediftheisolates arenotsubjectedtoteststhatdetectinducibleresistanceto clindamycin.11

Studies carried out intwo Brazilian stateswith clinical isolatesofStaphylococcusspp.reportedthecMLSBphenotype

asthe mostfrequent.12,13 _{Coutinhoet al.}13 _{have also}

eval-uated the occurrenceofthe erm genes amongthe isolates analyzed.However,thefrequencyofcMLSBandiMLSB

resis-tancevaries amongdifferenthospitalsand thereare other resistancemechanismsthatconferresistancetoonlyoneor twoclassesoftheMLSBcomplex.14,15

The objective of this study was to characterize the phenotypic (occurrence of cMLSB and iMLSB phenotypes)

and molecular (occurrence of ermA and ermC genes) pro-files of MLSB resistance of clinical isolates of susceptible

and methicillin-resistant Staphylococcus aureus and CNS (coagulase-negative Staphylococcus) from patients of a uni-versityhospitalinPernambuco,Brazil.Obtaininglocaldata relatingtoresistance,maybehelpfulinguidingtherapeutic approaches.

Materials

and

methods

Atotalof103clinicalisolatesweregatheredfromvarious sam-plesfrompatientsinfectedwithS.aureusorSCNofauniversity hospitalofPernambuco,Brazil,duringtheyear2012andwere stored inglycerol (25%)at−20◦_{C.To verify the purity, the}

colonieswereinoculatedintoBrainHeartInfusionbroth(BHI) andafterincubationat37◦Cfor48hwereplatedonbloodagar. Antimicrobialsusceptibilityprofile

Theantibiogramwasperformedbydiskdiffusiontechniquein Mueller–Hintonagar,usingantibioticclindamycin2␮g, eryth-romycin15␮g,cefoxitin30␮g,andoxacillin1␮g.Theresults were interpretedaccordingtothestandards determinedby CLSI.10

Screeningforoxacillinresistance

Isolateswithresistanceorintermediateresistancetooxacillin and/orcefoxitinweresubmittedtooxacillinscreening,as pro-posedbyRabeloetal.16

Dtest

S.aureusand SCNisolateswithresistancetoerythromycin andsusceptibilityorintermediateresistancetoclindamycin inthe antibiogramwere selected. Forthe executionofthis testadiskof2␮gofclindamycinwasplacedatadistanceof 15mm–26mmfromtheedgeofadiskof15␮goferythromycin inaplatecontainingMueller–Hintonagarsowninthesame wayasitwasfortheantibiogram.Afterincubationat35◦Cfor 16–18h,isolatesthatshowednoflatteningoftheinhibition zonearoundtheclindamycindiskwerereportedas suscepti-bletoclindamycin(negativeDtest)andisolatesthatshowed flatteningoftheinhibitionzonearoundtheclindamycindisk adjacenttoerythromycindisk(“D”zone)indicatedinducible clindamycinresistance(positiveDtest).10

ExtractionoftotalDNA

ToexaminethepresenceofermAandemrCgenes,therapid extractionofthetotalDNAofisolatesthatshowedphenotypes MLSBcandMLSBiwasperformedbyamodifiedtechniqueof

thermallysisdirectlyfromthecolony,accordingtoHuetal.,17

afterinoculationofacolonyofeachisolateinto5mLofBHI andincubatedat37◦Cfor24h.

Polymerasechainreaction(PCR)conditions

PCRwasperformedusingtheprimersdescribedbyLinaetal.18

for ermAand ermCgenes. For the detection ofermA gene, each amplificationreactionwaspreparedinafinalvolume of25␮Lforeachtubeandincludes:1␮L(40ng)oftotalDNA, 1␮L(20pmol)ofeachprimer,0.6␮Lofdeoxyribonucleotide triphosphate (dNTP) (8mM), 5.0␮L ofbuffer (5×), 1.5␮Lof

