Arachidonic acid affects biofilm formation and PGE2 level in Candida albicans and non-albicans species in presence of subinhibitory concentration of fluconazole and terbinafine

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

Arachidonic

acid

affects

biofilm

formation

and

PGE2

level

in

Candida

albicans

and

non-albicans

species

in

presence

of

subinhibitory

concentration

of

fluconazole

and

terbinafine

Nripendra

Nath

Mishra

_,

_Shakir

_Ali

_,

_Praveen

_K.

_Shukla

a_{MedicalMycologyLab,DivisionofFermentationTechnology,CSIR-CentralDrugResearchInstitute,}

B.S.10/1,Sector10,JankipuramExtension,SitapurRoad,Lucknow226021,India

b_{DepartmentofBiochemistry,JamiaHamdard,NewDelhi,India}

a

r

t

i

c

l

e

i

n

f

o

Received26May2013 Accepted13September2013 Availableonline2January2014

Keywords: Arachidonicacid Candidaalbicans AmBR Candidatropicalis ProstaglandinE2 FDA

a

b

s

t

r

a

c

t

Candidaalbicansutilizesarachidonicacid(AA)releasedduringthecourseofinfection (Can-didiasis)fromphospholipidsofinfectedhostcellmembranesandsynthesizesextracellular prostaglandin(s)whichplayanimportantroleinhyphaeformationandhostcelldamage. C.albicansbiofilmssecretesignificantlymoreprostaglandin(s)andevidencesuggeststhat Candidabiofilmshavedramaticallyreducedsusceptibilitytomajorityofantifungaldrugs. AAinfluencesthesaturationleveloflipidsandfluidityofyeastcellmembranes.Therefore theaimofthisstudywastoevaluatetheeffectofAAaloneorincombinationwith anti-fungalagentsonbiofilmformationandproductionofprostaglandin(PGE2)inC.albicans,C. parapsilosis,C.glabrata,C.tropicalis,andC.albicansamphotericinBresistantstrain(AmBR). MaximumbiofilmformationwasfoundtobeinthecaseofC.albicanscomparedtoC. non-albicansspecies.However,amongthenon-albicansspeciesC.tropicalisexhibitedhighest biofilmformation.TreatmentwithAAincombinationwithsubinhibitoryconcentrationsof fluconazoleandterbinafineseparatelyexhibitedsignificant(p<0.05)reductioninbiofilm formationagainstC.glabrata,C.parapsilosis,C.tropicalisandAmBRascomparedtotheir individualeffect.Further,thesetwoantifungalagentsincombinationwithAAcausedan increaseinproductionofprostaglandinfromfungalcellitselfwhichwassignificant(p<0.05) incaseofallthestrainstested.

©2013 ElsevierEditoraLtda.Allrightsreserved.

Introduction

The pathogenic species of the genus Candida have gained recognitionasnosocomialagentsinrecentyearswhichmay beattributedtospecificriskfactorsassociatedwithmodern therapeuticslikeimmunosuppressiveagents,cytotoxicdrugs,

∗ _{Correspondingauthor.}

E-mailaddress:pkshukla@cdri.res.in(P.K.Shukla).

andbroad-spectrumantibioticsthatsuppressthenormal bac-terialmicrobiota.Likewise,useofvariousmedicalimplants may result in Candida biofilm formation that often leads to aggravation ofinfection.Evidence suggests thatCandida biofilms have dramatically reduced susceptibility to clini-callyusedantifungaldrugslikefluconazoleandamphotericin B. Biofilm formation capacity ofC. albicans and its role in

