Molecular characteristics of methicillin-resistant Staphylococcus aureus isolates from hospital and community environments in northeastern 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

Brief

communication

Molecular

characteristics

of

methicillin-resistant

Staphylococcus

aureus

isolates

from

hospital

and

community

environments

in

northeastern

Brazil

Suzi

P.

de

Carvalho

_,

_Jéssica

_B.

_de

_Almeida

_,

_Yasmin

_M.F.S.

_Andrade

_,

Lucas

S.C.

da

Silva

_,

_Raiane

_C.

_Chamon

_,

_Kátia

_R.N.

_dos

_Santos

_,

Lucas

M.

Marques

a_{UniversidadeEstadualdeSantaCruz,CampusSoaneNazarédeAndrade,Ilhéus,BA,Brazil} b_{UniversidadeFederaldaBahia,InstitutoMultidisciplinardeSaúde,VitóriadaConquista,BA,Brazil} c_{UniversidadeFederaldoRiodeJaneiro,InstitutodeMicrobiologiaPaulodeGóes,RiodeJaneiro,RJ,Brazil} d_{UniversidadeFederalFluminense,FaculdadedeMedicina,CentrodeCiênciasMédicas,RiodeJaneiro,RJ,Brazil}

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Articlehistory:

Received21November2018 Accepted25April2019 Availableonline16May2019

Keywords: Methicillin-resistantStaphylococcus aureus CA-MRSA HA-MRSA Moleculartyping

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Thisstudycharacterized30MRSAisolatesfromintensivecareunit(ICU)environmentand equipmentsurfacesandhealthychildren.TheSCCmectypesI,IVaandVweredetectedin HA-MRSAisolateswhileCA-MRSAshowedtheSCCmectypeIVaandV.Mostisolateswere classifiedasagrgroupII.Allisolatespresentedtheseigene,andonlyHA-MRSAwerepositive foretbetstgenes.ThreegenotypeswererelatedtoPediatric(ST5/SCCmecIV)andBerlin (ST45/SCCmecIV)clones.ThepresentstudyshowedmolecularsimilaritybetweenCA-and HA-MRSAisolatesinhospitalandcommunitysettingsinaBrazilianregion.

©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Staphylococcusaureusisanimportanthumanpathogen asso-ciated with a broad spectrum of infections in community and hospital settings worldwide.1 Currently, MRSA is the maincause ofnosocomialinfections inLatinAmerica,and in many Brazilian hospitals the Brazilian Endemic Clone (BEC/ST239/CC8/SCCmecIII) remains the prevalent lineage in these settings.2 _{However, a number of studies report}

on a substitution of this well-established clone by the USA400(ST1/CC1/SCCmecIV)lineageandthePediatricclone

∗ _{Correspondingauthor.}

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

(USA800/ST5/CC5/SCCmecIV).2,3_{Meanwhile,theMRSA}

preva-lence incommunityacquired infections continuestogrow. The aim of the present study was to compare molecular characteristics ofHA-and CA-MRSAisolatesobtainedfrom intensivecareunits(ICUs)environmentandequipment sur-faces and fromhealthy childreninpublic daycarecenters (DCCs),fromVitóriadaConquista,Brazil.

Thirty isolates were identified as MRSA and had their genomicDNAextractedusingthePureLinkTM_GenomicDNA

https://doi.org/10.1016/j.bjid.2019.04.005

1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

PurificationKit(Invitrogen,Carlsbad,CA,USA).Amongthem, 19isolateswerecollectedfromICUsenvironmentand equip-ment surfaces oftwo Brazilian public hospitals.The sites, determinedbyfollowingtheroutinesofeachhospital,with agreater possibility ofcontaminationwere selectedand a total of 117 points were analyzed. S. aureus were isolated from98 sitesand ofthose 19(19.28%) isolateswereMRSA. Onehundred and forty-eightsamplesofnasal swabswere obtainedfromhealthychildrenagedonetosixyears attend-ingDCCs.Childrenwhohadreceivedcareinhospitalsand/or weretreatedwithantibioticsinthelast30dayspriortosample collectionwerenotincluded.Among148childrenanalyzed,S. aureuswasisolatedin70(47.3%).Eleven(15.7%)isolateswere MRSA. All institutions are locatedin Vitória da Conquista, Brazil.

