Community-acquired methicillin-resistant Staphylococcus aureus carrying SCCmec type IV and V isolated from healthy children attending public daycares in northeastern Brazil

brazjinfectdis2017;21(4):464–467

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

Brief

communication

Community-acquired

methicillin-resistant

Staphylococcus

aureus

carrying

SCCmec

type

IV

and

V

isolated

from

healthy

children

attending

public

daycares

in

northeastern

Brazil

Suzi

P.

de

Carvalho

_,

_Jéssica

_B.

_de

_Almeida

_,

_Yasmin

_M.F.S.

_Andrade

_,

Lucas

S.C.

da

Silva

_,

_Arianne

_C.

_de

_Oliveira

_,

_Flávia

_S.

_Nascimento

_,

Guilherme

B.

Campos

_,

_Márcio

_V.

_Oliveira

_,

_Jorge

_Timenetsky

_,

_Lucas

_M.

_Marques

a_{UniversidadeEstadualdeSantaCruz,DepartamentodeMicrobiologia,Ilhéus,BA,Brazil}

b_{UniversidadeFederaldaBahia,InstitutoMultidisciplinaremSaúde,NúcleodeTecnologiaemSaúde,VitóriadaConquista,BA,Brazil} c_{UniversidadedeSãoPaulo,InstitutodeCiênciasBiomédicas,DepartamentodeMicrobiologia,SãoPaulo,SP,Brazil}

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Articlehistory:

Received24August2016 Accepted17April2017 Availableonline5May2017

Keywords: CA-MRSA Antibioticresistance SCCmec MLST

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Nasalcolonizationwithmethicillin-resistantStaphylococcusaureus (MRSA)have increas-ingly been reported in healthy communities. This study aimed to assess the rate of

S.aureus ingeneralandMRSAinparticularfromnasalsecretionofchildrenindaycare centersinVitóriadaConquista,Brazil.Theisolateswereidentifiedbasedonmorphology, biochemicaltestsandbyPCR.Detectionofvirulencegenes,biofilmproduction,and suscep-tibilitytestbydiskdiffusionagarwereperformed.MRSAisolateswerecharacterizedbyspa,

SCCmec,andmultilocussequencetyping(MLST).S.aureuswererecoveredfrom70(47.3%)of 148children.Amongthe11MRSAstrains(15.7%),twoSCCmectypes(IVandV)weredetected. MLSTidentifiedfourSTsrelatedtothreeclonalcomplexes(CC):5,45,and398.Fourspatypes werefoundcirculatinginthissetting.ResistanceofS.aureusisolatestoampicillin, eryth-romycin,ciprofloxacin,clindamycin,andtetracyclinewas80%,32.8%,7.1%,7.1%and4.3%, respectively.OneisolatepresentedintermediateresistancetovancomycindetectedbyEtest methodology.Allstrainswerebiofilmproducers.Thevirulencegenesseb,sec,spa,andpvl

weredetectedinsomeisolates.ThisstudyrevealedahighrateofchildrencarryingMRSA amonghealthyattendeesindaycarecentersinVitóriadaConquista,Brazil.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](L.M.Marques).

http://dx.doi.org/10.1016/j.bjid.2017.04.001

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazj infect dis.2017;21(4):464–467

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Staphylococcus aureus may cause various infections with considerablemorbidityandmortalityinhealthyand immuno-compromised hosts.1 _{Methicillin-resistant Staphylococcus}

aureus(MRSA) iscommonlyassociatedwithsevere nosoco-mialinfections(HA-MRSA);however,ithasbeendetectedin individualswithout riskfactorsforinfection,referredtoas community-acquired MRSA (CA-MRSA).2 _Methicillin

resis-tance is carried on a staphylococcal cassette chromosome

mec(SCCmec).HA-MRSAusuallycarrySCCmectypesI,II,orIII, whereasCA-MRSA strainscommonlycarrySCCmectypes IV andV.3

