All isolates were considered susceptible (MIC ≤ 1.0

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

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

MRSA

from

Santa

Catarina

State,

Southern

Brazil:

intriguing

epidemiological

differences

compared

to

other

Brazilian

regions

Alessandro

C.O.

Silveira

,

Gabriela

R.

Cunha

,

Juliana

Caierão

,

Caio

M.

de

Cordova

_,

_Pedro

_A.

_d’Azevedo

a_{LaboratóriodeCocosGramPositivos,UniversidadeFederaldeCiênciasdaSaúdedePortoAlegre,PortoAlegre,RS,Brazil}

b_{DepartamentodeCiênciasFarmacêuticas,Fundac¸ãoUniversidadeRegionaldeBlumenau,Blumenau,SC,Brazil}

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Articlehistory:

Received30October2014 Accepted22April2015 Availableonline25June2015

Keywords:

Antimicrobialresistance Vancomycin

Oxacillin

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b

s

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Methicillin-resistantStaphylococcusaureus(MRSA)isoneofthemostfrequently isolated agentsinbothnosocomialandcommunitysettings.Itisaconstantchallengefor antibac-terialtherapy.Therefore,itbecomesessentialtounderstandtheepidemiologyofMRSA isolatesintheinstitutionand/orregiontoguideempiricaltherapy.Theobjectiveofthisstudy wastoevaluatetheepidemiologicalcharacteristicsofMRSAisolatesinthestateofSanta Catarina,Brazil,anddetermineifthereisaclonalspread.Weevaluated124clinicalisolates ofMRSAobtainedfromvariousanatomicalsitesfrompatientsinthestateofSantaCatarina inSouthernBrazil.Theantimicrobialsusceptibilityprofilewasevaluatedbydiskdiffusion andminimuminhibitoryconcentration(MIC)wasdeterminedbyEtestandbroth macrodi-lution.SCCmectypesweredeterminedbymultiplexPCRandtheclonalrelationshipamong isolateswasassessedbypulsedfieldgelelectrophoresis.Antimicrobialsthathave demon-stratedlowerratesofresistanceweretetracycline(20.2%),sulfamethoxazole–trimethoprim (20.2%)andchloramphenicol(12.9%).Wedidnotdetectanyresistancetoglycopeptides, daptomycin,linezolid,andtigecycline.SCCmectypeIIIwaspredominant(54%),followed bytypeII(21.8%),consistentwithotherBrazilianstudies.Twenty-sixcloneswereobserved grouping72(58%)isolatesandnoclonalrelationshipwasobservedbetweenourisolatesand themajorepidemicclonescirculatinginBrazil.AnintriguingdistinctMRSAepidemiology wasobservedinSantaCatarina,comparedtootherBrazilianregions.

©2015ElsevierEditoraLtda.Allrightsreserved.

Introduction

Staphylococcusaureusisamajoragentofcommunity-acquired infectionsandhealthcareassociatedinfections.Amulticenter

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](AlessandroC.O.Silveira).

studyamongBrazilianhospitalsduring2005–2008foundthat S.aureuswasthemainagentofbloodstreaminfections(20.2%) and infections of the skin and soft tissue (28.1%)and the second most common agentin pneumoniaofhospitalized

http://dx.doi.org/10.1016/j.bjid.2015.04.009

patients(24.9%).Oftheseinfections,approximately30%were resistanttomethicillin(MRSA).1

Staphylococcibecomeresistanttomethicillinbychanging transpeptidasesinthewall,calledpenicillin-bindingproteins (PBPs),andloseaffinityforall␤-lactamagents.This informa-tioniscontainedinthemecAgeneresponsibleforencoding analteredPBP(PBP2a).2,3

Until the early 1990s, MRSA isolates were restricted to hospitals.Currently,however,MRSAisnolongerexclusively associated with healthcare infections. During this period, increase in community-acquired MRSA (CA-MRSA) among patientswithoutassignableriskfactorsforacquiringMRSA wasobserved(i.e.,hadnodirectorindirectcontactwithhealth servicesthatcouldassociatetheMRSAinfectionwithhealth care).4

