Clonal dissemination of vancomycin-resistant Enterococcus faecium ST412 in a Brazilian region

brazjinfectdis2017;21(6):656–659

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

Clonal

dissemination

of

vancomycin-resistant

Enterococcus

faecium

ST412

in

a

Brazilian

region

Geraldo

da

Silva

Alves,

Monalessa

Fábia

Pereira,

Lais

de

Lima

Bride,

Ana

Paula

Ferreira

Nunes,

Ricardo

Pinto

Schuenck

∗

UniversidadeFederaldeEspíritoSanto,CentrodeCiênciasdaSaúde,DepartamentodePatologia,Vitória,ES,Brazil

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Articlehistory:

Received26April2017 Accepted9July2017 Availableonline29July2017

Keywords: Enterococcusfaecium VRE ST412 Biofilm

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Vancomycin-resistantEnterococcusfaecium (VREfm)hasemergedas animportantglobal nosocomialpathogen,andthistrendis associatedwith thespreadofhigh-riskclones. Here,wedeterminedthegeneticandphenotypicfeaturesof93VREfmisolatesthatwere obtainedfrompatientsin13hospitalsinVitória,EspíritoSanto,Brazil,during2012–2013. Alltheisolateswerevancomycin-resistantandharboredthevanAgene.Only6(6.5%)of theVREfmisolatesshowedtheabilitytoformbiofilm.The93isolatesanalyzedbelongtoa singlepulsed-fieldgelelectrophoresislineageandpresentedsixsubtypes.MLST genotyp-ingshowedthatallVREfmbelongedtoST412(thehigh-riskclone,hospital-adapted).The presentstudydescribesthedisseminationofST412cloneinthelocalhospitals.Theclonal spreadoftheseST412isolatesintheareaweanalyzedaswellasotherhospitalsin south-easternBrazilsupportstheimportanceofidentifyingandcontrollingthepresenceofthese microorganismsinhealthcare-relatedservices.

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

Withinthelasttwodecades,Enterococcusfaeciumhasemerged as an important global healthcare-associated pathogen becauseofitsabilitytocolonizeandcausediseasein high-riskpatients.1,2_{Thisemergencecanbeexplainedinpartasa}

resultoftheresistanceofE.faeciumtoseveralantimicrobial agents,bothintrinsicandacquired.3_{Vancomycinisusually}

requiredfortreatment,especiallyforinvasiveinfections. How-ever,datafromnosocomialinfectionsurveillanceworldwide hasrevealedagrowingpercentageofvancomycin-resistantE. faecium(VREfm)clones.4,5

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](R.P.Schuenck).

Antimicrobial resistance, together with virulence fac-tors, contributetothe developmentofhumanenterococcal infections.6_{Biofilmproductionhasanimportantroleinthe}

pathogenesisofbacterialinfections,sincethisfeatureallows thepermanencyofthemicroorganismbyprotectingitfrom the hostdefensemechanismsand canfacilitatehorizontal genetransfer,therebycontributingtoantimicrobialresistance spreading.7,8

Changes in the epidemiology of E. faecium infections have been associatedwiththe global dissemination ofthe

http://dx.doi.org/10.1016/j.bjid.2017.07.001

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

brazj infect dis.2017;21(6):656–659

657

high-riskclonesthathaveacquiredadaptiveelementssuch asantimicrobialresistanceandvirulencegenes,particularly inadefinedsubpopulationofE.faeciumthatisenrichedin hospitalisolates.1,2

Theaimofthepresentstudywastocharacterize93VREfm isolatesobtainedbetween2012and2013fromvariousclinical specimens:catheter(1),wound(3),blood(4),urine(19),and rectalswab(66)ofdifferentpatientsat13hospitalsinVitória, EspíritoSanto,locatedinsoutheasternBrazil,toincreasethe knowledgeofthemolecularepidemiologicalcharacteristicsof thisnosocomialpathogen.