The molecular characterization is important, since it enables studying the relatedness of MRSA strains, their geneticdiversity,andclonaldistribution.Variousmolecular typingtechniqueshavebeendeveloped,includingthe multi-locussequencetype(MLST),aswellastheSCCmectyping,spa

typing.3–5

NasalcolonizationofS.aureusiscommoninchildren,but thegeneticfindingssuggestedacausalrelationshipbetween carriers,chromosomecassettesofthoseharboringMRSA,and invasivestaphylococcaldisease.6_{Inaddition,theincidenceof}

pediatricinfectionsduetoCA-MRSA,includingchildrenwith noidentifiableriskfactors,hasincreasedworldwide.7_Daycare

centers(DCCs)arereservoirsofS.aureusingeneralandMRSA inparticular,andthesechildrenmayspreadthesebacteriato thecommunityandhospitals.6

InBrazillittleisknownaboutthedistributionand charac-teristicsofS.aureusandMRSAinchildrenasnasalcarriers, particularlyinthoseattendingDCCs.Theaimofthepresent studywastoassesstherateofS.aureusandMRSAnasal car-riageinhealthychildrenattendingpublicDCCsinVitóriada Conquista–BahiaState(BA),Brazil,andtoidentifythe resis-tanceprofile,genotypiccharacterization,andpathogenicity.

ThestudywasconductedfromOctobertoDecember2012, infourpublicDCCsinVitóriadaConquista,acityin North-easternBrazil.Onehundredandforty-eightsamplesofnasal swabswereobtainedfromhealthychildrenrangingfromone tosixyearsattendingDCCs.Childrentreatedwithantibiotics inthelast30dayswerenotincluded.Theisolatesobtained wereincubatedat37◦Cfor48h;Gram-positivecoccicolonies, positiveforcatalaseandcoagulasetestswereselectedas pre-sumptiveS.aureusandidentifiedbyPCR.

MRSA isolates and reduced vancomycin activity were screenedforallS.aureusisolatesbymicrodilutionmethod fol-lowingtherecommendationsoftheCLSIguidelines.8_Strains

ofS.aureusATCC29213andATCC43300wereusedas con-trols. Vancomycin-resistant isolates were confirmed using the E-test® (bioMérieux’s, Brazil) and the bacterial growth at≥16␮g/mLwas indicativeofresistance. Susceptibility to erythromycin, tetracycline, clindamycin, ciprofloxacin, and ampicillinwereperformedbydiskdiffusion.8_Inducible

resis-tance to clindamycin was tested by ‘D test’.8 _Multidrug

resistancewasconsideredwhenthestrainwasresistant to twoormoreantibiotics.

ThegenomicDNAofallstrainswereextractedusingthe GenomicDNAPurificationKit(Invitrogen,Carlsbad,CA,USA), andallisolateswerecharacterizedasSCCmectypes(I–V),3_and

forthepresenceofvirulencegenes,includingsea,seb,sec,pvl, clfA,andspabyPCR.9_{Moreover,MRSAisolatesweresubjected}

toMLSTandSPAtypingtechniques,asdescribedpreviously.4,5

Biofilm assays were performed in 96-well polystyrene microplates using trypticase soybroth(TSB/Difco) with1% (w/v) glucose (TSB-1% Glc).10 _{The samples were compared}

withculturesofStreptococcuspyogenesATCC75194(non-biofilm producersample).S.aureusisolateswere classifiedas non-biofilm producers, weak producers, moderate producers, producers,andstrongproducers.Statisticalanalysiswas per-formedbychi-squaretestandap-valuelowerthan0.05was consideredstatisticallysignificant.

Outof148childrenanalyzed,S.aureuswasrecoveredfrom 70 (47.3%).Eleven (15.7%)isolateswere MRSA,the majority (n=8;72.7%) were SCCmecIVA typeand three(27.2%)were SCCmecVtype.

ResistanceratesofS.aureusisolatestoampicillin, eryth-romycin, clindamycin, ciprofloxacin, and tetracycline were 80%,32.8%,7.1%,7.1%,and4.3%,respectively.One methicillin-susceptibleS.aureus(MSSA)isolateshowedreduced suscepti-bilitytovancomycinbyE-testandwasconfirmedwithaMIC of6␮g/mL.Therewerenosignificantdifferencesinresistance betweenMRSAandMSSAisolates.Therateofresistant iso-latestomorethantwoantibioticswas47.1%(33/70),andonly 15.7%(11/70)oftheisolatesweresusceptibletothestudied antibiotics.Atotalof63.6%ofMRSAisolatesweremultidrug resistant(Table1).