Sincethen,areversephenomenonbegantooccur: com-munity isolates, once characterized by the presence of a staphylococcalcassettechromosome(SCCmec)typeIV,began to be isolated in hospital settings and those types that were typically isolated in the hospital began to appear in outpatients.5,6

AstheepidemiologyofMRSAprovedtobedynamic,itis importanttogeneratedatatobetterunderstandtheevolution ofthisepidemiologicalbehaviorandtoassessitsimpacton clinicalsettings.7_{SantaCatarinaStatehasanintriguinglow} prevalenceofMRSAcomparedtootherBrazilianStatesand understandthisepidemiologicaldynamicswouldbevaluable. Theobjectiveofthisstudywastoevaluatethe epidemio-logicalcharacteristicsofMRSAisolatesinthestateofSanta Catarina.

Methods

Bacterialsamples

Weused 124 clinical MRSA isolatesobtained from various anatomicalsitesfrompatientsattendedintwocities (Blume-nauandFlorianopolis),locatedintheStateofSantaCatarina, Southern Brazil. Samples were collected from November 2009 through February 2013. One isolate per patient was considered.Sampleswereselectedbyconvenience. Identifica-tionwasdoneusingstandardizedphenotypicmethodology8 and isolateswere maintained frozen(−20◦C) inskim milk

(DIFCO®_{)plus10%glycerol.}

Antimicrobialsusceptibilitytesting

Antimicrobialsusceptibilitytestingwasperformedusingthe disk diffusion method, according to the recommendations and interpretative criteria of the Clinical and Laboratory Standards Institute9 _{and European Committee on} Antimi-crobial Susceptibility Testing (to tygecycline only).10 _The antimicrobialstestedweregentamicin(10␮g),ciprofloxacin (5␮g), erythromycin (15␮g), clindamycin (2␮g), trimetho-prim/sulfamethoxazole (1.25␮g/23.75␮g), chloramphenicol (30␮g), tetracycline (30␮g), teicoplanin (30␮g), tigecycline (10␮g),and linezolid(30␮g).MRSAwascharacterizedusing thecefoxitindisk(30␮g),andconfirmedbyPCRforthegene mecA.11_{Todetect␤-lactamaseproduction,weusednitrocefin}

disk(BDBBLTM_DrySlideTM_{Nitrocefin),usingbacterial} suspen-sionsinphysiologicalsaline.S.aureusATCC29213wasused asapositivecontrol,andS.aureusATCC25923wasusedasa negativecontrol.

MICdetermination

Vancomycin MICs were determined by macrodilution method9 _{and byEtest}® _{(BioMérieux,Marcy l’Etoile,France)} followingCLSIinterpretativecriteria9_{andthemanufacturer’s} instructions, respectively. MICs for teicoplanin and dapto-mycin were determined by Etest®_{. S. aureus strains ATCC} 29213(MSSA),ATCC43300(MRSA),ATCC700698(hVISA)and ATCC700699(VISA)wereusedforqualitycontrol.

MultiplexPCR

TheSCCmectypewas determined usingthe multiplexPCR methodaccordingtotheprotocoldevelopedbyZhangetal.12 Theamplicons thatwere formedhadthe followingsizes:I (613bp),II(398bp),III(280bp),IVa (776bp),IVb(493bp),IVc (200bp),IVd(881bp),andV(325bp).12,13

Forisolatespresentingphenotypicinducibleresistanceto clindamycin,ermgenewasassessedbyamultiplexPCR proto-col,describedelsewhere.14_{ThePCRproduct(610bpforermA} and520bpforermC)wasanalyzedbyelectrophoresisthrough a1.5%agarosegel.14,15

Pulsed-fieldgelelectrophoresis(PFGE)