TheisolateswerepreviouslyidentifiedasVREfmusingthe Vitek2®system(bioMérieux,France)andconfirmedbyPCR,as describedbyDepardieuetal.9_{Theminimuminhibitory}

con-centration(MIC)forvancomycinwasdeterminedusingEtest (bioMérieux).

Biofilmformation was measured in 96-well polystyrene microtiter plates (Costar, USA), following 24h of incuba-tion at35◦C,and stainingwithcrystal violetas previously describedbyStepanovicetal.10 _{Theopticaldensity(OD)of}

each crystal violet-stained well was measured at 570nm (TP-ReaderSpectrophotometer,ThermoPlate,China). Entero-coccus faecalis ATCC 29212 and Staphylococcus aureus 111711

were used as negative and positive controls, respectively. All tests were carried out in experimental and biological triplicates.Basedonthebacterial biofilmOD,isolateswere classified into four categories: non-biofilm producer, weak, moderate, or strong biofilm producer. Thecutoff OD(ODc) wasdefinedas threestandard derivationsabovethe mean ODofthenegativecontrol.Isolateswereclassifiedasfollows: OD<ODc=non-biofilm producer; ODc<OD<2ODc=weak biofilm producer; 2ODc<OD<(4ODc)=moderate biofilm producer;andOD>4ODc=strongbiofilmproducer.

GenomicDNAfromE.faeciumwasextractedfollowingthe methoddescribedbythermallysisandusedastemplatefor multiplexPCRofthevangenes, aspreviouslydescribed by Depardieuetal.9

Pulsed-fieldgelelectrophoresis(PFGE)wasperformedafter macrorestrictionwithSmaIinaCHEF-DRIIIsystem(Bio-Rad, USA),accordingtoSaeedietal.,12_{andanalyzedwith}

Bionu-mericsv6.5(AppliedMaths,Belgium)usingtheunweighted pair-groupmethodwiththearithmeticmean(UPGMA) apply-ingtheDicecorrelationcoefficient.Isolatesweredesignated asthesamepulsotypeiftheysharedatleastan80% similar-ityinthebandpatternsandthesamesubtypeifshowedan identicalbandpattern.

Oneisolateofeach subtypewas characterizedbyMLST methodaccordingtotherecommendationsdescribedinthe

E.faeciumMLSTdatabase(http://efaecium.mlst.net/). All isolates harbored the vanA gene and showed van-comycinMICs>256␮g/mL(Fig.1).Inaddition, 87(93.5%)of theisolatescouldnotformbiofilmonapolystyrenesurface. However,four (4.3%) ofthe isolates showed aweak ability toproduceabiofilm,1(1.1%)presentedamoderate biofilm-forming ability,and the remaining strain presentedstrong biofilmproduction.

The93isolatesbelongedtothesamePFGElineageand pre-sentedsixsubtypes(A1-A6)(Fig.1).TheMLSTresultsshowed thatallVREfmsubtypesoflineageApresentedST412.

Our study describes the microbiological and epidemio-logical characteristics ofVREfm clinical isolates that were obtained indifferent hospitalsin a southeastern regionof Brazil.ThevanA-andvanB-mediatedglycopeptideresistance occurs frequentlyinVREfm,andbothtypesarecarriedout by transposons(Tn1546 and Tn1547, respectively).13 _{In the}

present study, vancomycin resistance was common in all isolates, and they all harbored the vanA gene. This result is consistent with the vancomycin-resistance phenotype, as all isolates were high-level resistant to vancomycin. In Brazil, clinical studies after outbreaks in different states havereportedtheemergenceandprevalenceofVREfm iso-latescarryingthevanAgene.6,14 _{Thewidespreadprevalence}

of the vanA gene in E. faecium has also been observed in Canadianand Europeanstudies.2,5 _{Theformationof}

multi-layeredbiofilminenterococciisacomplexandmultifactorial process.15_{StudiesonE.faeciumisolateshaveshownthelow}

or moderate ability of this species to form biofilm.15,16 _In

the present study,biofilm formation was observed inonly six (6.5%) isolates. Paganelli et al.16 _{observed that E.}

fae-cium strains of different phylogenetic clades form biofilm with distinct properties and suggested that under differ-ent ecological conditions, different types of biofilms are produced, possibly contributing to adaptation to different niches.

