The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata i.docx
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TheincreasinguseofazoleantifungalsforthetreatmentofmucosalandsystemicCandidaglabratai
MechanismsofAzoleResistanceinClinicalIsolatesofCandida
glabrataCollectedduringaHospitalSurveyof
AntifungalResistance
TheincreasinguseofazoleantifungalsforthetreatmentofmucosalandsystemicCandidaglabratainfectionshasresultedintheselectionand/oremergenceofresistantstrains.ThemainmechanismsofazoleresistanceincludealterationsintheC.glabrataERG11gene(CgERG11),whichencodestheazoletargetenzyme,andupregulationoftheCgCDR1andCgCDR2genes,whichencodeeffluxpumps.Inthepresentstudy,weevaluatedthesemolecularmechanismsin29unmatchedclinicalisolatesofC.glabrata,ofwhich20isolateswereresistantand9weresusceptibledosedependent(S-DD)tofluconazole.Theseisolateswererecoveredfromseparatepatientsduringa3-yearhospitalsurveyforantifungalresistance.Fourofthe20fluconazole-resistantisolateswereanalyzedtogetherwithmatchedsusceptibleisolatespreviouslytakenfromthesamepatients.Twentyotherazole-susceptibleclinicalC.glabrataisolateswereincludedascontrols.MICdataforallthefluconazoleresistantisolatesrevealedextensivecross-resistancetotheotherazolestested,i.e.,itraconazole,ketoconazole,andvoriconazole.Quantitativereal-timePCRanalysesshowedthatCgCDR1andCgCDR2,aloneorincombination,wereupregulatedathighlevelsinallbuttwofluconazole-resistantisolatesand,toalesserextent,inthefluconazole-S-DDisolates.Inaddition,slightincreasesintherelativelevelofexpressionofCgSNQ2(whichencodesanATP-bindingcassette[ABC]transporterandwhichhasnotyetbeenshowntobeassociatedwithazoleresistance)wereseeninsomeofthe29isolatesstudied.Interestingly,thetwofluconazole-resistantisolatesexpressingnormallevelsofCgCDR1andCgCDR2exhibitedincreasedlevelsofexpressionofCgSNQ2.Conversely,sequencingofCgERG11andanalysisofitsexpressionshowednomutationorupregulationinanyC.glabrataisolate,suggestingthatCgERG11isnotinvolvedinazoleresistance.Whentheisolatesweregrowninthepresenceoffluconazole,theprofilesofexpressionofallgenes,includingCgERG11,werenotchangedorwereonlyminimallychangedintheresistantisolates,whereasmarkedincreasesinthelevelsofgeneexpression,particularlyforCgCDR1andCgCDR2,wereobservedineitherthefluconazole-susceptibleorthefluconazole-S-DDisolates.Finally,knownABCtransporterinhibitors,suchasFK506,wereabletoreversetheazoleresistanceofalltheisolates.Together,theseresultsprovideevidencethattheupregulatlonoftheCgCDR1-,CgCDR2-,andCgSNQ2-encodedeffluxpumpsmightexplaintheazoleresistanceinoursetofisolates.
Candidaglabratahasrecentlyemergedasasignificantpathogeninvarioushospitalsettings,whereitisresponsibleforanincreasingnumberofsystemicinfectionsandcandiduria(2,16).Inarecentstudy,C.glabratawasthesecondmostcommonnon-C.albicansspeciesasacauseoffungemiaintheUnitedStatesandwasfoundtoaccountfor21%ofallCandidabloodstreamisolates(26).SecondonlytoC.albicans,C.glabrataisalsotheCandidaspeciesmostcommonlyrecoveredfromtheoralcavitiesofhumanimmunodeficiencyvirus-infectedpatients(13,16,40).
TheriseinthenumberofC.glabratasystemicinfectionsdeservesagreatdealofconcernduetothehighmortalityrateassociatedwithC.glabratafungemiaandtothepropensityofthismicroorganismtorapidlydevelopresistancetoazoleantifungalagents(10,19).SeveralstudieshaverevealedthatasignificantpercentageofC.glabrataclinicalisolatesareresistanttofluconazole(approximately9%)anditraconazole(37to40%)(3,16,25).Morerecently,inasurveillancestudyconducedbypfalleretal.(27)toexaminetheantifungalsusceptibilitiesofCandidaspeciesisolatedfrompatientswithbloodstreaminfectionsstratifiedbypatientage,atrendofdecreasingsusceptibilitiestofluconazoleanditraconazolewithincreasingpatientagewasobserved.Infact,noneoftheC.glabrataisolatesfromindividuals:
51yearoldwereresistanttofluconazole,whereasahigherproportion(5to9%)ofresistantisolateswasfoundinadultpatients.Similarly,among347bloodstream,invasive,andcolonizingstrainsOfC.glabrataisolatedfrompatientsatthreeurbanteachinghospitalsinNewYorkCity,theoverallratesofresistancetofluconazoleanditraconazolewere10.7and15.2%,respectively(33).ThemechanismsofresistancetoazoleantifungalagentshavebeenwellelucidatedinC.albicansandcanbemainlycategorizedas(i)changesinthecellwallorplasmamembrane,whichleadtoimpairedazoleuptake;(ii)alterationsintheaffinityofthedrugtargetErgl1p(lanosterol14a-demethylase)toazolesorinthecellularcontentofErgl1pduetotargetsitemutationoroverexpressionoftheERG11gene;and(iii)theeffluxofdrugsmediatedbymembranetransportproteinsbelongingtotheATP-bindingcassette(ABC)transporterfamily(CDR1andCDR2)ortothemajorfacilitatorsuperfamily
RIandFLU1).Inthelastcase,theCDR1andCDR2sandtheMDR1genewereshowntobeoverexpressedinsresistantisolates,anddeletionofthesegenesresultedininsensitivitytoazoles(34).Inaddition,compensatorypath-thatinvolvealterationsofspecificstepsinergosterolrithesishavebeendocumentedasmechanismsofresisetotheazoleandpolyeneantifungalclasses(39).