PFGEwasperformedaccordingtoMcDougaletal.16_andPinto et al.17 _{The fragments were subjected to PFGE using 1%} agarosegels(PulsedFieldCertifiedAgarose;Bio-Rad)in0.5×

Tris–borate–EDTAbufferwithaCHEF-DRIIIsystem(Bio-Rad). Gels were stained with 0.5␮g/mL ethidium bromide, visu-alizedunderUVlight,and photographedusing aGelDocTM XR System (Bio Rad). PFGE patterns were analyzed using Bionumericsversion6.1(AppliedMaths,Sint-Martens-Latem, Belgium)andclusteredbyUPGMA.Adendrogramwas gen-erated from a similarity matrix calculated using the Dice similaritycoefficientwithanoptimizationof0.5%anda tol-eranceof1%.PFGEclustersweredefinedasisolateswitha similarityof80%orhigheronthedendrogram.18_SCCmec con-trolstrainswereincluded[typeI(NCTC10442);typeII(N315); typeIII(85/2082);typeIVa(CA05);typeIVb(8/6-3P);typeIVc (MR108);typeIVd(JSC4469)andtypeV(WIS)],aswellas iso-latesofthemaincirculatingepidemicclonesinLatinAmerica (BrazilianEpidemicClone,NewYork/Japan,USA300,USA400, Pediatric,OSPC,Cordobean,E-MRSA15,E-MRSA16).

Results

MIC Vancomycin

A

B

Number of isolates Number of isolates

MIC (μg/mL) MIC (μg/mL)

60

50

40

30

20

10

0

10 20 30 35 40 50

45

25

15

0 5

0.5 0.75 1.0 1.5 2.0 3.0 0.19 0.25 0.38 0.5 0.75 1.0 1.5 2.0 3.0 4.0 6.0 8.0 12.0

Fig.1–MICoftheglycopeptidesto124MRSAdeterminedbyEtest.In(A),thevaluesobtainedforvancomycin.Theseven isolateswithMICof3.0␮g/mLwereconsideredsusceptiblebecauseallMICswere≤2.0␮g/mLbymicrodilution.In(B),the resultsforteicoplanin.Oneisolate(MIC12␮g/mL)whentestedbymicrodilutionyieldedanMICof4.0␮g/mL(susceptible).

(13.7%)wereconsideredcommunityisolatesand107(86.3%) hospitalisolates.

MRSApresentedthefollowingresistancerates:amikacin (35.5%),gentamicin (33.1%),chloramphenicol (12.9%), tetra-cycline (20.2%), ciprofloxacin (79%), norfloxacin (72.6%), sulfamethoxazole–trimethoprim(20.2%), clindamycin(75%), and erythromycin (81.5%). Also, 93 strains (75%) showed productionof␤-lactamase.All isolates were susceptible to linezolidandtigecycline.

AllisolatesweresusceptibletovancomycinandMICsare presentedinFig.1.SevenisolatesshowedMICof3.0␮g/mLby Etest.BymacrodilutionthreeofthemshowedMICof1.0␮g/mL andfourhadMICof2.0␮g/mL.Etestalsooverestimated(above CLSIsusceptibilitybreakpoint)teicoplaninMICforoneisolate butmacrodilutiondefineditsMICas4␮g/mL(susceptibleby CLSIinterpretativecriteria).

Daptomycin,analternativetoglycopeptidesforthe treat-mentofinfectionscausedbyMRSA,showedMCIvalueswithin the susceptibility range (Fig. 2), even though four isolates presentedMICof1.0␮g/mL(upperlimitofsusceptibility cate-gory).

AlthoughpredominantSCCmecwastypeIII(54%,67/124), therewasaheterogeneousdistributionofSCCmectypes:type II(21.8%),typeIVa(10.5%),typeIVc(3.2%)andtypeIVb(2.4%). FiveisolatescarriedtypesIandIItogether.AmongCA-MRSA

MIC Daptomycin

Number of isolates

MIC (μg/mL)

0 5 10 20 30 40 45

35

25

15

0.125 0.19 0.25 0.38 0.5 0.75 1.0

Fig.2–MICsfordaptomycindeterminedbyEtest.All isolateswereconsideredsusceptible(MIC≤1.0␮g/mL).