PFGEprofilesandMLSTdataindicatethatthereisaclonal dispersion among the VREfm clinical isolates analyzed in thepresent study.Thestrainsshowedahomogeneous pat-tern that wasassociatedwithaconserved presenceofthe

vanAgeneforallisolates.Notably,the93VREfmwere clus-teredintoonlyonelineage,theST412,whichbelongstothe high-riskclonescomplex.Molecularepidemiologicalstudies haveshown theglobalspread ofhigh-riskclones,whichis associated withthe majorityof nosocomial outbreaks and clinical infections in all continents. The wide distribution ofspecificsubpopulationsseemstohavebeenfacilitatedby thecumulativeacquisitionofantibioticresistance,virulence characteristics,andtheabilitytoacquiredifferentgenetic ele-mentsviahorizontaltransfer.14

Damanietal.17_{describedST412,whichbelongstoclonal}

complex17(CC17),forthefirsttimeduringanepidemiological studyofVREfmisolatesfromGreecein2010,wherethisclone waspredominantinGreekclinicalsettings.

VREfm isolates belonging to CC17 are predominant in sporadiccasesand duringoutbreaksinBrazil.Studieshave shown that ST412 was the most frequent sequence type inhospitalenvironmentsinBrazilandfourother countries in South America, including Colombia, Ecuador, Peru, and Venezuela.6,18,19 _{Theemergence ofVREfm ST412 has been}

observedinsoutheasternBrazil,indicatingastrong correla-tionbetweenthisstrainandthehospitalenvironment.6,19,20

ThepresentstudyfoundthatST412isawell-establishedclone inhospitalsofVitória,EspíritoSanto,Brazil.Theclonalspread ofST412amonghospitalsindifferentareas ofthecountry indicatesaninter-hospitalspreadandemphasizestheneed fortheapplicationofstringentcontrolmeasurestodecrease theriskofdisseminationofthebacteria,suchastheisolation ofinfectedpatients, increasedenvironmentalcleaning,and improvedantimicrobialtherapy.

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braz j infect dis.2017;21(6):656–659 80 90 100 PFGE type/ST (N° of isolates) A1/412 (37) A >256 N (35) W (01) S (01) N (11) W (02) M (01) N (16) N (18) W (01) N (03) N (04) >256 >256 >256 >256 >256 A A A A A A2/412 (19) A3/412 (03) A4/412 (04) A5/412 (14) A6/412 (16) MIC Van1 (µg/mL) Biofilm2 Van gene

Fig.1–Moleculartypingandgeneralcharacteristicsofthe93vancomycin-resistantEnterococcusfaeciumfromVitória, EspíritoSanto,Brazil.1–vancomycin;2–biofilmproduction:N,non-producer;W,weak;M,moderate;S,strong.

Ethics

statement

The present research received ethical and methodological approvalfromtheResearchEthicsCommitteeoftheCenterof HealthSciencesoftheUniversidadeFederaldoEspíritoSanto (Protocol65/2011).

Financial

support

ThisworkwassupportedbyFundac¸ãodeAmparoàPesquisa doEstadodoEspíritoSanto(FAPES)andConselhoNacionalde DesenvolvimentoCientíficoeTecnológico(CNPq).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WeacknowledgethecontributionofPhDNazarethMagnago Klein(FederalUniversityofEspíritoSanto)forthesupplyofthe bacterialstrainsandPhDThiagoCésarNascimento(Federal UniversityofJuizdeFora)forthedataanalysis.

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