1413-8670/$–seefrontmatter©2013 ElsevierEditoraLtda.Allrightsreserved. http://dx.doi.org/10.1016/j.bjid.2013.09.006

virulence and antifungal resistance has been studied in detail.1_{Candidanon-albicansspecieslikeC.parapsilosisandC.}

tropicalishavealsobeenfoundasbiofilmformingpathogens inrecentpast.2,3

Candidaalbicanshasbeenreportedtoberesponsibleforthe release ofarachidonicacid (AA)from the hostcells during infections4,5 _{whichmaymodulatethecellgrowth,}

morpho-genesisand invasivenessofcausal agentbyseveralmodes. AAisaprecursorfortheproductionofeicosanoidswhichplay animportantroleinmorphogenesisandbiofilmformation. ProstaglandinE2(PGE2)isaprimaryproductofAAmetabolism

inmostoftheeukaryoticcellsthathasalsobeenreportedin pathogenicfungiaswell.6,7_{Enhancedprostaglandin}

produc-tionduringfungal infectionscouldbeoneoftheimportant factorsinpromotingcolonizationaswellaschronicinfections. Theshiftinhostimmuneresponsetowardincreased coloniza-tionandchronicinfectionsisduetoPGE2,whichhasabilityto

elicitbothpro-andanti-inflammatoryresponsesdepending uponthehostcells.8,9

Exogenous AAhasbeen reported toincrease PGE2 level

significantlyinC. albicans whereas thebehavior ofCandida non-albicansspeciesandresistantstrainsinpresenceofAAis notmuchstudiedtillnow.8–10Thestudiesrelatedto determin-ingthelevelofPGE2inC.albicansandC.non-albicansspeciesin

presenceofAAmayhelpinunderstandingthebiofilm form-ingcapacity.Thepurposeofthepresentstudywastocompare thebiofilmformationcapacityandthelevelofprostaglandin secretionoftheC.albicansandCandidanon-albicansspeciesin thepresenceofAAandknownantifungalagents.Thestudy alsoexploresthepatternandamountofbiofilmformationand theamountofprostaglandinsecretedbyalaboratoryinduced amphotericinBresistantC.albicansstrain(AmBR).11

Materials

and

methods

Candidastrainsused

FivespeciesofCandidawereusedinthisstudy.Outofthese fourspeciesviz.C.albicans(ATCC-10231),C.glabrata (ATCC-MYA2950),C.parapsilosis(ATCC-22019),andC.tropicalis (ATCC-750)werepurchasedfromATCC(Americantypecellculture) whilethefifthwasanamphotericinBresistantstrainofC. albicans(AmBR)developedearlierinourlaboratory.11

Mediausedandgrowthconditions

AlltheteststrainsmaintainedonSabourauddextroseagar were grown in yeast extract peptone and dextrose (YPD) medium(DifcoBD,USA)for24hat28◦Cinanincubatorshaker (180rpm).Theyeastcellswereharvested,washedtwicewith 0.15Mphosphate-bufferedsaline(PBS,pH7.4)andsuspended in10mlofsamebuffer.

Antifungalsusceptibilityinvitro

Theminimalinhibitoryconcentrations(MICs)offluconazole (SigmaAldrich,USA)andterbinafine(Biomole,USA)against allthefivestrains(asabove)weredeterminedbybroth micro-dilutionmethodasperguidelinesofCSLI(formerlyNCCLS)12

using RPMI1640 (SigmaAldrich, USA)buffered withMOPS (Sigma Chemical Co.) in 96-welltissue culture plates with positive and negativegrowthas wellas drugcontrols.The test antifungalsterbinafineand fluconazolewere dissolved in DMSO (Merck, Germany) at a concentration of 2mg/ml and 0.64mg/ml respectively, diluted twofold and tested against2×103_{cellspermlofthetestfungi.Themicro-titer}

plateswereincubatedat35◦Cinamoist,darkchamberand MICs were recorded spectrophotometrically (Spectra Max, Moleculardevices,USA)at490nmafter48hincubation.