All isolates were characterized for SCCmec types (I–V) and subtype of SCCmecIV (IVa, IVb, IVc and IVd),4 _agr

polymorphism (I–IV),5 _{and for the presence of 14}

viru-lence genes, including nine enterotoxins (sea,seb, sec, sed, see, seg,seh, sei,sej), two exfoliatins (eta and etb), the toxic shocksyndrometoxin(tsst),Panton–Valentineleukocidin(pvl), and icaA.6–9 _{Moreover, all isolates were analyzed by PFGE}

after digestion with SmaI (New EnglandBiolabs, Inc., Bev-erly, MA).10 _{USA300/ST8/IV, USA400/ST1/IV, USA800/ST5/IV,}

USA600/ST45/II,USA1100/ST30/IV,andUSA100/ST5/IIstrains wereusedasstandardstrainsforcirculatingpandemicclones characterization.OnerepresentativeisolateofeachPFGEtype wassubjectedtoMLSTandSPAtypingtechniques.11,12

Sta-tistical analysis: Fisher’s exact test was used to compare categoricalvariablesbetweengroups.

Thegeneralandmolecularcharacteristicsofthe30MRSA isolatesare shown in Table1. Amongthem,three SCCmec typeswereidentified.HA-MRSAisolatescarriedtheSCCmec typesI(37%;7/19),IVa(37%;7/19)andV(26%;5/19)while CA-MRSAisolatescarriedSCCmectypesIVa(73%;8/11)andV(27%; 3/11).Moreover,despitethatonlytwoagrpolymorphismswere foundamongHA-MRSAstrains,agrgroupII(79%;15/19)andI (21%;4/19),threeagrweredetectedinCA-MRSAstrains,most isolatesrelatedtoagrgroupII(64%;7/11)(Table1).

Out ofthe30 MRSA isolates,13 differentPFGE patterns wereidentifiedgroupedinto10genotypes,fromAtoJ(Fig.1). Amongthem,10STswereidentifiedasbeingrelatedtothree clonalcomplexes (CC): 5, 45 and 398. For all 19 HA-MRSA isolates,sevendifferentclones(>80%similarity)werefound (A–G)toberelatedtoCC5(53%;10/19)andCC45(47%;9/19) andnine spatypeswerecharacterized. Meanwhile,for CA-MRSAisolates, fivelineages were described (A,C,H,I and J)belongingtoCC5(64%;7/11), CC45(18%; 2/11)andCC398 (18%;2/11)aswellasfourspatypes,themostprevalentbeing t242(73%,8/11). Interestingly,twolineageswere commonly foundamongHA-andCA-MRSAisolates(n=14;47%),sharing thesamemolecularcharacteristics,relatedtoAandC geno-types. Overall, the Pediatric Clone (USA800/ST5/SCCmecIV) wasrelatedtonineisolates(30%;9/30),bothCA-MRSA(n=6) andHA-MRSA(n=3),eightofthemsharingthesamePFGE pro-file(C).Moreover,theSTs45/CC45,characterizedastheBerlin Clone(USA600/ST45/SCCmecIV), and 2228/CC45were found amongbothgroups,relatedtogenotypesA1(2/30;6.7%)and A2(4/30;13.3%),respectively.

In regards to virulencegenes, the sei gene was present inall30MRSAisolates(Table1).Despitethattheseg(100%) and sej(64%; 7/11)geneswere foundtobeassociatedonly with CA-MRSA isolates (p-value<0.05), the etb (63%; 12/19) (p-value<0.05)and tst(21%;4/19)geneswere onlydetected amongHA-MRSAisolates.Theetagenewaspresent in CA-MRSA(27%)andHA-MRSA(10.5%)isolates.TheicaDgenewas related to both HA- and CA-MRSA, whereas icaA/icaD was detectedonlyamongHA-MRSAisolates(p-value<0.05).

TheepidemiologyofMRSAinfectionsisverydynamicand thesubstitutionofwelladaptedhospitalclonesby commu-nityoneshasbeenshownbyseveralauthors.2,3,13_However,

therearefewstudiescomparingHA-andCA-MRSAisolatesin Brazilianstudies.

AprevalenceofSCCmectypesIVandV,typicallyfromthe community,werereportedinallCA-MRSAisolates,andwere themaintypesfoundamongHA-MRSAisolates(63%)aswell. ThedistinctionbetweenMRSAisolatesfromcommunityand healthcarefacilitiesbecamecloudywiththereplacementof HA-MRSAlineagesbyCA-MRSAinthehospitalsetting.Ina studyconductedinBrazildescribingthemolecular character-isticsofisolatescollectedfromhealthychildren,mostisolates wererelatedtoSCCmecIIIandonlythreeisolatesshowedthe SCCmectypeIVandonetypeV.14 _{Ontheotherhand,}