Table1–Susceptibilityprofiletoerythromycinandclindamycinofsusceptibleandmethicillin-resistantS.aureusand CNS. Phenotypes MSSA n(%) MRSA n(%) MSCNS n(%) MRCNS n(%) Total n(%)

ERY-S,CLI-S(susceptible) 25(67.6) 5(22.7) 4(28.6) 7(23.3) 41(39.8) ERY-S,CLI-I 1(2.7) 0(0) 1(7.1) 0(0) 2(1.9) ERY-S,CLI-R 0(0) 0(0) 0(0) 0(0) 0(0) ERY-I,CLI-S 1(2.7) 0(0) 2(14.3) 0(0) 3(2.9) ERY-I,CLI-I 0(0) 0(0) 0(0) 0(0) 0(0) ERY-I,CLI-R 0(0) 0(0) 0(0) 1(3.3) 1(1) ERY-R,CLI-R(cMLSB) 4(10.8) 15(68.2) 3(21.4) 17(56.7) 39(37.9)

ERY-R,CLI-S/I,+DTest(iMLSB) 4(10.8) 1(4.5) 0(0) 0(0) 5(4.9)

ERY-R,CLI-S/I,–TestD(MSB) 2(5.4) 1(4.5) 4(28.6) 5(16.7) 12(11.6)

Total 37(35.9) 22(21.4) 14(13.6) 30(29.1) 103(100) ERY,erythromycin;CLI,clindamycin;S,susceptible;I,intermediary;R,resistant;MSSA,methicillin-susceptibleStaphylococcusaureus;MRSA, methicillin-resistantS.aureus;MSCNS, methicillin-susceptiblecoagulase-negativeStaphylococcus; MRCNS,methicillin-resistant coagulase-negativeStaphylococcus;MLSB,macrolides,lincosamidesandtypeBstreptogramins;cMLSB,phenotypeofconstitutiveMLSBresistance;iMLSB,

phenotypeofinducibleMLSBresistance;MSB,phenotypeofresistancetomacrolidesandtypeBstreptogramins.

MgCl2 (25mM), 0.4␮Lof TaqDNA polymerase (5U).For the

detectionofermCgene,eachamplificationreactionwas pre-paredinthesamemannerasforermAgene,exceptinrelation tothequantityofTaqDNApolymerase(5U),whichinthis reac-tionwas0.3␮L.Amplificationreactionswereperformedinthe thermocycleratthefollowingconditions:30cyclesof1minat 94◦Cinthedenaturingstep,30sat49◦Cintheannealingstep and30sat72◦Cintheextensionstep,accordingtothe condi-tionsdescribedbyFranc¸aetal.19withmodification.InallPCR anegativecontrolwasincluded,correspondingtoatube con-tainingallcomponentsofthemixturetowhichtemplateDNA wasnotadded.IdentifiedpositiveisolatesfortheermAand ermCgeneswereusedaspositivecontrols.ThePCRproducts weresubjectedtoelectrophoresison1.5%agarosegelin0.5× TBEbuffer.Theseproductswerestainedwithblue,visualized onanultraviolettransilluminatorandphotodocumented. SequencingofermAandermCgenes

APCR productpositive forthe ermAand ermCgenes were purifiedthroughthekit Wizard® SVGeland PCRClean-Up System(Promega),accordingtomanufacturer’sprotocoland then,quantifiedbyspectrophotometry.Thereaftertheywere submittedtosequencingandanalysisoftheresultswas per-formedthroughthesoftwaresChromasLite2.1.1,BasicLocal Alignment Search Tool(BLAST), and ExpertProtein Analy-sisSystem (ExPASy).The analyzedsequences of ermAand ermCgenesweredepositedintheGenBankwiththefollowing accessnumbers,respectively:KT599443andKU232395.

Results

Fifty-nine(57.3%)clinicalisolatesofS.aureusand44(42.7%) of SCN were analyzed, totaling 103 isolates. Of these, 37 (35.9%),22(21.4%),14(13.6%),and30(29.1%)wereclassifiedas methicillin-susceptibleS.aureus(MSSA),methicillin-resistant S.aureus(MRSA),methicillin-susceptiblecoagulase-negative Staphylococcus (MSCNS) and methicillin-resistant coagulase-negativeStaphylococcus(MRCNS),respectively(Table1).