(n=17),onlyfourshowedSCCmectypeIV:twoIVaandtwoIVb. Amongtheother13isolates,eighthadSCCmectypeIIIandfive hadtypeII.

Ofthe124isolates,23(18.5%)hadapositiveDtest:20(87%) had ermAgeneandthree(13%)carriedermC.Twelve(9.7%) isolatespresentedresistancetoerythromycinand suscepti-bilitytoclindamycin(negativeDtest)andhadnopositivePCR reaction.Fifty-six(45.1%)isolateshavehadconstitutive resis-tancemechanismandonly33(26.6%)weresusceptibletoboth antibiotics.

Twenty-sixclones(Fig.3)wereobserved,grouping72 iso-lates(withfivebeingthehighestnumberofisolatesperclone). Indeed, 52 isolates presented a unique pulsotype. Isolates belongingtoallclonescarriedSCCmectypeIII(Table1).

Discussion

The prevalence of MRSAin the state ofSanta Catarina is extremely lowandiscomparabletoratesfoundin Scandi-naviancountries.2,19,20_{Isolationratesareintherangeof4–8%} forallisolatedS.aureusandtheseratesareevenlowerwhen onlynosocomialisolatesareconsidered(lessthan2%).Such lowoccurrenceofMRSAjustifiestheintriguinglownumberof MRSAincludedinthisstudy,asthetwomostpopulouscities ofSantaCatarinawereincluded.Inareportpublishedbythe WorldHealthOrganization(WHO),severalEuropeancountries havenationalsurveillancedatawithverylowratesofMRSA: Denmark(1.2%),Estonia(1.7%),Finland(2.8%),Iceland(2.8%), Netherlands(1.4%),Norway(0.3%)andSweden(0.8%).21_These countriesmayhavesucceededinmaintaininglowMRSArates becauseofeffectivesearch-and-destroypoliciesand/or con-trolled antibioticoveruse.22 _{Ofnote, occurrenceofMRSAin} Santa Catarina differsgreatly from other regions ofBrazil, whichhave,ingeneral,MRSAratesofaround29%.23,24_Asfar asweknow,thosesearch-and-destroypoliciesarenot prac-ticedinSantaCatarinaand,surprisingly,MRSAisolationrates aresimilartothosefoundintheScandinaviancountries.

SI 2 pfge pfge SI 8 SI 12 SI 10 SI 29 SI 14 SI 3 SI 15 SI 30 SI 21 SI 5 SI 9 SI 1 SI 16 SI 28 SI 6 L 78 L 79 L 35 L 77 L 58 L 86 L 61 L 62 L 40 L 64 L 60 L 49 L 81 L 22 L 32 L 46 L 39 L 47 L 48 L 76 L 55 L 88 L 66 L 56 L 85 L 27 L 94 L 23 L 31 L 73 C1 C2 L5 L 75 C4b C4a C4c C3 L 38 L 25 L 59 L 19 L 57 L 91 L 93 L 71 L 20 L 50 L 26 L 29 L 24 L 28 L 53 L 83 L 12 L 21 L 70 L 13 L 14 L 17 L 11 L 16 L 8 L 37 L 7 L 52 L 3 L 6 L 1 L 15 L 18 L 82 L 10 L 43 L 51 L 84 L 67 L 9 C5 L 87 L 74 L 2 L 4 L 44 L 63 Mu 3 SI 11 SI 4 L 54 L 69 L 92 L 36 L 80 L 41 L 42 L 45 SI 17 SI 20 L 89 SI 19 SI 13 SI 22 SI 23 SI 7 SI 18 SI 24 C4d SI 26 SI 27 SI 25 L 65 L 72