MICs of fluconazole and terbinafine were determined againstbiofilmformationbyusingthestandardCLSImicro dilution protocol M27-A.12–14 _{Briefly, 1–5×10}3_{cells/ml were}

incubated inRPMI1640buffered withMOPS undershaking condition at37◦C for 90min and the plates were washed three times with PBS to remove non-adherent cells. The test antifungals fluconazole(32–0.25␮g/ml) and terbinafine (100–0.39␮g/ml)weredilutedtwofoldinareplicaplate and thewholecontentstransferredtotheplateshavingtheculture ofbiofilmsandincubatedfor48hat37◦C.Subsequentlythe plates containingCandidabiofilmswerewashedthricewith PBSand50␮lofXTTsaltsolution(1mg/mlinPBS)and4␮lof menadionesolution(1mMinacetone;Sigma)addedtoeach well,incubatedat37◦Cfor5h,andXTTformazanmeasured inthesupernatantmediumat490nmbyusinga spectropho-tometer (Molecular Devices, USA)where ≥80% decrease in formazan(ODat490nm)wastakenasMIC.Theexperiment wasdoneinduplicateandrepeatedthreetimesondifferent occasions.

Biofilmformation

Allthestrainsweresubjectedtobiofilmformationunderthe followinggrowthconditionsseparately:YNBwith50mM glu-cose, YNB with50mM Glucose supplementedwith 500␮M AA (Sigma Aldrich, USA), YNB with 50mM glucose added subinhibitoryconcentration(1/2MIC)offluconazole,YNBwith 50mMglucoseadded1/2MICterbinafine,YNBand50mM glu-cosesupplementedwith1/2MICfluconazoleandAAandYNB supplemented with50mM glucose, 1/2MIC terbinafine and AA.Hereitmaybementionedthatsubinhibitory concentra-tion (1/2MIC) ofboththedrugsfluconazoleand terbinafine against biofilm were used (Table 1). Biofilmformation was carriedoutin96wellcultureplates.15_{Two-hundred␮l}

fun-gal suspensions (1×106_{cells) ofall the test strains in the}

abovemediumwerepouredin96welltissuecultureplates separately,keptundermildshakingconditionfor90minand subsequently thenon-adherentcellswerewashedoutwith PBS.Then200␮lmediacontainingappropriategrowth condi-tionsasabovewasaddedineachwellandincubatedat37◦C

for48h.16,17

Biofilmquantitation

Quantitation of Candida biofilms was performed by using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT; Sigma Aldrich, USA) reduction assay where the water-soluble formazan productwasmeasuredat490nm.17,18 _{Following thebiofilm}

Table1–Minimalinhibitoryconcentration(MIC)ofantifungalagentsagainstdifferentspeciesandstrainsofCandida.

Strains Terbinafine(MICin␮g/ml) Fluconazole(MICin␮g/ml)

Planktonic Biofilm Planktonic Biofilm

MIC 1/2MIC MIC 1/2MIC MIC 1/2MIC MIC 1/2MIC

C.albicans 18.3 9.2 >100 100 0.36 0.18 4.0 2.0

AmBR 0.23 0.12 50.0 25.0 0.61 0.30 32.0 16.0

C.glabrata 72.0 36.0 >100 100 3.31 1.65 32.0 16.0

C.parapsilosis 0.29 0.14 50.0 25 0.74 0.37 2.0 1.0

C.tropicalis 68.0 34.0 >100 100 0.19 0.09 0.25 0.12 XTTreductionassayofbiofilm.

werewashedthricewithPBSand50␮lofXTTsaltsolution (1mg/mlinPBS)and4␮lofmenadionesolution(1mMin ace-tone;Sigma)addedtoeachwell.Theplateswereincubatedat 37◦Cfor5handXTTformazanmeasuredinthesupernatant mediumat490nmbyusingaspectrophotometer(Molecular Devices, USA). Theexperiment was done induplicate and repeatedthreetimesondifferentoccasions.