Fig.1–PositiveDtest,showingtheflatteningofthe inhibitionzonearoundtheclindamycindiskadjacentto erythromycindisk(called“D”zone).

Thesusceptiblephenotype(ERY-S,CLI-S)wasdetectedin25 (67.6%),five(22.7%),four(28.6%),andseven(23.3%)ofMSSA, MRSA, MSCNSandMRCNS,respectively(Table1).The phe-notypecMLSB(ERY-R,CLI-R)wasdetectedrespectively,infour

(10.8%)15(68.2%),three(21.4%),and17(56.7%)ofMSSA,MRSA, MSCNSandMRCNS.TheiMLSBphenotype(ERY-R,CLI-S,

pos-itiveDtest)(Fig.1)wasfoundinonlyfour(10.8%)oftheMSSA andinone(4.5%)oftheMRSA.TheMSBphenotype(ERY-R,

CLI-S,negativeDtest)wasdetectedintwo(5.4%),one(4.5%),four (28.6%),andfive(16.7%)ofMSSA,MRSA,MSCNS,andMRCNS, respectively(Table1).

FortheidentificationoftheermAandermCgenes,44 iso-lateswithcMLSBandiMLSBphenotypesunderwentPCR.Four

(9.1%) isolatesshowed onlyermAgene, 17(38.6%)hadonly ermCgene,one(2.3%)hadbothgenes,and22(50%)didnot havethereferredgenes(Table2).

Discussion

Inthisstudy,thefrequencyofMSSA(25;67.6%)wasgreater thanMRSA(five;22.7%)andofMSCNS(four;28.6%)waslower

Table2–DistributionofermAandermCgenesamong

Staphylococcusspp.isolateswithMLSBresistance.

Genes Isolates(%) ermA 4(9.1) ermC 17(38.6) ermA+ermC 1(2.3) None 22(50.0) Total 44(100.0)

thanMRCNS(seven;23.3%).InastudyconductedinIndia,313 isolatesofStaphylococcus spp.there were 83(64.84%) MSSA and45(35.15%) MRSA,inlinetothepresent study;in con-trast,therewere124(67.02%)MSCNSand61(32.97%)MRCNS.5

Atotalof1687isolatesofStaphylococcusspp.investigatedin Turkeyconsistedof419(24.8%)MSSAand464(27.5%)MRSA, unlike this study, and the rest of the isolates, similar to thisstudy,consistedof196 (11.6%)MSCNSand608 (36.1%) MRCNS.11

The susceptible phenotype (ERY-S, CLI-S) found in 41 (39.8%)isolates in this study prevailed amongthe isolates tested.Thisphenotypewasalsoprevalentinstudiescarried outinIndia,Libya,andTurkeywherethereferredphenotype wasdetectedin192(51.5%),87(54.7%),and688(40.8%)isolates, respectively.11,20,21

Inthisstudy,the39(37.9%)cMLSBphenotypeisolates

pre-vailedoverthefive(4.9%)iMLSBphenotypeandoverthe12

(11.6%)MSB phenotype resistant tomacrolides and type B

streptogramins,whichisconsistentwithotherstudies con-ductedinBrazil.12,13,22 _{Oneofthestudieswasperformedin}

SãoPaulorevealed37(25.17%)andnine(6.12%)isolateswith cMLSBandiMLSBphenotypes,respectively,whiletheMSBwas

notdetected.12_{Otherstudieswereconductedinthestateof}

RioGrandedoSul;oneofthemidentified71(46.7%),five(3.3%), andfive(3.3%)isolateswithcMLSB,iMLSB,andMSB

pheno-types,respectively.13_{Theotherstudydetected25(17.9%),11}

(7.9%),and two(1.4%)isolateswithcMLSB,iMLSB,and MSB

phenotypes,respectively.22

InthisstudythecMLSBphenotypeprevailedamongthe

iso-latesresistanttomethicillin,whichisconsistentwithstudies conductedinothercountries3,5,11,15,21_{aswellinBrazil.}12,22