Cabo-cloetal.15_{showeddisseminationoftheUSA400/SCCmecIV,a}

communitylineage,atamilitaryhospitalinRiodeJaneiro.2_In

Brazil,thereportofisolatescarryingtheSCCmectypeVisstill rare,butinthepresentstudythiscassettetypewaspresent inbothgroupsevaluated.ThepresenceofbothSCCmectypes IVandVamonghealthyandhospitalizedpeopleseemstobe relatedtotheexposureofhealthcareprofessionals,hospital staff,visitors,andpatientstoawiderangeofpathogens.16

TheMLSTrevealedawidediversityofSTsinboth environ-ments.ST5,ST45andST2228(singlelocusvariantST45)were foundamongCA-MRSAandHA-MRSAstrains. TheST5 iso-lateshasemergedinhospitalandcommunityisolates,while ST45 has mostoftenbeen foundin the CA-MRSA carrying SCCmecIV.1,17_{Thespatypingalsoshowedgreatgenetic}

vari-ation amongthe analyzedisolates.MostCA-MRSA isolates sharedcommonspatypeswithHA-MRSAisolates.

TheClonalcomplexes–CC5andCC45–whichrepresented 100%ofHA-MRSAand82%ofCA-MRSAisolatesinthisstudy, areamongtheclonalgroupsknowntobeinvolvedinaglobal pandemiccausedbyMRSA.1_{AlthoughCA-MRSAisolates}

usu-allyhavedifferentmolecularcharacteristicsthanHA-MRSA, mostcommunityisolatesinthisstudysharedthesame geno-typeofthosefromhospitals,mainlyinpulsotypesAandC (n=14; 47%). TheC and I cloneswere related tothe Pedi-atricclone(ST5/CC5-SCCmecIV).TheA1andA2subtypeswere related to the Berlinclone (ST45/CC45-SCCmecIV). Thereby, bothPediatricandBerlinclonesarepresentinchildren attend-ingdaycarecenters(55%and18%,respectively)andonICUs environmentalsurfaces(16%and 21%respectively),sharing thesamegeneticbackground.Thissimilaritybetweengenetic groups ofCA-and HA-MRSAmay indicatean eventual co-transmissionbetweenthecommunityandhospitalsettings.

TheBerlinclone(CC45),recentlydescribedinthenortheast ofBrazilamongMSSA(2%)isolatesfromnosocomial infec-tions,hastheabilitytocausehighmortalityinpatientswith

Table1–Generalcharacteristicsof30methicillin-resistantStaphylococcusaureusisolatesfromhospitalandcommunity environmentsinVitóriadaConquista,Brazil.

Isolate number Origin Isolation source MRSA origin SCCmec type agr type Virulence geneprofile