Fluorescencemicroscopy

Tostudythebiofilmformationfluorescencemicroscopywas performedinsixwell tissuecultureplates and the images were obtained on Leica DM2500 Fluorescence microscope. Briefly, biofilms of above strains were grown in six well tissue culture plates in RPMI 1640 with l-glutamine and 0.165MMOPSunderdifferentconditionsofmediaas men-tionedabove. Afterincubationeach well was washed with DPBS, stained with 100mg/ml fluorescein diacetate (FDA; SigmaAldrich,USA)and50mg/mlpropidiumiodide(PI;Sigma Aldrich,USA)for3handviewedundermicroscope.19

ProstaglandinE2quantitation

Supernatantobtained(after48h)fromthebiofilmcultureof allthestrainsunderdifferentgrowthconditionsasmentioned abovewere subjected toPGE2 estimation.Prostaglandin E2

ExpressEIAkit(Cayman Chemicals)wasusedaccording to manufacturer’sinstructions.Briefly,cellculturesupernatant was useddirectly for prostaglandin estimation and appro-priate standards and controls were run simultaneously to obtainastandardcurve.Eachsamplewasassayedintriplicate andtheexperimentswererepeatedthreetimesondifferent occasions.10 _{Sample concentrationswere calculated onthe}

basisofstandardcurve(plottedagainststandardsversusPGE2

concentration)andthedatarepresentedistheaverageofthree experiments.

Statisticalanalysis

All experimentswere performed intriplicateunless stated otherwise.Thet-testwasperformedtodeterminethe signifi-canceofthedatasets.

Results

and

discussion

Candidiasisisoneofthecommonfungalinfectionsinhumans and C. albicans being endogenous has been considered a

majorcausative agentbut inrecentpast,but Candida non-albicansspecieshavealsomarkedtheirpresenceasfrequent pathogens.Themajorclinical impactgeneratedbyCandida species is their ability to form biofilms which results in increasedresistancetoantifungaltherapyandtheabilityof yeast cells withinthe biofilms to withstand host immune defences.Overandaboveitisinterestingtonotethatthere arespecies-specificvariationsinCandidabiofilmmorphology andformationcapacity.1–3_{Consideringthis,twoantifungals,}

fluconazoleandterbinafinewereincludedinthisstudy know-ingthefactthatthetwoarechemicallydifferentandyetblock thesynthesisofergosterol.

Antifungalsusceptibilityagainstfluconazoleand terbinafine

MICsweredeterminedbyCLSImethod12_{forfluconazoleand}

terbinafine against both planktonic and biofilm cells of C. albicans,C.glabrata,C.parapsilosis,C.tropicalisandAmBR.In thepresentstudyfluconazole(0.19–32␮g/ml)exhibited over-all better spectrum of efficacy as compared to terbinafine (0.23–>100␮g/ml)againstplanktoniccells aswellasbiofilm cells(Table1).Howeverterbinafineexhibitedanedgeover flu-conazoleincaseofAmBRandC.parapsilosis(Table1).

Biofilmformation

Inthepresentstudy,biofilmformationwasstudiedinC. albi-cansandCandidanon-albicansspecies(C.glabrata,C.tropicalis, andC.parapsilosis)andalaboratorydevelopedamphotericin Bresistant(AmBR)strainofC.albicans.11_{Polystyrene96-well}

cell culture plates were usedfor biofilmformation inYNB media,whichwasmeasuredbyXTTreductionassay(Fig.1).It isknownthatbiofilmsexhibitresistanceagainstantifungals comparedtoplanktoniccells;thus,thesub-inhibitory concen-trations(1/2MIC,Table1)ofantifungalagentsmayhaveseveral importantimplicationsonplanktoniccellsdestinedtoform biofilms.14,18,19_{Theinfluenceofdifferenttreatments(growth}

conditions)wascomparedwiththeformationofbiofilmsin YNB alone which was considered as100% for each of the fivestrainstested. Inthepresentstudy YNBsupplemented with AA (500␮M) did not affect the biofilm formation so theresultsindicatethat500␮MAAhasnoinfluenceonthe viability ofthebiofilms. Thesubinhibitoryconcentrationof fluconazolecausedreduced biofilm formationincaseofC. parapsilosis(71.9%±2.9,p<0.05)andtheresistantstrainAmBR

Table2–Biofilmformation(in%)undertheinfluenceofdifferentmediatreatment.

