Molecular epidemiology of heteroresistant vancomycin-intermediate Staphylococcus aureus in Brazil

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The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Molecular

epidemiology

of

heteroresistant

vancomycin-intermediate

Staphylococcus

aureus

in

Brazil

Alessandro

Conrado

de

Oliveira

Silveira

a,b,∗

_,

_Gabriela

_Rosa

_da

_Cunha

a

_,

_Juliana

_Caierão

a

_,

Caio

Mauricio

Mendes

de

Cordova

b

_,

_Pedro

_Alves

_d’Azevedo

a

a_{LaboratóriodeCocosGramPositivos,UniversidadeFederaldeCiênciasdaSaúdedePortoAlegre,PortoAlegre,RS,Brazil} b_{DepartamentodeCiênciasFarmacêuticas,Fundac¸ãoUniversidadeRegionaldeBlumenau,Blumenau,SC,Brazil}

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Articlehistory: Received23April2015 Accepted12June2015

Availableonline21August2015

Keywords: MRSA

Smallcolonyvariants Antimicrobialresistance

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Todeterminetheepidemiologicalandmolecularcharacteristicsof12Staphylococcusaureus isolatespresentingheteroresistancetovancomycininlaboratoriesoftwocitiesinSanta Catarina,southernBrazil.Epidemiologicaldata,includingthecityofisolation,health insti-tution,anddateofisolationwereconsidered,aswellastheassociatedclinicalspecimen.For molecularcharacterization,weanalyzedthestaphylococcalcassettechromosometypes, theermgenepresence,andthegenomicdiversityofisolatesusingpulsed-fieldgel elec-trophoresis.The12isolatesofS.aureuswerepreviouslyconfirmedasheteroresistanceto vancomycinusingthepopulationanalysisprofile–areaundercurve.Regardinggenetic vari-ability,twoclonesweredetected:themainone(cloneA)composedoffourisolatesandthe clonesB,withtwoisolates.ForcloneA,twoisolatespresentedidenticalbandpatternsand wererelatedtothesamehospital,withanintervalof57daysbetweentheirisolation.The otherisolatesofthiscloneshowednoepidemiologicallinkbetweenthembecausetheywere isolatedindifferenthospitalsandhadnotemporalrelationship.Theothercloneshowed nodetectableepidemiologicalrelationship.Theheteroresistancetovancomycinrecovered inSantaCatarinaStatefrom2009to2012had,ingeneral,heterogeneous genomic pat-ternsbasedonpulsed-fieldgelelectrophoresisresults,whichisinaccordancewiththefact thattheseisolateshadlittleornoepidemiologicalrelationshipamongthem.Duetothe characteristicphenotypicinstabilityandoftenprolongedvancomycintherapyforselection, clonalspreadisnotascommonasforotherresistancemechanismsdisseminatedthrough horizontalgenetransfer.

©2015ElsevierEditoraLtda.Allrightsreserved.

∗ _{Correspondingauthor.}

E-mailaddress:alessandrocosilveira@gmail.com(A.C.O.Silveira). http://dx.doi.org/10.1016/j.bjid.2015.06.013

Introduction

Staphylococcus aureus can acquire, during prolonged ther-apywithglycopeptides,averypeculiarresistantphenotype. Through selective pressure and apparently without trans-ferofgeneticmaterial,itmay undergo mutationsingenes responsibleforcellwallproduction,makingitthickerandless susceptibletotheactionofantimicrobials.1