Fig.3–DendrogramillustratingthePFGEpatternsof124 strainsandcontrols.Twenty-sixcloneswereobserved.

studieswithsimilarcharacteristics,whichdemonstratea pre-dominanceofbloodstreaminfections(39%)25_{orskinandsoft} tissueinfections(61.5%).26

Compared tothe antimicrobialsurveillance in the mul-ticenter study SENTRY,1 _{resistance rates in this study} were much lower: ciprofloxacin (91.4–79%), tetracycline (46.7–20.2%), trimethoprim–sulfamethoxazole (68.1–20.2%), clindamycin(87.9–75%)anderythromycin(94–81.5%).A pos-sible explanation for the large differences in rates of susceptibility could be due to the epidemiological profile. Cavalcante et al. proposed phenotypic markers associ-ated with SCCmec types. Resistance to tetracycline and trimethoprim–sulfamethoxazole may be associated with SCCmectypeIII,inwhich100%resistancewasfoundtoboth drugs.SCCmectypeIVhadabsolutesusceptibility,withonly 2%oftheisolatesresistanttotrimethoprim–sulfamethoxazole and100%susceptibilitytotetracycline.27_Wefounda predom-inanceofSCCmectypeIII,whichjustifieswhytheresistance ratesinourstudywerelowerthanthosefoundintherestof Brazil.

Thepresenceof␤-lactamasemayresultinunusual phe-notypesknown asBORSA (BorderlineOxacillin ResistantS. aureus).Thesebacteria,despitelackingthemecAgene,may be resistant to oxacillin and are phenotypically character-ized asMRSA.28 _{They mayalsoberesponsible forelevated} MICsofoxacillin.Aftertherapywith␤-lactams,these bacte-riamay inducethedevelopmentofvancomycinresistance, aphenotypecalled␤-lactamantibiotics-inducedvancomycin resistantS.aureus(BIVR).29

AllMRSAweresusceptibletovancomycin,teicoplaninand daptomycin.Althoughthephenotypeofresistancetothese drugs is uncommon.30 _{31.45% of the isolates showed MIC} of2.0␮g/mLforvancomycin,whichmaysuggestthe devel-opmentofaphenomenonofanupwardtrendforMICsfor vancomycin,called“MICcreep”.31

TheisolatesdemonstratedhigherprevalenceofgeneermA thanermC.TheDtestprovedtobeamethodologycapableof detectingallisolateswithinducibleclindamycinresistance. Therefore,thetestiseasytoperform,inexpensiveandis clin-ically relevant becauseit reducesthe riskof inappropriate antibioticsuse.32

Inourstudy,therewasahighprevalenceofSCCmectypeIII (54%).In2010,astudyconductedattheHospitaldeClinicasde PortoAlegre(capitaloftheneighboringstateofRioGrandedo Sul),ahighprevalenceofSCCmectypeIIIwasdetected(49%),33 similartoourfindings.In2005,incontrasttothelocal epi-demiology,astudyconductedattheHospitaldeClinicasde SãoPaulofoundthat65%ofMRSAcarriedSCCmectypeIV.34

Notsurprising,ourisolatesdidnotpresentaclonal dis-semination,asbothcities(FlorianopolisandBlumenau)are separatedbyapproximately120kmandtheisolateswere col-lectedduringaperiodofthreeyears.

Table1–Epidemiologicalandantibioticsusceptibilitycharacteristicsofthe26clones.