Strains %growthofbiofilmundertheinfluenceofdifferentmedia

YNBa_{+AA YNB}a_{+1/2FLZ YNB}a_{+1/2TER YNB}a_{+AA+1/2FLZ YNB}a_+AA+1/2TER

C.albicans 95.9±1.2 96.4±1.02 93.6±1.4 91.0±.2.4 86.1±1.8

AmBR 94.1±1.7 80.8±3.2 47.2±1.6 61.4±1.9 26.5±1.2

C.glabrata 93.6±1.4 91.9±2.9 62.3±1.4 82.6±2.3 31.6±1.4

C.parapsilosis 90.3±1.8 71.8±2.9 42.7±1.6 60.6±2.2 23.0±1.3

C.tropicalis 99.1±1.1 96.6±1.2 93.1±1.4 88.6±2.3 48.9±2.4

a _{BiofilmformationinYNBalonewasconsideredascontrol(100%).}

(81.9%±3.2,p<0.05).ThebiofilmsofC.albicansaswellasC. non-albicans speciesare known toexert resistance against manyantifungalagentsincluding fluconazole,2,3 _whichhas

alsobeenreportedtoreducebiofilmformationinclinically isolated fluconazole susceptible and resistant strains ofC. albicans.17_{However,inthepresentstudyareductioninbiofilm}

formationwasevidentincaseofAmBRonly(Fig.1andTable2). Thesubinhibitoryconcentrationofterbinafinecaused signif-icantly (p<0.0005) reduced biofilm formation in case ofC. parapsilosis(42.7%±1.6,),AmBR (47.8%±1.6) and C.glabrata (61.2%±1.4) while in case of remaining two strains non-significantreductionwasnoticed(p>0.05,Table2).

Itisencouraging tonotethatboththeantifungalswhen testedincombinationwithAAresultedinfurtherinhibition inbiofilmformationascomparedtoeitherofthetwoagents (antifungalandAA)testedalone.Ourresultsexhibitthat flu-conazoleand terbinafine whentested incombination with AAcausedleastbiofilm formationincaseofC. parapsilosis followedbyAmBRandC.glabrata.However,thiseffectwas morepronounced(p<0.0005)withterbinafineinC.parapsilosis (23%±1.3),AmBR(26.5%±1.2)andincaseofC.glabrataitwas

0 0.5 1 1.5 2 2.5 3

YNB AA FLZ TER FLZ+AATER+AA

X TT re d u c tion O.D C.albicans C.glabrata C.parapsilosis C.tropicalis AmBR

Fig.1–XTTreductionassayofbiofilm.

BiofilmformationbyCandidaalbicans,non-albicans,and AmBRundertheinfluenceofdifferentmediatreatmentwas estimatedbyXTTassay.BiofilmformationinYNBalone wasconsideredascontrol.Further1/2MICoffluconazole (FLZ)andterbinafine(TER)weresupplementedinmedium individuallyorincombinationwitharachidonicacid(AA). ResultsexhibitthatFLZandTERaffectsbiofilmformation butitwasmorepronouncedwhentestedincombination withAAincomparisontocontrol(p<0.005)andleast biofilmformationwasobservedincaseofC.parapsilosis followedbyAmBRandC.glabrata.However,thiseffectwas morepronouncedwithTER(p<0.005)incomparisontoFLZ.

31.6%±1.4(Fig.1andTable2).Inadditiontothisterbinafine withAAalsoreducedbiofilmformationofC.tropicalis indi-catingthatterbinafinewithAAwasthebestcombinationin reducingbiofilmformationagainstteststrainsaswellas resis-tantstrainAmBR(Table2).