PFGEtype ST/CC spatype

17A H1 Telephone–ICU HA IVa 1 etb,sei,icaD A1 45/45 t371

C137 DCC4 NasalSwab CA IVa 1 seg,sei,icaD A1 45/45 t371

52 H2 Controlpanel HA IVa 1 sei,icaA/icaD A2 2228/45 t004

59 H2 Drug’sdilution

countertops

HA IVa 1 etb,sei,icaD A2 2228/45 t004

85.1 H2 Bed-drawerknob –PICU

HA IVa 1 eta,se,icaA/icaD A2 2228/45 t004

C60 DCC2 NasalSwab CA IVa 1 eta,seb,seg,sei,sej,icaD A2 2228/45 t004

C18 DCC1 NasalSwab CA V 4 eta,seg,sei,icaD J 398/398 t242

C83 DCC2 NasalSwab CA V 4 eta,seb,seg,sei,sej,icaD J 398/398 t242

12D H1 Controlpanel– ICU

HA V 2 etb,seitst,icaA/icaD B1 676/5 t9784

47.1 H2 Drug’sdilution countertops

HA V 2 sei,icaD B2 461/5 t5085

43.1 H2 Floor HA V 2 sei,icaA/icaD B3 5/5 t311

91 H2 Floorofdrug’s dilutionroom

HA IVa 2 sei,tst,icaD C 5/5 t242

101 H2 SinkPICU HA IVa 2 sei,icaA/icaD C 5/5 t242

27A H1 BabyCrib HA IVa 2 etb,sei,tst,icaA/icaD C 5/5 t242

C138 DCC4 NasalSwab CA IVa 2 seg,sei,sej,icaD C 5/5 t242

C48 DCC2 NasalSwab CA IVa 2 seg,sei,sej,icaD C 5/5 t242

C54 DCC2 NasalSwab CA IVa 2 seg,sei,sej,icaD C 5/5 t242

C77 DCC2 NasalSwab CA IVa 2 seg,sei,icaD C 5/5 t242

C94 DCC3 NasalSwab CA IVa 2 seg,sei,sej,icaD C 5/5 t242

16A H1

Weighing-machine

HA V 2 etb,sei,icaD D 2226/5 t3374

28 H1 Controlpanel–

BabyCrib

HA I 2 etb,sei,icaD E 1427/5 t5046

29 H1 Controlpanel

ICU

HA I 2 etb,sei,tst,icaA/icaD E 1427/5 t5046

33A H1 Telephone–ICU HA I 2 etb,etb,sei,icaD E 1427/5 t5046

39 H2 Medicine’sfridge HA I 2 etb,sei,icaD F 3050/45 t3374

41 H2 Prescription

paper

HA I 2 etb,sei,icaD F 3050/45 t3374

62 H2 Prescription

paper

HA I 2 etbsei,icaD F 3050/45 t3374

74 H2 Emergency

stretcher

HA I 2 eta,etb,sei,icaA/icaD F 3050/45 t3374

67 H2 Controlpanel HA V 2 sei,icaA/icaD G 3019/45 t11545

C152 DCC4 NasalSwab CA V 2 seg,sei,sej,icaD H 5/5 t242

C80 DCC2 NasalSwab CA IVa 2 seg,sei,icaD I 5/5 t002

H1,HospitalMunicipalEsaúMatos;H2,HospitalGeraldeVitóriadeConquista;DCC,daycarecenter;HA-MRSA,hospital-acquiredMRSA; CA-MRSA,community-acquiredMRSA;ICU,intensivecareunit;PICU,pediatricintensivecareunit;SCCmec,Staphylococcalcassetechromosome mec;agr,accessorygeneregulator;PFGE,pulsedfieldgelelectrophoresis;ST,sequencetype;CC,clonalcomplex.

bloodstreaminfectionsandalargeglobalspreadcapacity.18

Ontheotherhand,thePediatricclone(CC5)hasemergedin Brazilianhospitals,19_{andthisisthefirstdetectioninhealthy}

childrenandobjectsinICUsandequipmentsurfaces, suggest-ingthatthiscloneisspreadingfromBrazilianhospitalstothe community,whichcanactasareservoirandcontributetothe spreadofthispathogen.

The accessory gene regulator group I and II predomi-natedinthecommunityandhospitalisolates.Thesegroups have been associated with endocarditis and suppurative infections.20_{Thisstudyrevealedthatenterotoxingeneswere}

moreprevalent inCA-MRSAisolates,aresultalsofoundby Xie et al.17 comparing HA- and CA-MRSA in hospital iso-latesinChina,whiletheexfoliatinb(etb)genepredominated

among HA-MRSA isolates. The presence of superantigen genes inMRSAisolatesfrom hospitalsurfacesand healthy individualsimpliesthepossibilityofincreasedbacterial dis-semination and more severe infections. Genes related to biofilm production were detected in all strains. The find-ing ofthisgene isimportantbecause infectionsassociated withbiofilmproductionareusuallyrecurrent,aggravate noso-comial infections, and act as a barrier to antimicrobial action.

Our results demonstrated high genetic diversityamong MRSAisolates, althoughmostoftheisolatesare relatedto theCC5andCC45showingtheimportanceoflocalstudiesto betterunderstandthedynamicsinvolvedinthespreadand pathogenicityofMRSAlineages.

Similarity coefficient 60 80 100 PFGE type DCCs D E F J B1 B2 B3 G H I b b C K 3/5 ATCC 0/1 0/2 0/1 1/0 1/0 1/0 3/1 1/1 1/0 4/0 3/0 1/0 A1 A2 a a Hospital/

Fig.1–DendrogramofthePFGEpatternsrelatedto30CA-andHA-MRSAisolates.Isolatesshowingasimilaritycoefficient ≥80%wereconsideredgeneticallyrelated.DCC,daycarecenters.Oneisolate,S.aureusstrainATCC29213/ST5wasusedas control.a_{RelatedtoUSA600lineage;}b_{RelatedtoUSA800lineage.}

Inconclusion,theCA-andHA-MRSAisolatessharing simi-larmolecularcharacteristicsirrespectiveoftheirenvironment originaswellasthediscoveryofinternationalclonallineages demonstratethedisseminationabilityofMRSAandtherisk ofcommunityandhospitalinfections.Itunderscorestheneed forpublichealthofficialstomonitorthesepopulations,sites andtodevelopstrategiestoreducetheprevalenceofthese MRSAclonesonhospitalsurfacesandDCCs.These environ-mentscouldactasimportantreservoirsforfuturecommunity andhospitalinfections.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

We thank the Hospitals and DCCs, children and par-ents/guardiansforallowingthisresearchtobeconducted,and AcademicEnglishSolutions.comforproofreading.Thisstudy wassupportedbyProgramadeapoioapesquisadores emer-gentesdaUFBA(PRODOC02/2011).

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