The
Brazilian
Journal
of
INFECTIOUS
DISEASES
w w w. e l s e v ie r . c o m / l o c a t e / b j i d
Original
article
Distribution
of
serotypes
and
evaluation
of
antimicrobial
susceptibility
among
human
and
bovine
Streptococcus
agalactiae
strains
isolated
in
Brazil
between
1980
and
2006
Tatiana
Castro
Abreu
Pinto
∗,
Natália
Silva
Costa,
Aline
Rosa
Vianna
Souza,
Ligia
Guedes
da
Silva, Ana
Beatriz
de
Almeida
Corrêa, Flavio
Gimenis
Fernandes,
Ivi
Cristina
Menezes
Oliveira,
Marcos
Corrêa
de
Mattos,
Alexandre
Soares
Rosado,
Leslie
Claude
Benchetrit
InstitutodeMicrobiologiaProfessorPaulodeGóes,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil
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r
t
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c
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e
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f
o
Articlehistory:
Received6August2012 Accepted24September2012 Availableonline28February2013
Keywords:
Streptococcusagalactiae
Pulsedfieldgelelectrophoresis Epidemiology
Serotyping Resistanceprofile
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s
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Streptococcusagalactiaeisacommonagentofclinicalandsubclinicalbovinemastitisand
animportantcauseofhumaninfections,mainlyamongpregnantwomen,neonatesand nonpregnantadultswithunderlyingdiseases.Thepresentstudydescribesthegeneticand phenotypicdiversityamong392S.agalactiaehumanandbovinestrainsisolatedbetween 1980and2006inBrazil.ThemostprevalentserotypeswereIa,II,III andVandall the strainswere susceptibletopenicillin,vancomycin andlevofloxacin. Resistanceto clin-damycin,chloramphenicol,erythromycin,rifampicinandtetracyclinewasobserved.Among theerythromycinresistantstrains,mefA/E,ermAand,mainly,ermBgeneweredetected, and a shift ofprevalence from the macrolide resistancephenotype to the macrolide-lincosamide-streptograminBresistancephenotypeovertheyearswasobserved.The23 macrolide-resistantstrainsshowed19 differentpulsed-field gelelectrophoresisprofiles. Regardingmacrolideresistance,amajorconcerninS.agalactiaeepidemiology,thepresent studydescribesanincreaseinerythromycinresistancefromthe80stothe90sfollowed byadecreaseinthe2000–2006period.Also,thegeneticheterogeneitydescribedpointsout thaterythromycinresistanceinBrazilisratherduetohorizontalgenetransmissionthanto spreadingofspecificmacrolide-resistantclones.
©2013 ElsevierEditoraLtda.Allrightsreserved.
Introduction
Streptococcus agalactiae (Group B Streptococcus; GBS) is
a common agent of clinical and subclinical bovine
∗ Correspondingauthorat:InstitutodeMicrobiologiaProfessorPaulodeGóes,CentrodeCiênciasdaSaúde-BlocoI,Av.CarlosChagas
Filho,373,CidadeUniversitária,IlhadoFundão,RiodeJaneiro,RJ21941-590,Brazil. E-mailaddress:[email protected](T.C.A.Pinto).
mastitis, accounting for great economic losses in dairy industryworldwide.1,2Additionally,itisanimportantsource
ofseverediseasesinnewbornsandpregnantwomen,andan emergingcauseofinvasiveinfectionsinnonpregnantadults, mostlyinthosepresentingunderlyingconditions.3,4
1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2012.09.006
Asymptomaticcolonizationcanevolvetoinvasivediseases insusceptiblehumanhosts;inanimalhosts,non-treated sub-clinicalmastitisusuallyturnsinto chroniccasesleading to permanentdamages.2,4Forthisreason,high-quality
preven-tionandearlytreatmentarecrucialgoals,whicharegenerally achievedthroughtheusageofantimicrobials.Incattle, com-mercial solutions (mostly including macrolides, penicillins andtetracyclines)areusuallyemployedasoneofthe mas-titiscontrolstrategies.2,5 Inhumans,penicillinremainsthe
drugofchoiceforchemoprophylaxisaccordingtoCentersof DiseaseControl(CDC).6
Macrolidesandlincosamidesrepresentthealternativein casesoftherapeuticfailureandofpatientsallergicto beta-lactams,althoughhumanandbovine GBS strainsresistant to those drugs have been described in the last decades, varyingfrom2.0%to54.0%.7,8Likewise,ratesof
tetracycline-resistantGBSisolatesfromhumanandbovinehostshavebeen increasing.8–10
TenS.agalactiaecapsulartypesweredescribedsofar(Ia,Ib,
II–IX),andserotypingisstillthefirstepidemiologicalapproach to characterize GBS strains.11 In addition, pulsed-field gel
electrophoresis(PFGE)hasbeenusedtoevaluatethegenetic diversity and clonal relatedness within this species.8,12,13 In Brazil, few data about the genetic diversity, serotyping andantimicrobialsusceptibilityofS.agalactiaeareavailable. Therefore,hereweprovidelongitudinaldataabout distribu-tionofcapsulartypes,profilesofantimicrobialsusceptibility, includingmajormechanismsoferythromycinresistance,and geneticdiversityoferythromycinresistantstrainsamongS.