MLST performed on MRSA isolates identified four STs, relatedtothreeclonalcomplexes(CC):5(63.5%),45(18.2%), and398(18.2%).AmongthefourSTsidentified,theST5clone (63.6%,7/11)wasthemostprevalent(Table2).ProteinAwas characteredinisolatesoffourspatypes.Thet242(72.7%,8/11) wasdominantamongallisolates(Table1).

TwoMRSAisolates(2.8%)hadthesebgene,andallhadspa

genes.TwostrainsofMSSA(2.8%)showedthesecgene,three (4.2%)werepositiveforpvlgene,andallhadspa(Table1).All

S.aureusisolatesproducedbiofilm(Table2)andtherewasno statisticaldifferenceinbiofilmproductionbetweenMRSAand MSSAisolates(p>0.05).

The detection of S. aureus(47.3%) and MRSA(15.7%) in nasal swabsofthe studiedchildrendemonstrated ahigher rateofthesestapphylococcithaninstudiesconductedinother DCCswithratesrangingfrom17%to31.1%and0.8to13.2%, respectively.7,11,12

Although the prevalence of MRSA among healthy chil-dren was high,no correlationwas foundbetween carriage of MRSA and antibiotic use. In addition, the close con-tact among DCC attendees has also been considered a risk factor for carring MRSA.6 _{A similar Brazilian study}

showed a MRSA prevalence of 1.2%12 _{confirming the}

vari-ations between regions. The high incidence of MRSA in healthy childrenisthe mostrelevantand worryingfinding for the susceptible hosts and even for the healthy car-rier children if immunosuppressed. This context involves atleasttheriskforcoagulase-positivestaphylococcal infec-tions in elderly hosts, hospitalized individuals, neonates, and the community. Furthermore,mostCA-MRSA reported worldwidehave beenshown tocarry SCCmectypes IVand V, as found in our results.11,13 _{The isolates detected by}

Ho et al. had SCCmec IV (46.4%) or V (53.6%),13 _unlike

Lamaro-Cardoso et al. who found SCCmec IIIA (57%) in most Brazilian children, which is widespread in hospi-tals in many countries worldwide, and only three MRSA

466

Table1–Microbiologicalaspectsandgeneticcharacteristicsof11MRSAisolatescarriagefrominfantsattendingDCCsin VitóriadaConquista,Brazil.

Isolates SCCmec typing

Resistanceprofilea _{MIC(␮g/mL) Phenotype}b _Virulence

genes profiles

ST/CC spatype

(MRSA) OXA VAN

C18 V amp,ery 4 – Moderate spa 398/398 t242 C48 IVA amp,ery 4 2 Strong spa 5/5 t242

C54 IVA amp 4 2 Strong spa 5/5 t242

C60 IVA amp 4 2 Strong seb,spa 2228/45 t004 C77 IVA amp,ery 4 – Strong spa 5/5 t242

C80 IVA – 4 – Strong spa 5/5 t002

C83 V amp,ery 4 2 Producer seb,spa 398/398 t242

C94 IVA – 8 2 Strong spa 5/5 t242

C137 V amp,cli,ery 4 – Strong spa 45/45 t371 C138 IVA amp,ery,tet 4 – Strong spa 5/5 t242 C152 IVA amp,ery,cip 4 – Strong spa 5/5 t242

a _{amp,ampicillin;ery,erythromycin;cip,ciprofloxacin;cli,clindamycin.}

b _{Moderate,moderatebiofilmproducer;producer,biofilmproducer;strong,strongbiofilmproducer.}

strainsshowedSCCmec IVand oneSCCmec typeVgenes.12

ThereforethedistributionoftheMRSAclonevaries through-outtheworld.

TheSCCmecIVhasbeenstronglyassociatedwithstrains causingMRSAinfectionsinpatientswithoutriskfactors in Brazil14_{andelsewhere.}15_{Ourfindingspointoutthepotential}

ofthisspecificDCCstobeareservoirofemergingMRSA geno-typesandhighlighttheneedtoenhancesurveillanceofthese bacteriaandcontroltheirtransmission.