TheheteroresistanceofS.aureustovancomycin(hVISA) causeschangesinthemacro-morphologicalfeaturesofthe colonies,that present with heterogeneousappearance and pigmentation,giving the impressionofcontamination and may confuse the microbiologist.2 _{Its mechanism of} resis-tance is associated with activation of cell wall synthesis, whichincreases the productionofwastemucopeptide and reduces the amount of antibiotic that reaches the site of action,thuscausingcellwallthickeningandsubsequentdrug imprisonment.3_{SpeculationthathVISAcouldberegardedas} precursorsofVISAstrainsonce,afterprolongedexposureto antimicrobialcouldselectahomogenouspopulationofcells expressingthephenotypeVISA.Thisphenotypeisunstable.4 ThedetectionofinfectionscausedbyhVISArepresentsa challengeformicrobiologists,sincethesestrainsare consid-eredsusceptibletovancomycininvitro(minimuminhibitory concentration(MIC)≤2␮g/mL),andthereforecategorizedas susceptiblebytheusuallaboratorymethods.1_However,asit containssubpopulations1ineach106_{bacterialcells,thatcan} growinthepresenceof4␮g/mLofvancomycin,itmayleadto treatmentfailure5,6_{withvancomycin,.}

Reference methods used to evaluate susceptibility as broth microdilution, Etest and automated methods fail to detect hVISA. Because the phenotype is a heterogeneous phenomenon,reliablemolecularmarkerstoconfirmthis phe-notype have not yet been found. Partly due to the small inoculum,relativelypoorgrowthonMueller-Hintonagar,or incubationforonly24h.Theinoculumsizeiscriticaltothe detection of subpopulation of resistant cells; furthermore hVISAstrains arenotoriouslyslow growing,withcell walls thickerandpleomorphicuniquecharacteristics,withcolonies ofvaryingsizesandnutritionallyexacting.7

The population analysis profile–area under the curve (PAP–AUC) has been the most reliable and reproducible approach,beingconsideredthegold-standardtestforhVISA.8 It was specifically designed for discriminating hVISA and VISA.Itisamethodofanalysisofmodifiedsub-populations usingserialconcentrationsofvancomycin,inorderto quan-tifytheviablebacterialpopulationsinsuchconcentrations.It isaverylaborious,expensive,andinappropriateforroutine useinclinicallaboratories.9

Inameta-analysispublishedin2011,the ratesof treat-mentfailure(designatedaspersistentinfectionorbacteremia) relatedtohVISAisolatesweretwotimesmorecommonthan ininfections causedbyS.aureussusceptibletovancomycin (OR:2.37,95%CI:1.53–3.67).6_{Therefore,anaccurateand} prac-ticallaboratorymethodforthedetectionofhVISAisolatedin clinicalpracticeisofincreasingimportance.10

Despite the controversy between studies regarding the association ofhVISA and mortality, knowledge ofthe epi-demiologicalprofileisveryimportantinassistingtheclinician

when choosing the appropriate antibacterial therapy. The objective ofthisstudy was toevaluatethe phenotypic and molecularepidemiology characteristicsofhVISAisolatesin thestateofSantaCatarina,Brazil.

Methods

Bacterialsamples

Weused12clinicalisolatesofhVISAobtainedfromvarious anatomicalsitesofpatientsinhospitalsinFlorianópolisand hospitalinBlumenau,alllocatedinthestateofSantaCatarina insouthern Brazil.Sampleswere collected fromNovember 2009toOctober2012.

Antimicrobialsusceptibilitytesting

Antimicrobialsusceptibilitytestingwasperformedusingthe diskdiffusionmethod,accordingtotherecommendationsand interpretivecriteriaoftheClinicalandLaboratoryStandards Institute.11 _{Wealsoperformedthe D testforthe detection} ofinducibleresistancetoclindamycin.Disksoferythromycin andclindamycinwereplaced26mmapart,andaflattening ofthe inhibitionzone indicated apositivetest, which was reportedasclindamycinresistance.VancomycinMICswere determinedbymacrodilutionmethod.11