agalactiaeisolatedfromhumansandcattleinBrazil.
Materials
and
methods
Bacterialstrains
Atotalof392GBSstrains,including363fromhumansand29 frombovineorigin,wereanalyzed.Onehundredfortythree strainswereisolatedbetween1980and1989,87between1990 and1999and 162between2000and2006. Mostofthe iso-lateswere recoveredduringdifferent studiesperformed by ourgroup,including289isolatespreviouslyserotyped3,14–16
andwerestoredat−20◦Cinourcollectionofcultures. Thehumanoriginatedstrainswereisolatedbetween1980 and2006intheStatesofRiodeJaneiro,SantaCatarina,and SãoPaulo,frompatientsandhealthycarriers,including chil-dren, pregnant women and nonpregnant adults, and were mainlyfromthroat(n=69),urine(n=58),vagina(n=51),anus (n=22),cervix(n=18),outerear(n=9)andliquor(n=7). Oth-erslesscommonclinicaloriginsincludedblood(n=4),surgical wounds(n=4),peritoneum(n=1),skin(n=3),placenta(n=4), lungs(n=2),sperm(n=4)andurethra(n=1).Thebovine origi-natedstrainswereisolatedfrommilkofclinicalorsubclinical mastitiscases at 5different herds in the States of Riode Janeiro,SantaCatarina,andSãoPaulo,between1987and1989 andbetween2003and2006.
Determinationofcapsularserotypes
Onehundred and threestrains isolated between 2001and 2002inRiodeJaneirofromhumanhostswereserotypedby
immunoprecipitationusingspecificantiserafortypesIa,Ib, and II–VIII,whichwerepreparedinhouseusingrecognized referencestrains.3
Antimicrobialsusceptibilitytesting
Antimicrobial susceptibility testing of 392 strains was per-formedbyKirby–Bauermethodusingclindamycin, chloram-phenicol, erythromycin, levofloxacin, penicillin, rifampicin, tetracyclineandvancomycindisks(Cecon,SãoPaulo,Brazil).
StreptococcuspneumoniaeATCC49619wasusedasquality
con-trolandCLSIbreakpointsastheinterpretativecriteria.17
Determinationoferythromycinresistancephenotypesand genotypes
Theerythromycin-clindamycindouble-disktestwascarried out for the determination of the resistance phenotypes amongall theerythromycinresistant strains,aspreviously described.18 The presence of ermA, ermB, mefA/E and lnuB
geneswasalsoinvestigatedamongthesestrainsusing poly-merasechainreaction(PCR).DNAextractionwasperformed accordingtoSambrooketal.19Thereactionswereperformed
inaGeneAmpPCRSystem2400(AppliedBiosystems)using primers andcycles previouslydescribed.20–22 PCR-amplified
productswererunon1%agarosegelsandstainedwith ethid-iumbromide.The100-bpDNAladderkit(Invitrogen)wasused astheDNAsizemarker.