Clones Isolates Time Source City Resistance SCCmec

1 SI2,SI8 2–4/09 HOS BLU ER,CL,SU,CIP,N0,TE,CLO,AM,GE II 2 SI10,SI29,L79 5/09–1/12 HOS BLU,FLO ER,CL,CIP,NO II

3 L35,L77 9/10–12/11 HOS FLO ER,CL,CIP,NO II

4 SI3,SI14 2/09–4/10 COM BLU ER,CL,CIP,NO II

5 SI15,SI30 4/10–1/12 HOS BLU ER,CL,CIP,NO II

6 SI1,SI5,SI9,L58,L86 1/09–3/12 HOS BLU,FLO ER,CL,CIP,NO III 7 L40,L61,L62 10/10–4/11 HOS FLO ER,CL,CIP,NO II 8 SI16,SI28,L72 5/10–11/11 HOS BLU,FLO CIP,NO IVa 9 L22,L32,L46,L49,L81 7/10–1/12 HOS FLO ER,CL,CIP,NO III 10 L39,L47,L48 8/10–11/10 HOS FLO ER,CL,CIP,NO III 11 L55,L66,L88 6/11–5/12 HOS FLO ER,CL,CIP,NO III 12 SI25,L27,L94 2/10–10/12 COM,HOS BLU,FLO ER,CL,CIP,NO IVa

13 L5,L75 8/08–11/11 COM,HOS FLO – IVa

14 L19,L25,L59 7/10–2/11 HOS FLO ER,CL,CIP,NO IVa

15 L91,L93 7/12–10/12 HOS FLO ER,CL,CIP,NO IVb

16 L20,L50 7/10–11/10 HOS FLO ER,CL,SU,CIP,NO,AM.GE III 17 L24,L26,L28,L29 7/10–8/10 HOS FLO ER,CL,CIP,NO,AM,GE I 18 L12,L21,L70 12/09–6/11 HOS FLO ER,CL,CIP,NO,CLO,AM,GE III 19 L13,L14,L17 1/10–5/10 COM,HOS FLO ER,CL,SU,CIP,NO,TE,AM,GE III 20 L3,L6 7/08–1/09 HOS FLO ER,CL,SU,CIP,NO,TE,CLO,AM,GE III 21 L43,L51 10/10–11/10 HOS FLO ER,CL,SU,CIP,NO.AM.GE III 22 SI19,L67 8/10–1/11 COM,HOS BLU,FLO ER,CL,CIP,NO III

23 L2,L4 5/08–6/08 COM,HOS FLO – IVa

24 L44,L63 10/10–7/11 HOS FLO ER,CL,CIP,NO,AM,GE III 25 L54,L69,L92 5/11–8/12 HOS FLO ER,CL,SU,CIP,NO,TE,CLO,AM,GE II 26 L36,L41,L42,L80 9/10–1/12 HOS FLO ER,CL,CIP,NO II

HOS,hospital;COM,community;BLU,blumenau;FLO,florianopolis;ER,erythromycin;CL,clindamycin;SU,trimethoprim/sulfamethoxazole; CIP,ciprofloxacin;NO,norfloxacin;TE;tetracycline;CLO,chloramphenicol;AM;amikacin;GE,gentamycin.

Conclusions

AlthoughweidentifiedtheSCCmecthatwerepresentand eval-uatedthepresenceofclones,thepresentstudydidnotprovide consistentscientificevidenceforthelowprevalenceofMRSA inSanta Catarina, whichwas very different from the data foundinotherBrazilianstatesandsomeEuropeancountries. We conclude that the MRSA epidemiological profile in SantaCatarinaissimilartothatfoundintherestofBrazil, withapredominanceofSCCmectypeIII,but theresistance rateswerelowerthanthosefoundintherestofBrazil.

A wide variety of complexes with clonal isolates were detected without evidence of clonal spread, and a small amountofhighgeneticdiversitywasfound.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsthankCoordenac¸ãodeAperfeic¸oamentode Pes-soaldeNívelSuperior(CAPES),ConselhoNacionaldePesquisa e Desenvolvimento Tecnológico (CNPq) and Fundac¸ão de AmparoàPesquisadoRioGrandedoSul(FAPERGS)for finan-cialsupport.ThankstoLaboratórioSantaLuzia(CassiaZoccoli and Nina Tobouti) and Laboratório Santa Isabel (Reginaldo SimõesandMarceloMolinari)forgrantingclinicalsamples.

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