PAP–AUC

Afterincubationonsolidmedium,thebacteriawerediluted insterilesalineatdilutionsrangingfrom 10−1 to10−8 and subsequentlyspottedas10-␮LspotsonBHIagarplates con-taining 0, 0.5, 1, 2, 3, 4, 5, 6 and 8␮g/mL of vancomycin. The plates were incubated for 48h, and the colonieswere countedtodeterminethelog10CFU/mL;thesedatawerethen plottedonagraphasafunctionofthevancomycin concen-tration.TheAUCwascalculatedusingthestrainMu3(ATCC 700698)as acontrol. Toconfirmthe designation ashVISA, theratiooftheAUCoftheisolatetothatoftheMu3strain mustbegreaterorequalto0.9andnon-hVISAisolateshada PAP–AUC<0.9.7,8

MultiplexPCRforthedetectionofthestaphylococcal cassettechromosome(SCCmec)

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

PCRforermgenedetection

For isolateswithpositiveresultsinthe phenotypictestfor inducibleresistancetoclindamycin,ermgenePCR amplifica-tionwasperformedaccordingtothemultiplexPCRprotocol developedbyKhanetal.14_{ThePCRproducts(610bpforermA} and520bpforermC)wereanalyzedbyelectrophoresisthrough a1.5%agarosegel.14,15

12.00 10.00 8.00 6.00 4.00 2.00 0.00 0 0.5 Log 10 CFU/mL 1 2 3 4 5 6 8 Isolate S13 Isolate S4 Isolate S11 Mu3 Isolate 43 Isolate 10 Isolate 36 Mu3 Vancomycin concentration (μg/mL) 0 0.5 1 2 3 4 5 6 8 Vancomycin concentration (μg/mL) PAP-AUC PAP-AUC

A

12.00 10.00 8.00 6.00 4.00 2.00 0.00 Log 10 CFU/mL

B

12.00 10.00 8.00 6.00 4.00 2.00 0.00 0 0.5 Log 10 CFU/mL 1 2 3 4 5 6 8 Isolate 74 Isolate 54 Isolate 69 Mu3 Isolate 84 Isolate 92 Isolate 80 Mu3 Vancomycin concentration (μg/mL) 0 0.5 1 2 3 4 5 6 8 Vancomycin concentration (μg/mL) PAP-AUC PAP-AUC

C

12.00 10.00 8.00 6.00 4.00 2.00 0.00 Log 10 CFU/mL

D

Fig.1–GrowthcurvesofthetwelveisolatescharacterizedashVISAcomparedtothestandardstrainMu3.Allisolates showedanAUC/Mu3AUCratiogreaterthan0.9.Forclarity,bacterialgrowthwasexpressedinlog10CFU/mL.Nine

concentrationsofvancomycin(0,0.5,1.0,2.0,3.0,4.0,5.0,6.0and8␮g/mL)wereused.GraphAdepictsisolatesS4,S11and S13.GraphBshowsisolatesL10,L43andL36.GraphCshowsisolatesL54,L69andL74.GraphDshowsisolatesL80,L84 andL92.ThereferencestrainMu3isshowninallgraphs.

Pulsed-fieldgelelectrophoresis(PFGE)

PFGEwasperformedaccordingtoMcDougaletal.16_andPinto etal.17_{ThefragmentsweresubjectedtoPFGEusing1%agarose} gels (Pulsed Field Certified Agarose; Bio-Rad) in 0.5× Tris-borate-EDTAbufferwithaCHEF-DRIIIsystem(Bio-Rad).The gelswerestained with0.5␮g/mLethidiumbromide, visual-izedunderUVlight,andphotographedusingaGelDocTM_XR System (Bio Rad). The PFGE patterns were analyzed using Bionumericsversion6.1(AppliedMaths,Sint-Martens-Latem, Belgium).ThePFGEpatternswereclusteredbyUPGMA.A den-drogramwasgeneratedfromasimilarity matrixcalculated usingtheDicesimilaritycoefficientwithanoptimizationof 0.5%andatoleranceof1%.PFGEclustersweredefinedas iso-lateswithasimilarityof80%orhigheronthedendrogram.18