iorerecently,increasedlevelsofexpressionoftheABC/TonergenesC.glabrataCDR1(CgCDR1)andCgCDR2itbeenalsoshowninazole-resistantisolatesofC.glabrata'15,35,36).SimilartoC.albicans,geneticevidencesupport-*roleofmultidrugtransportersintheazoleresistanceof?
'bratawasprovided(36).Moreover,Marichaletal.(14)euslyshowedincreasedlevelsofexpressionofERG11invole-resistantC.glabratastrainwhicharosefromachronalduplication.Incontrast,ithasyettobewellexplorederpointmutationsintheERG11genearealsoimpli-0intheresistanceofC.glabratatoazoles.
`6epurposeofthepresentstudywastodetermineifthe)cularmechanismsdescribedabove,aloneorincombinaweresufficienttoexplainthephenotypeofazoleresisinunmatchedclinicalC.glabrataisolatesobtainedfrompusclinicalspecimensduringa3-yearhospitalsurveyof`tingalresistanceorifother(notwell-established)mechamightcorrelatewithazoleresistance.Inaddition,pairs;saceptibleandresistantC.glabrataisolatesthathadbeentinedfromthesamepatientandthathadthesamegenotwerealsoexamined.
MATERIALSANDMETHODS
!
isolatesandgrowthconditions.TheisolatesofC.glabrataincludedinmotstudywerefromacollectionofclinicalisolatesrecoveredduringan.miologiealsurveyofantifungalresistanceconductedat-ourinstitution,auniversityhospitalinRome,overa3-yearperiod(January2000throughber2003).Theywereidentifiedbystandardmethods(43)andtestedfor
/7
ceptibilitiestoamphoterieinB,flucytosine,fluconazole,ketoconazole,1,.azole,andvoriconazolebytheSensititreYeastOnecommercialmethodurlicalService,Milan,Italy),asrecommendedbythemanufacturer.TwenaisolatesforwhichfluconazoleMICsexceededtheestablishedsusceptiilireakpoint(MIC5Spg/mt)(17,32)wereretestedfortheirantifungalnbilitiesbytheNCCLSreferencemethodforconfirmationandwerethendformolecularstudies(seebelow).Theseisolateswererecoveredfrombodysitesof29separatepatients(Table1).Amongtheisolatesstudied,
(
were4fluconazole-susceptibleisolatesthatmatched4fluconazole-resis'Elatesobtainedfromthesamepatient,aswellas20randomlyselectedrtflisolatesofC.glabratasusceptibletoazoles(Table2).Inaddition,twoidsracterizedC.glabrataisolates,asusceptibleisolate(isolateDSY562136j)rresistantisolate(isolateDSY5651361),kindlyfurnishedbyDominiquebird,wereusedascontrols.All53isolateswerekeptat-80°Cas20%palstocksandweresubcultured,asrequired,onYEPD(1%yeastextract,splone,2%glucose)agarplatesat30°C.Forliquidculture,theisolates]gowninYEPDbrothat30°Cunderconstantagitation(240rpm).
dolgalsusceptibilitytestingbytheNCCLSreferencemethod.Reference'iogalsusceptibilitytestingofthestudyisolateswasperformedbythebrothIliilutionmethoddescribedinNCCLSdocumentM27-A2(17).Powdersof!
eiikingalagentsamphotericinB,flucytosine,ketoconazole.fluconazole,!
anzole,anditraconazolewereobtainedfromtheirrespectivemanufacturHy,MICsweredeterminedwithRPMI1640with2%glucosebyuseofMitunsizeof1.5(±1.0)x103cells/nilandincubationat35°Cfor48h.ScontrolwasensuredbytestingtheNCCLS-recommendedstrainsC.ATCC6258andC.parapsilosisATCC22019(4).Alltestswerecarriedoutlicate.Theinterpretivecriteriatotsusceptibilitytofluconazole,itraconundflucytosinewerethosepublishedbyRexetal.(32)andtheNCCLS4dwereasfollows:
(i)forfluconazole,susceptible,58pg/ml;susceptible-!
Ipendent(S-DD),16to32pg/m1;andresistant,64µg/m1;(ii)forzuole,susceptible,50.125pg/m1;S-DD,0.25to0.5pg/ml;andresistant,
Quantitativereal-time.RT-PCR.Forquantitativereal-timereversetranscription(RT)-PCRanalysis,totalRNAwasextractedfromC.glabrataculturesgrowntothemid-exponentialphase(opticaldensityattwonm[OD,,,„)],approximately0.6)withanRNAeasyProtectminikit(Qiagen,Hilden,Germany),accordingtotheinstructionsofthemanufacturer,bymechanicaldisruptionofthecellswithglassbeadsandanRNase-freeDNasetreatmentstep.RNAintegritywasassessedbydeterminationoftheOD.26„/OD-,,,,,,,absorptionratio,andth