The MLST and spa typing revealed a small diversity betweenCA-MRSAisolates.ST5cloneshaveemergedin hos-pitalandcommunityisolates,whileST45ismostoftenfound inthe communitycarrying SCCmecIV.16,17 _TheClonal

com-plexes–CC5andCC45–whichrepresented81.8%ofCA-MRSA isolatesinthisstudy,areamongtheclonalgroupsknownto beinvolvedinglobalpandemiccausedbyMRSA.Meanwhile, invasiveinfectionbyCC398rarelyoccurs.18

HighratesofMRSAandMSSAisolatesresistantto ampi-cillin (80%) and erythromycin (32.8%) were found, as well as low resistance rates to clindamycin, tetracycline, and ciprofloxacin. Our findings agree with previous studies in the literature, except for higher susceptibility to erythro-mycinelsewhere (14–20%).7 _{Thus,isolatesofCA-MRSAhad}

limitedresistancetonon-␤-lactams.ThemajorityofMRSA isolates(63.6%)detectedhereinwereresistanttomorethan twoantibiotics andthis is achallenge incontrolling these infections.Oneisolate(1.43%)withreducedsusceptibilityto vancomycin among MSSA strains is an important finding.

MostcasesofreducedvancomycinsusceptibilityinS.aureus

(vancomycin-intermediateS.aureus,orVISA)reportedinthe literaturearealsomethicillin-resistant.However,the develop-mentofVISAinMSSAisolateshavebeenreported.19,20_Thus,

reduced vancomycin susceptibility can occur in S. aureus

irrespective of background methicillin susceptibility. There arenodataintheliteratureofCA-MRSAstrainssusceptibleto orwithreducedsusceptibilitytovancomycin,sothisfactisof particularconcerninahealthyyoungpopulation.Therefore, additional studies and continuous monitoring ofS. aureus

andMRSAarerecommended.

Herein,allS.aureusisolateswereabletoproducebiofilm, and the percentageof MRSA and MSSA strains producing biofilmwassimilar.Therearenostudiesonbiofilm produc-tionofS.aureusrecoveredfromhealthyindividuals.Thereis nodirectrelationshipofthemethicillinresistanceprofilewith greaterorlesserabilitytoproducebiofilmbythestrains.The majorityofbiofilmproducerisolateswereclassifiedasstrong producers. Thisfeaturemayactasanadditionalbarrierto controlaninfectionwithantimicrobiotics.2

ProteinAencodedbyspageneisahallmarkofS.aureus.15

Othervirulencegenessuchasseb,sec,andpvlweredetected inafewisolates.Inastudyofstrainsisolatedfromhealthy students inTurkey,thesec genewas foundtobethemost frequent,followedbyseaandnoneoftheS.aureusisolates had pvl.21 _{Thepvlgenehasbeenshown tooccurr less}

fre-quentlyinCA-MRSA isolatesassociatedwithasymptomatic nasalcolonization,consistentwiththeresultsofthepresent

Table2–BiofilmproductionbyMRSAandMSSAisolatesrecoveredfromnasalsecretionofhealthychildrenattending daycarecentersinVitóriadaConquista,Bahia,Brazil.

Samples n Non-producers Weakproducers Moderateproducers Producers Strongproducers

n % n % n % n % n %

MRSA 11 0 0 0 0 1 9.1 1 9.1 9 81.8

MSSA 59 0 0 4 6.7 7 11.8 12 20.3 36 61

Total 70 0 0 4 5.7 8 11.4 13 18.5 45 64.2

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study.22_{Thegenesofstaphylococcalenterotoxinshavebeen}

implicatedasimportantdeterminantsofS.aureusvirulence2_;

however,theyhavebeenpoorlystudiedinhealthychildrenin thecommunity.

Inconclusion,thisstudyshowedhighdetectionratesofS. aureusandMRSA(7.4%)inhealthyBraziliandaycarechildren. Moreover,thecharacterizedisolateswithreducedsensitivity tovancomycinandbiofilmproductionareofspecialconcern. Thus,continuoussurveillanceofS.aureusandMRSAinVitória daConquistashouldbeencouragedtogain abetter under-standingofthecirculatingstaphylococciinordertoestablish bettercontrolofpossibleinfections.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

This study was supported by CAPES.We thank the DCCs, childrenandparents/guardiansforallowingthisresearchto beconducted,andAcademicEnglishSolutions.comfor proof-reading.

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