Results

ThetwelvehVISAisolateswererecoveredfromtracheal aspi-rates(n=5),osteomyelitis(n=4),blood(n=1),skinlesion(n=1), and surgicalwound(n=1).Resistance ofthe 12 hVISA iso-latestoantimicrobialagentswasassessedbydiskdiffusion, withthefollowingresults:clindamycin(92.7%),erythromycin (100%),trimethoprim/sulfamethoxazole(16.7%),ciprofloxacin (92.7%),tetracycline(16.7%),chloramphenicol(8.3%),and gen-tamicin (33.3%). All isolates were considered susceptible

to linezolid and teicoplanin. The vancomycin MICs were 1.0␮g/mL(33.3%)and2.0␮g/mL(66.7%)(Fig.1).

Only two isolates (10 and 54) exhibited inducible clin-damycinresistance,asdeterminedbyapositiveDtest.These isolatesweresubjectedtoPCRforermgenedetection,andboth containedtheermAgene.Isolate84,whichwasresistantto erythromycinandsensitivetoclindamycin,yieldedanegative Dtest,andtheermgenewasnotamplified.Allotherisolates showedconstitutiveclindamycinresistance.

IsolateSI4containedtwoSCCmectypes(IandII).Isolate 84 containedSCCmec typeI.IsolatesS11,36,54,69,80 and 92 containedSCCmec typeII. SCCmec typeIIIwasobserved inisolatesSI13,10and 43.Althoughisolate80 was consid-eredtobeacommunityisolate,itdidnotcontainSCCmectype IV,whichisfrequentlyassociatedwithcommunity-acquired methicillin-resistantS.aureus(CA-MRSA).Onlytheisolate74 showedSCCmectypeIV(IVc).Theisolationofthe microorgan-isminthefirst48hofadmissionwasusedasthecriterionfor classificationasCA-MRSA.

PFGE was used to assess the degreeof genetic similar-ity, andtheresultsarepresentedinFig.2.Twocloneswere observed:aprimaryclone(A),comprisingfourisolates(SI4, L54,L69,andL92);andcloneB,comprisingisolatesL36and L80.IsolatesSI11,SI13,L10,L43,L74,andL84didnotdisplay geneticsimilarity withany ofthe other isolates.The crite-riaestablishedbyTenoverwereusedforclassification,with asimilarityindexof80%.

pfge pfge L 43 L 84 L 74 L 54 L 69 L 92 L 36 L 80 Mu 3 SI 11 Cordobean 012 Cordobean 023 OSPC E-MRSA 16 E-MRSA 15 BEC NY/Japan Pediatric USA 300 USA 400 SI 4 L 10 SI 13 75.0 65.5 50.0 38.4 35.1 91.7

A

B

78.6 67.6 67.1 78.3 66.7 50.5 45.2 36.6 47.6 60.0 56.0 53.4 32.7 24.9 30 40 50 60 70 80 90 100

Fig.2–Dendrogramshowingthesimilaritiesbetweenthe12hVISAisolates.Twocloneswereobserved:onecomprising fourisolates(A),twoofwhichdisplayed100%similarity(L54andL69);andcloneB,whichcomprisedtwoisolatesthatwere geneticallyindistinguishable(L36andL80).TheSI11,SI13,L10,L43,L74andL84isolatesdidnotexhibitanysimilaritywith theotherisolates.

In addition to the molecular characteristics described above,Table1presentsthecity ofisolation,attended insti-tution, biological sample, and isolation date. The samples wereisolatedintwodifferentcitiesthatare120kmapart.In thecityofFlorianópolis,thestatecapitalofSantaCatarina, threehospitalsandtwoclinicswereincluded.Approximately 33monthsseparatedthe firstandlastisolates.IsolatesL54 andL69wereisolatedinthesamehospital,had100%genetic similarity,andwereisolatedfrompatientswithosteomyelitis

approximately57daysapart,whichmightindicateacommon routeofinfection.