TheMSBphenotypewasmorefrequent(12;11.6%)thanthe

iMLSB(five;4.9%)inthisstudy.Divergingfromthisfinding,the

studydonebyKumaretal.8_{inIndiafoundthesamefrequency}

(33;16.9%)ofMSBandiMLSBphenotypesamongtheS.aureus

isolatesanalyzed.Merino-Díazetal.23_{inSpainidentifiedthe} msrAgeneinallStaphylococcusspp.isolateswithMSB

pheno-type.InStaphylococcusspp.thisgeneisresponsibleforanefflux mechanismwhichconfersresistancetomacrolidesandtype Bstreptogramins,butnottoclindamycin.5,24

OnlyS.aureusshowedthe iMLSBphenotypeand its

fre-quencywashigheramongtheMSSA(four;10.8%)thanamong the MRSA (one; 4.5%) in this study. Astudy conducted in ChicagodetectedtheiMLSBphenotypein59(20%)MSSA

iso-latesandin14 (7%)MRSAisolatesinonehospital,andthe iMLSBphenotypein94(19%)andin30(12%)MSSAandMRSA

isolates,respectively,inanother hospital.25_{In astudy}

con-ductedinTurkey,eight(5.8%)MSSAisolatesandtwo(1.7%) MRSAisolatespresentedtheiMLSBphenotype.26Inthestudy

conductedinSãoPaulo,theiMLSBphenotypewasobserved

inseven(6.73%)MSSA and intwo(4.65%)MRSAisolates.12

These data from previouslyreported studiesare similar to the findings ofthe present study.However,thereareother studiescarriedoutabroad,inwhichtheiMLSBphenotypewas

prevalentamongMRSAisolates.3,8,15,27,28

Relevant datafor antimicrobialsusceptibility are signif-icant inestablishing anappropriatetherapy; therefore, the availabilityoftheDtestresultsareimportant.20,25_Reporting

anisolatedStaphylococcusspp.assusceptibletoclindamycin withoutverifyingifitpresentsinducibleresistance,maylead toinadequatetherapywiththisdrug.Incontrast,anegative resultforinducibleresistancetoclindamycin,confirmsthe susceptibilityofthisantimicrobial,providingaverygood ther-apeuticoption.3,8

In this study, the ermA and ermC genes were detected individuallyinfour(9.1%)and17(38.6%)isolateswithMLSB

resistancephenotypes,respectively.Insomestudies,contrary tothis,theermAgenewasidentified morefrequentlythan theermCgene,asinastudyconductedinIran,inwhichthe ermAandermCgeneswerefoundin76(60.3%)and69(54.8%)S. aureusisolates,respectively.29_{Schmitzetal.,}30_when

analyz-ing S.aureusisolatesfrom 24Europeanuniversityhospitals detectedthe ermAgenein571 (67%)isolatesand theermC in192(23%).InKorea,Junget al.31_{identified among280S.} aureusisolates,theermAgenein250andtheermCin14 iso-lates.InIran,Moosavianetal.32_{detectedtheermAandermC}

genes,respectivelyin41.1%and17.7%oftheS.aureusisolates studied.

Inotherstudies,consistentwiththis,theermCgenewas predominantrelativelytotheermAgene,asinastudyfrom Spain,inwhichtheermCgenewasmorefrequentlydetected, in both S. aureus and SCN isolates with constitutive and induciblephenotypes.23_{InGreece,Spiliopoulouetal.}33

iden-tifiedtheermAandermCgenesin22%and70%ofS.aureus isolates,respectively.Vallianouetal.34_{havereportedthatthe} ermCgenewasfoundmorefrequentlyamongS.aureusand SCNisolatesofauniversityhospitalalsoinGreece.

