The increasing use of azole antifungals for the treatment of mucosal and systemic Candida glabrata i.docx

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

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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