Discussion

Asfar asweknow,thisisthe firstdescription ofhVISAin Brazil.TwoBrazilianstudiesreporteddecreasedsusceptibility tovancomycin,buthavenotconfirmedhVISAphenotype.In

Table1–Epidemiologicalandclinicalcharacteristicsofthe12hVISAisolates.

Isolatenumber City Institution Clinicalsample SCCmec Clone Date

10 Florianópolis HospitalA Osteomyelitis III Nonclonal 23/11/2009

36 Florianópolis HospitalA Trachealaspirate II B 03/09/2010

43 Florianópolis HospitalA Surgicalwound III Nonclonal 14/10/2010

54 Florianópolis HospitalA Osteomyelitis II A 04/05/2011

69 Florianópolis HospitalA Osteomyelitis II A 01/07/2011

74 Florianópolis HospitalB Skinlesion IVc Nonclonal 22/12/2011

80 Florianópolis ClinicA Trachealaspirate II B 09/01/2012

84 Florianópolis HospitalA Trachealaspirate I Nonclonal 25/02/2012

92 Florianópolis HospitalC Trachealaspirate II A 18/08/2012

SI4 Blumenau HospitalD Blood I,II A 28/09/2010

SI11 Blumenau HospitalD Trachealaspirate II Nonclonal 20/02/2011

2001,Oliveiraandcolleaguesreportedfiveclinicalisolatesof MRSAwithMICof8␮g/mL,whichwerenamedVRSA,basedon availablecriteria(NCCLS-1997).However,theisolatesdidnot harborVangenesand,aspreviouslydemonstratedbyother authors,presentedacellwallthickening.19_{In2006,Lutzand} Barthdescribed18clinicalisolatesaspossiblehVISA,since theywerepositiveforscreeningtest(BHIwith4␮g/mLof van-comycin).However,thePAP–AUCconfirmatorytestforhVISA wasnotperformed,especiallybecause,onthattime,the cur-rentacceptedcriteriaforconfirmationofthisphenotypewere notknown.20,21_{Basedonthesedata,webelieveourstudy} pro-videsthefirstreportofhVISAisolationinBrazil.

The most frequently anatomical sites associated with hVISAinfectionsarethosewithahigherbacterialinoculum (abscesses, pneumonia) and those associated with chronic infections(endocarditis,osteomyelitis)forwhichthe useof vancomycinforprolongedperiodsisquitecommonandthe lowpenetrationoftheantibioticinthesesitesfavorsthe devel-opmentofresistance.7_{Severalstudies}3,22–25_{haveestablished} thatblood,lowerrespiratorytract,skinwounds,abscessesand osteomyelitisarethemostcommonsitesofhVISAisolation. Thesefindingsaresimilartothoseofthepresentstudy.

Theisolatesexhibitedaprofileofheterogeneous suscepti-bility,characterizedbylowresistancetochloramphenicoland trimethoprim/sulfamethoxazole,drugs that are used infre-quently to treat MRSA infections. Other studies22,24 _have reportedlowratesofresistancetosulfonamidesinhVISA iso-lates,rangingfrom9to9.9%.Bycontrast,studiesofsamples fromKorea26_andChina23_{revealedmuchhighervalues,58and} 54%,respectively.Achloramphenicolresistancerateof16.7% wasobservedinourstudy,whileHuandcolleaguesobserved a rate of 47.6% in 2013.23 _{Resistance rates to macrolides,} lincosamides,aminoglycosides,fluoroquinolones,and tetra-cyclinesare high,withvaluesgreater than64%,precluding itsempiricaluse.22–24,26_{Knowledgeofthelocalsusceptibility} profileisessentialforadequateempiricaltherapy.