Inthisstudy,one(2.3%)isolatepresentedbothermAand ermCgenes.TheassociationofthesegenesinStaphylococcus spp.isolates,29,35,36_{wasalsoobservedinotherstudies.}

TheermBgenewasnotinvestigated,becauseitisusually detectedinStaphylococcusspp.isolatesofanimalorigin13,23,30

anditisspreadmainlybetweenstreptococciandenterococci.4

InRioGrandedoSul,Coutinhoetal.13_{reportedlowfrequency}

of the ermB gene. They foundthat among152 Staphylococ-cusspp.isolatesanalyzed,77hadoneormoreermgene,the ermA, ermC,and ermBgeneswere foundrespectivelyat49, 29,and threeisolatesand,thecombination ofthesegenes was foundinfourisolates,13 _{datawhich differedfrom this}

study.However,inaworkdevelopedinTexas,withS.aureus isolatesofpediatricorigin,amongthe67isolatesevaluated, the ermB genewas identified in31 ofthese,and the ermA and the ermC genes were detected in 12 and 24 isolates, respectively.37

Thenitwaspossibletoobservethefrequencyvariationof thephenotypesoftheMLSBresistanceandoftheermgenes

amongsthospitalsandgeographicregions,alreadyreportedby otherauthors.13,15,21,29_{Therefore,theimportanceof}

Thegood correlationbetweenthe phenotypicand geno-typicmethodsallowsustoinferthemechanismofresistance to erythromycin and clindamycin, to establish the most appropriateantimicrobialtreatmentandtoappreciatethe epi-demiologicaldifferencesintheirdistribution.23

Conclusion

TheermAgenewaslessfrequentthantheermCgeneamong Staphylococcus spp. isolates with cMLSB and iMLSB

pheno-types.DespitethelowerfrequencyofiMLSBthancMLSBand MSB phenotypes, it isimportant to perform the D test in ordertoidentifyitandguidetherapeuticprocedures.Asthe phenotypic and molecular data about a particular mecha-nismofantimicrobialresistancevarybetweenhospitalsand geographic regions, obtaining local data is useful, since it canbe helpfultoemphasize the importanceofthe imple-mentationofproceduresthataimatcontrollingthespread of this mechanism, in the hospital where the study was conducted.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisstudywassupportedbytheNationalCounselof Techno-logicalandScientificDevelopment(CNPq).

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1. DaurelC,HuetC,DhalluinA,BesM,EtienneJ,LeclercqR. Differencesinpotentialforselectionofclindamycin-resistant mutantsbetweeninducibleerm(A)anderm(C)Staphylococcus aureusgenes.JClinMicrobiol.2007;46:546–50.

2. FiebelkornKR,CrawfordSA,McElmeelML,JorgensenJH. Practicaldiskdiffusionmethodfordetectionofinducible clindamycinresistanceinStaphylococcusaureusand coagulase-negativestaphylococci.JClinMicrobiol. 2003;41:4740–4.

3. PrabhuK,RaoS,RaoV.Inducibleclindamycinresistancein Staphylococcusaureusisolatedfromclinicalsamples.JLab Physicians.2011;3:25–7.

4. LeclercqR.Mechanismsofresistancetomacrolidesand lincosamides:natureoftheresistanceelementsandtheir clinicalimplications.ClinInfectDis.2002;34:482–92. 5. MaheshCB,RamakantBK,JagadeeshVS.Theprevalenceof

inducibleandconstitutiveclindamycinresistanceamongthe nasalisolatesofStaphylococci.JClinDiagnRes.2013;7:1620–2. 6. DrinkovicD,FullerER,ShoreKP,HollandDJ,Ellis-PeglerR.

ClindamycintreatmentofStaphylococcusaureusexpressing inducibleclindamycinresistance.JAntimicrobChemother. 2001;48:315–6.

7. FokasS,FokasS,TsironiM,KalkaniM,DionysopouloyM. Prevalenceofinducibleclindamycinresistancein

macrolide-resistantStaphylococcusspp.ClinMicrobiolInfect. 2005;11:337–40.