Pulsed-fieldgelelectrophoresis(PFGE)analysis
PFGE profiles from all erythromycin resistant strains were obtained aspreviouslydescribed.23 Thegenomic DNA was
digestedwiththeSmaIrestrictionenzyme(NewEngland Bio-labs,Ipswich,MA,USA)andelectrophoresiswasperformed inaCHEFDRIIIsystem(Bio-RadLaboratories,USA)usingthe followingprogram:switchtime1–30sduring23hwitha120◦ angle ata temperatureof11.3◦C anda voltagegradientof 6V/cm.TheLambdaLadderPFGmarkerkit(NewEngland Bio-labs,USA)wasusedastheDNAsizemarker.Thegelswere stained with ethidiumbromide and digitallyphotographed usingaScorpionSCOR-14SOMscanner(DNRBioimaging Sys-tem,Jerusalém,Israel)underultravioletlight.Theimageswere analyzedusingGelComparII®software(AppliedMaths, Bel-gium). Electrophoretic profiles werecompared accordingto theguidelinesproposedbyTenoveretal.24TheDicecoefficient
(95%)anda1%positiontolerancewereusedtoanalyzethe similaritiesinthebandpatterns amongtheelectrophoretic profiles.Theunweightedpairgroupmethodusingthe arith-metic average was used to gather electrophoretic profiles intopolymorphismpatterns,alsoreferredtoasclusters.The polymorphism patternswere definedgroupingprofiles that showed>70%dendrogramidentity.
Results
Analyzing our serotyping data together with data previ-ously described,3,16,25 thestrainswere classifiedasfollows:
30 25 20 15 10 5 0 1980 - 1989 (n = 143) 1990 - 1999 (n = 87)
Time (year) and number of strains
2000 - 2006 (n = 162) 1980 1989 1990 1999 2000 2006 Ia Ib II III IV V NT 18.9% 5.6% 17.5% 18.9% 0.7% 16.1% 22.3% 26.4% 2.3% 13.8% 27.6% 3.4% 6.9% 19.6% 25.9% 6.8% 13.6% 14.2% 4.3% 17.9% 17.3%
Distribution of capsular serotypes (%)
Fig.1–DistributionofserologicaltypesamongS.agalactiaestrainsfromhumanandbovineoriginsaccordingtotheperiod ofisolation.NT,nontypeable.
(74), IV=2.8% (11)and V=14.8% (58). Serotype IIIprevailed amongthehumanstrainsandtypeVamongthebovineones. Seventy-sevenstrains(19.7%)werenon-typeable.Fig.1shows thedistributionofserotypesalongtime.
Allthebovineandhumanisolatesweresusceptibleto peni-cillin,vancomycin and levofloxacin.However,resistance to clindamycin(20.7% and1.9%inbovineandhumanstrains, respectively),erythromycin(27.6and4%inbovineandhuman strains, respectively), and tetracycline (89.6 and 89.2% in bovineandhumanstrains,respectively)wasdetectedamong strains from both origins. Resistance to chloramphenicol andrifampicinwasdetectedonlyamonghumanoriginated strains, with rates of 1.5% and 0.7% respectively. All the erythromycin-resistantGBSstrainsfromhumanoriginwere isolatedfromcarriersorcasesofnoninvasiveinfections.All theclindamycin-resistantstrainswerealsoresistantto eryth-romycin. The distribution of resistant strains according to origin(bovineorhuman)andalongtimeisshowninFig.2.
Constitutive macrolide-lincosamide-streptogramin B (cMLSB) and macrolide (M) phenotypes were equally dis-tributedamongthe23erythromycin-resistantisolates,with 12 and 11 strains each onerespectively, although the first prevailedamongthebovine(75.0%)and thesecond among
thehuman(60.0%)erythromycin-resistantGBSisolates.The inducibleMLSBphenotypewasnotobserved.TheermBgene wasthemostcommonresistancedeterminantinbothhuman andbovineerythromycin-resistantisolates,whilelnuBgene was not detected. Fourteen erythromycin-resistant strains analyzed(60.8%)had2or3erythromycinresistance determi-nants,andthecombinationermB/mefA/Ewaspredominantly foundin26.1%(6strains).OnlyoneS.agalactiaestrain,isolated fromahumanhost,didnotharboranyoftheerythromycin resistancegenestested.AllMphenotypestrainsharboredthe
mefA/Egene,eitheraloneorincombinationwithanerm(A
orB)gene,whileallthecMLSBphenotypestrainshadanerm gene,eitheraloneorincombinationwiththemefA/Egene.