Clindamycin is a therapeutic option for the treatment ofserious infections caused by S. aureus, including MRSA, particularlyforisolateswithSCCmectypeIV,whichusually areresistantonlytobeta-lactams.27_{However,onecommon} mechanism of resistance is the macrolide-lincosamide-streptograminB(MLSB)phenotype,whichexpressesinducible clindamycinresistance,which cannotbedetectedby tradi-tionalphenotypictestsandrequirestheDtest.28_Inourstudy, twoisolatesexhibitedinducibleclindamycinresistance,and bothpossessedtheermAgene.Bothwerereportedtobe resis-tanttoclindamycin,andthus inappropriatetreatmentwas avoided.

TherewasapredominanceofSCCmectypeII,whichis asso-ciatedwithhVISA;inMRSA,thistypeisassociatedwithhigher mortalityrates.23_{Otherstudieshavealsodemonstrateda} pre-dominanceofSCCmectypeIIamonghVISAisolates.22,23,29,30 Somestudies havedemonstrated apredominance ofother typesofSCCmecbetweenhVISAisolatessuchastypeI31_and typeIII.32–34_{Itisinterestingtonotethatamongthe12isolates,} onlyonehadSCCmectypeIV(IVc).Althoughsomestudiesdid notfindtypeIVSCCmecamongtheirMRSAisolates,31,34_others demonstratedratesashighas16.8%22_to26.3%.32

Fromanepidemiologicalperspective,itisdifficultto per-formfurtheranalysesduetothegeographicaldistanceandthe

timeintervalbetweentheisolationofthefirstandlastisolates. Themicroorganismswereisolatedoveraperiodofthreeyears indifferentcitiesandeveninseveraldifferentinstitutionsin thecityofFlorianópolis.ExceptforisolatesL54andL69,which arethesamecloneandwereisolatedinthesameinstitution, thehVISAisolatesdidnothaveanycharacteristicsthatwould enableanepidemiologicallinktobemadebetweenthem.

ThehVISArecoveredinSantaCatarinaStatefrom2009to 2012had,ingeneral,heterogeneousgenomicpatterns accord-ingtothePFGEresults,whichisinaccordancewiththefact thattheseisolateshadlittleornoepidemiologicalrelationship amongthem.

It should be noted that due to its continental dimen-sionsandtouristvocation,Brazilpresentsagreatdiversityof resistancemechanisms,andS.aureuswithresistanceto van-comycin(VRSA)wasfirstdescribedin2014inSouthAmerica.35 Thisfactdemonstratestherealneedforthesurveillanceof bacterialresistanceinourcountry.

Epidemiologicalinformationfromeach healthfacility,as wellaseachgeographicalregion,iscriticaltothe implemen-tationofappropriateempiricaltherapy,especiallywithregard tohVISAisolates.Suchbacteriahavephenotypic characteris-ticsthataredifficulttobecorrectlyidentifybyconventional methods (MIC determinationand molecular tests)and are associatedwithworseclinicaloutcomes.Duetothe charac-teristicinstabilityphenotypeandoftenprolongedvancomycin therapyforselection,clonalspreadisnotascommonasfor otherresistancemechanismsdisseminatedthrough horizon-talgenetransfer.

It is imperative that clinical microbiology laboratories detect the hVISAphenotype and associateit with the epi-demiologicalcharacteristicsofthepatients(e.g.,age,dateof isolation,anatomicalsite,prioruseofvancomycin)toprovide important information for research laboratories. Through appropriatemethodologies,associationsbetweenthe micro-biologicaldataandtheclinicalcharacteristicsofpatientsmay enablethedetectionofpossibleassociatedriskfactorsandaid inassessingtheprognosisofpatients.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

The authors thanks to Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Pesquisa e Desenvolvimento Tecnológico (CNPq) and Fundac¸ão de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS)forfinancialsupport.ThankstoLaboratórioSanta Luzia(CassiaZoccoliandNinaTobouti)andLaboratórioSanta Isabel(MarceloMolinari)forthegrantingofclinicalsamples.

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