8. KumarS,BandyopadhyayM,BhattacharyaK,etal.Inducible clindamycinresistanceinStaphylococcusisolatesfroma

tertiarycarehospitalineasternIndia.AnnTropMedPublic Health.2012;5:468–70.

9.ShouvalDS,SamraZ,ShalitI,etal.Inducibleclindamycin resistanceamongmethicillin-sensitiveStaphylococcusaureus infectionsinpediatricpatients.IsrMedAssocJ.2011;13: 605–8.

10.ClinicalandLaboratoryStandardsInstitute.Performance standardsforantimicrobialsusceptibilitytesting;

twenty-secondinformationalsupplement,M100-S22.Wayne, PA:CLSI;2012.

11.YilmazG,AydinK,IskenderS,CaylanR,KoksalI.Detection andprevalenceofinducibleclindamycinresistancein staphylococci.JMedMicrobiol.2007;56:342–5.

12.AmorimDMR,PersonOC,AmaralPJ,TanakaII.Resistência induzívelàclindamicinaentreisoladosclínicosde Staphylococcusaureus.OMundodaSaúde.2009;33:401–5. 13.CoutinhoVL,PaivaRM,ReiterKC,de-ParisF,BarthAL,

MachadoAB.Distributionofermgenesandlowprevalenceof inducibleresistancetoclindamycinamongstaphylococci isolates.BrazJInfectDis.2010;14:564–8.

14.RobertsMC,SutcliffeJ,CourvalinP,JensenLB,RoodJ,Seppala H.Nomenclatureformacrolideand

macrolide–lincosamide–streptograminBresistance determinants.AntimicrobAgentsChemother. 1999;43:2823–30.

15.SeifiN,KahaniN,AskariE,MahdipourS,NaderiNM.Inducible clindamycinresistanceinStaphylococcusaureusisolates recoveredfromMashhad,Iran.IranJMicrobiol.2012;4:82–6. 16.RabeloMA,NetoAMB,LoibmanSO,etal.Theoccurrenceand

disseminationofmethicillinandvancomycin-resistant Staphylococcusinsamplesfrompatientsandhealth professionalsofauniversityhospitalinRecife,Stateof Pernambuco,Brazil.RevSocBrasMedTrop.2014;47:437–46. 17.HuY,ZhangQ,MeitzlerJC.Rapidandsensitivedetectionof EscherichiacoliO157:H7inbovinefaecesbyamultiplexPCR.J ApplMicrobiol.1999;87:867–76.

18.LinaG,QuagliaA,ReverdyME,LeclercqR,VandeneschF, EtienneJ.Distributionofgenesencodingresistanceto macrolides,lincosamides,andstreptograminsamong staphylococci.AntimicrobAgentsChemother.1999;43:1062–6. 19.Franc¸aCA,PeixotoRM,CavalcanteMB,etal.Antimicrobial

resistanceofStaphylococcusspp.fromsmallruminant mastitisinBrazil.PesqVetBras.2012;32:747–53.

20.JuyalD,ShamanthAS,PalS,SharmaMK,PrakashR,Sharma N.Theprevalenceofinducibleclindamycinresistanceamong Staphylococciinatertiarycarehospital–astudyfromthe GarhwalHillsofUttarakhand,India.JClinDiagnRes. 2013;7:61–5.

21.ZorganiA,ShawerfO,TawilK,El-TurkiE,GhengheshK. Inducibleclindamycinresistanceamongstaphylococci isolatedfromburnpatients.LibyanJMed.2009;4:104–6. 22.BottegaA,RodriguesMA,CarvalhoFA,etal.Evaluationof

constitutiveandinducibleresistancetoclindamycinin clinicalsamplesofStaphylococcusaureusfromatertiary hospital.RevSocBrasMedTrop.2014;47:589–92. 23.Merino-DíazL,CantosdelaCasaA,Torres-SánchezMJ,

Aznar-MartínJ.Detecciónderesistênciainduciblea

clindamicinaemaisladoscutâneosdeStaphylococcusspp.por métodosfenotípicosygenotípicos.EnfermInfeccMicrobiol Clin.2007;25:77–81.