PFGErevealedahighheterogeneouspopulation,asshown inFig.3,with19distinctelectrophoreticprofilesdistributed amongthe23erythromycin-resistantGBS strainsanalyzed, suggesting a polyclonal origin for erythromycin resistance in our region. Four major clusters were observed, and the predominant one (cluster A) harbored 7 strains from both human and bovine origins. Similar genetic profiles were observed in different States of Brazil, as well as among strains belonging to different serotypes. In addition, one humanoriginatedserotypeIIIstrainfromRiodeJaneiroand 100 90 80 70 60 50 40 30 20 1980-1989 1990-1991 2000-2006 10 0 Time (years)
Distribution of resistant strains (%)
Erythromycin - human Erythromycin - bovine Clindamycin - human Clindamycin - bovine Chloramphenicol - human Chloramphenicol - bovine Rifampicin - human Rifampicin - bovine Tetracycline - human Tetracycline - bovine
Cluster B
Strain PFGE
20 30 40 50 60 70 80 90 100
Year Origin Serotype
II III III Ia IV V III V V V IV V Ia V V V II Ia Ia III II NT III Human Human Human Human Human Bovine Human Bovine Bovine Human Human Human Human Bovine Bovine Bovine Human Bovine Human Human Bovine Human Bovine Resistance phenotype Resistance genes Cluster A Cluster C Cluster D Cluster E 1981 1989 1990 1990 1996 2006 1990 2006 2006 2002 1996 2002 1990 2006 2006 2006 1980 1987 1988 1981 1987 1989 1989 cMLSB M M M cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB M cMLSB cMLSB cMLSB M M M M M M M ermA ermA ermB mef mef ermB mef ermB ermA ermB ermB mef ermA ermB ermB ermA mef ermB mef ermB mef ermB mef ermA ermB mef ermA ermB mef ermA ermB mef ermA mef ermA mef ermB mef ermA ermB mef
81376 89232 90003 901841 96008 06006 90184 06001 06002 02065 96009 02031 90180 06004 06005 06003 80358 87155 88606 81055 87169 89228 89646
Fig.3–GeneticdiversityandphenotypiccharacterizationofS.agalactiaemacrolide-resistantstrains.Pulsed-fieldgel electrophoresis(PFGE)profiles,strainidentificationnumbers,yearsofisolation,origins,serotypes,resistancephenotypes andresistancegenes.cMLSB:constitutivemacrolides,lincosamidesandstreptograminsB-typeresistance;M,macrolides resistance;NT,non-typeable.
onebovine serotype IIstrain from São Paulohad identical profiles.
Discussion
InUnitedStates,EuropeandAustraliaserotypesIa,II,IIIand Vaccountfor80–90%oftheisolatedstrains,whileserotypes IV, VI, VII, VIII and IX remain rarely described.11,12,26,27 In
thepresentstudyserotypesIa,II,IIIandVwerecollectively detectedinapproximately70%ofthestrains.Thisfindingis consistentwithpreviouslyreporteddata.Around20%ofour strainswere non-typeableand similar datawere described recentlyinourregion.23Ontheotherhand,lowerrates,
vary-ingbetween4and15%,havebeenreportedbyothers.8,28The
useofmoleculartechniquestodetectthecapsulargeneinthe lattermayexplainthisdiscrepancy.
Susceptibilityto vancomycin,penicillinand levofloxacin and a high level of resistance to tetracycline (89%) were observedinthepresentstudy.Thesedatawerealsodescribed previously in other countries13,29 and in Brazil.15,23,30,31
Althoughstrainsresistanttopenicillinandlevofloxacinhave beendescribed,theyremainasexceptions.32,33
Thetetracyclineresistanceratescanreachorovercomethe 90%index.TetracyclineresistanceratesobservedamongGBS isolatesfrom humans agreewithpreviouslocaland global studies.9,23,32However,therateamongGBSfromcattleis
con-siderablyhigherthanpreviousdata,whichhadrevealedonly 44.7%and14.5%oftetracyclineresistanceinBrazil30andin
theUnitedStates,9respectively.Theriseoftetracycline
resis-tanceisofconcernsinceitisoneofthemainantimicrobials usedinmastitiscontrol.2,5Moreover,accordingtoourresults,
thegeneralrateoftetracyclineresistance(includingstrains fromhumanandbovineorigins)hasincreasedfrom86%to 91%from80sto90sandhasbeenconstantlyhighsincethen. Although the rate oferythromycin resistance amongS.
agalactiae from humanhostsanalyzedinthepresent study
has increased from the 80s to the 90s but decreased by 2006, it is still importantto track erythromycinresistance amongBrazilianS.agalactiae strains.Recently,ratesaround 13% and 11%, which would rule out this drug asan alter-native option, have been described in Rio de Janeiro.23,33
Among strains from bovine origin, resistance to erythro-mycin increased from 10.5% in 1987 and 1988 to 60% in 2003 and 2006. This isprobably also due tothe proximity among the collection herds investigated in2006; hereafter mentioned.
PrevalenceofcMLSbphenotypeincreasedalongtheyears, whileprevalenceofM phenotypedecreased. The predomi-nanceofMLSbphenotypeamongS.agalactiaestrainsobtained in the last ten years was reported in Brazil and in other countries.23,33–35 Thepredominantresistancegenotype was
ermB+ mefA/E+,suggesting that themajority ofour strains
havebothresistancemechanisms,effluxandribosomal mod-ification.Thethreestatesincludedinthestudy(RiodeJaneiro, SantaCatarina,andSãoPaulo)wererepresentedamongthe 23 erythromycin-resistantGBS, andno correlationbetween erythromycinresistanceandaspecificserotypewasobserved,
sinceresistantstrainsbelongedtoavarietyofthem(Ia,II,III, IVandV,andNT).
Theincreasingresistanceto macrolidesamonggroup B streptococcihasbeenobservedduringrecentyearsinmany countries.Itisparticularlyimportantfromthe epidemiolog-icalpointofviewtoperform specificcharacterizationofS.
agalactiaeisolates,focusedon,amongothers,answeringifthe
phenomenonoccurredduetothespreadofaspecificS. agalac-tiaecloneinthepopulationorifit istheresultofacquired resistanceorboth.Someauthorshaveassociatedmacrolide resistancetotheintroductionofspecificS.agalactiaeclones, includingmostlyserotypeVstrains.28,34,36Thepresentstudy,
however,didnotshowassociationbetweenspecificS. agalac-tiaeclonesorserotypesandthemacrolideresistanceprofile amongstrains fromhumanorigin,indicatingthat horizon-talgenetictransferisthemainforcebehindtheappearance oferythromycinresistanceinourregion.Similardatawere describedpreviouslyforS.agalactiaestrainsisolatedin2008in RiodeJaneiro.23Indeed,someauthorshaveshownthat
eryth-romycinresistancedeterminantsare carriedbyconjugative transposons,whichareeasilyself-transferableandcommonly foundamongS.agalactiaestrains.37,38
Regardingthebovinestrains,however,it isimportantto highlighttheserotypeVcluster(clusterC)observedin2006. TheseisolateswererecoveredfromherdsintheSouth Cen-tralregionofRiodeJaneiroand,althoughmorestudiesare necessarytoevaluatethishypothesis,itseemsthatthey rep-resentasuccessfulclone,well-adaptedtothelocalcattle,and disseminationtothe metropolitanregion, which isnearby, couldeasilyhappen.Moreover,thefinding ofamajor clus-ter,clusterA,comprisinghumanandbovineoriginatedGBS strains suggests that, although erythromycin resistance is ratheremergingduetohorizontalgenetictransfer, success-fulclones,whichcanbefoundamongstrainsfromhumans andcattle,havetheabilitytocaptureandexchangethiskind ofmobilegeneticelements.
AGBSstrainfromhumanorigin,isolatedinRiodeJaneiro in1981,andonefromcattle,isolated inSão Pauloin1987, showedindistinguishableelectrophoreticprofiles(clusterE). Thegeneticrelationshipbetweenbovineand human origi-natedstrainshasbeenforalongtimespeculatedandprevious studiesaround theworldhave,as well,demonstratedvery similaroridenticalgenotypesamongthem.39–41 Infact, we
ourselveshaveearlierreportedhumanandbovineGBSstrains sharinggenetic(samePFGEprofile,ribotypeandrelatedST) andphenotypic(invivovirulenceprofile)characteristics, indi-cating a common evolutionary origin and suggesting that strainsfromdifferenthostscouldpresumablytransfergenetic materialbetweeneachother.25,42Nevertheless,thereisstilla
lottoelucidateaboutthishypothesis.
Conclusion
Inthepresentstudy,almostfourhundredS.agalactiaehuman and bovine strains collected duringa 27-year period were characterizedbyphenotypicandgeneticaspects,generating originalandvaluable dataon serologicaltyping, antimicro-bialsusceptibilityprofileandgeneticdiversity,inordertohelp elucidatingGBSepidemiologyinBrazil.Furthermore,ourdata
aboutmacrolideresistantstrains,amajorconcernregarding theepidemiologyofS.agalactiae,pointoutthatitisratherdue tohorizontalgenetransferthantomonoclonaldissemination amongBrazilianstrains.
Conflict
of
interest
Allauthorsdeclaretohavenoconflictofinterest.
Acknowledgements
ThisworkwassupportedbyCNPq,FAPERJ,CAPES,PRONEX, andTheThrasherResearchFund.
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