24.ZelaznyAM,FerraroMJ,GlennenA.Selectionofstrainsfor qualityassessmentofthediskinductionmethodfor detectionofinducibleclindamycinresistancein staphylococci:aCLSIcollaborativestudy.JClinMicrobiol. 2005;43:2613–5.

25.SchreckenbergerPC,IlendoE,RistowKL.Incidenceof constitutiveandinducibleclindamycinresistancein Staphylococcusaureusandcoagulase-negativestaphylococciin

acommunityandatertiarycarehospital.JClinMicrobiol. 2004;42:2777–9.

26.TekinA,DalT,DeveciO,TekinR,AtmacaS,DayanS. Assessmentofmethicillinandclindamycinresistance patternsinStaphylococcusaureusisolatedfromatertiary hospitalinTurkey.InfezMed.2013;21:111–6.

27.ShresthaB,PokhrelBM,MohapatraTM.Phenotypic characterizationofnosocomialisolatesofStaphylococcus aureuswithreferencetoMRSA.JInfectDevCtries. 2009;3:554–60.

28.PhukanC,AhmedGU,SarmaPP.Inducibleclindamycin resistanceamongStaphylococcusaureusisolatesinatertiary carehospitalofAssam.IndianJMedMicrobiol.2015;33: 456–8.

29.SaderiH,EmadiB,OwliaP.Phenotypicandgenotypicstudyof macrolide,lincosamideandstreptograminB(MLSB)

resistanceinclinicalisolatesofStaphylococcusaureusin Tehran,Iran.MedSciMonit.2011;17:48–53.

30.SchmitzFJ,SadurskiR,KrayA,etal.Prevalenceof macrolide-resistancegenesinStaphylococcusaureusand Enterococcusfaeciumisolatesfrom24Europeanuniversity hospitals.JAntimicrobChemother.2000;45:891–4. 31.JungYH,KimKW,LeeKM,etal.Prevalenceand

characterizationofmacrolide–lincomycin–streptogramin B-resistantStaphylococcusaureusinKoreanhospitals.J AntimicrobChemother.2008;61:458–60.

32.MoosavianM,ShojaS,RostamiS,TorabipourM,

FarshadzadehZ.Inducibleclindamycinresistanceinclinical isolatesofStaphylococcusaureusduetoermgenes,Iran.IranJ Microbiol.2014;6:421–7.

33.SpiliopoulouI,PetinakiE,PapandreouP,DimitracopoulosG. erm(C)isthepredominantgeneticdeterminantforthe expressionofresistancetomacrolidesamong

methicillin-resistantStaphylococcusaureusclinicalisolatesin Greece.JAntimicrobChemother.2004;53:814–7.

34.VallianouN,EvangelopoulosA,HadjisoteriouM,AvlamiA, PetrikkosG.Prevalenceofmacrolide,lincosamide,and streptograminresistanceamongstaphylococciinatertiary carehospitalinAthens,Greece.JChemother.2014;27:319–23. 35.YildizO,C¸obanAY,SenerAG,etal.Antimicrobial

susceptibilityandresistancemechanismsofmethicillin resistantStaphylococcusaureusisolatedfrom12hospitalsin Turkey.AnnClinMicrobiolAntimicrob.2014;13:44. 36.AleksandraAD,MisicMS,MiraZV,etal.Prevalenceof

inducibleclindamycinresistanceamong

community-associatedstaphylococcalisolatesincentral Serbia.IndianJMedMicrobiol.2014;32:49–52.

37.Chavez-BuenoS,BozdoganB,KatzK,etal.Inducible clindamycinresistanceandmolecularepidemiologictrends ofpediatriccommunity-acquiredmethicillin-resistant StaphylococcusaureusinDallas,Texas.AntimicrobAgents Chemother.2005;49:2283–8.