Fluorescencemicroscopyforbiofilmevaluation

ThebiofilmcellsofthetestCandidastrainswereinvestigated bystainingwithFDAandPIandviewedunderfluorescence microscope.Theliveanddeadcellsofbiofilmswere distin-guishedbygreen(FDA)andred(PI)fluorescencerespectively indicatingthepresenceofbothviableanddeadcellsinvaried amounts with regard to various treatments. The biofilm formation ofallthe teststrains inYNBalone(Fig. 2A1–E1) were used as respective control where green fluorescent images were observed suggesting the viability of biofilm cells.TreatmentofbiofilmswithvarioustestagentslikeAA alone,1/2MICfluconazole,1/2MICterbinafineand1/2MICof antifungalscombinationwithAAcausedvariedfluorescence (Fig.2).Thesubinhibitoryconcentration(1/2MIC)of flucona-zoleandterbinafinealsoexhibitedsomechanges(Fig.2A3–E3 and A4–E4)influorescenceascomparedtotheirrespective controls which is in agreement with the results of XTT assay(Fig.1).Asexpectedthesubinhibitoryconcentrationof fluconazoleandterbinafinesupplementedwithAAresulted insignificant suppression ofbiofilm formation(Fig.2A5–E5 and A6–E6). Fluorescence microscopy of test strains with different treatments exhibited that 1/2MIC of antifungals in combination withAAaffect the biofilmformation ofall theteststrains,whichwasgreaterincaseofterbinafineas comparedtofluconazole(Fig.2).

PGE2productionbybiofilmsofC.albicansandC.

non-albicansstrains

The planktonicand biofilm formsofC. albicans have been reported toproduce eicosanoids particularly prostaglandin itself and/or in the presence of AA which is greater in biofilms.10 _{Prostaglandins have been known to be}

poten-tial molecules to mediate cross talk between host and pathogenresultingindown-regulationofhostimmunological responses.Prostaglandinsalsoplayanimportantroleinthe persistenceoffungalpathogen inimmunocompetenthosts resulting in chronic infections. As stated above enhanced prostaglandin production has been observed in C. albicans biofilm10_{whereasitsproductioninbiofilmsproducedby}

C. albicans A1 YNB YNB+ AA 1/2 MIC FLZ 1/2 MIC TER 1/2 MIC FLZ+ AA 1/2 MIC TER+ AA A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 D5 D6 E1 E2 E3 E4 E5 E6

C. glabrata C. parasilosis C. tropicalis AmBR

Fig.2–Fluorescencemicroscopyofbiofilm:fluorescencemicroscopycomparisonofbiofilmsgrowninpresenceand absenceofarachidonicacid(AA)treatedwithsubinhibitoryconcentrationoffluconazoleandterbinafineexhibitedthatleast biofilmformationwasobservedincaseofC.glabrata(Figs.B5andB6),C.parapsilosis(Figs.C5andC6)andAmBR(Figs.E5 andE6)comparedtothatincaseofC.albicans(Figs.A5andA6).Theteststrainswithdifferenttreatmentsexhibitedthat 1/2MICofantifungalswithAAaffectthebiofilmformationofallteststrainsandamongalltreatments1/2MICofterbinafine withAAhasgreatereffectthan1/2MICfluconazolewithAA.Thisresultalsosuggeststhatterbinafineincreasesthe susceptibilitywithAAaswellasreducesthebiofilmformation.

yetbeen studied.TheamountofPGE2 secretedbythetest

strains under control condition (YNB alone) for C. albicans (141pg/ml,±6.2),C.glabrata(57.3pg/ml,±1.3),C.parapsilosis (28.1pg/ml, ±1.1), C. tropicalis (29.6pg/ml, ±2.4) and AmBR (100.2pg/ml,±2.1)wasconsideredasbasalconcentrationand theamountofPGE2wascalculatedaftersubtractingthebasal

concentration(Fig.3).AnincreaseinPGE2synthesiswasnoted

forC. albicans (1387±15.0pg/ml), AmBR (1878±11.0pg/ml), C.parapsilosis(1189.3±13.2pg/ml), C.glabrata (1123.0±15.5) and C. tropicalis (1846±15.3pg/ml) when grown in YNB

supplementedwithAA(p<0.005).Theseresultssuggestthat AAcouldhavebeenincorporatedinto thephospholipidsof yeasts,influencingthe saturationlevelandfluidityofyeast cellmembranes.InfluenceofAAoncellmembrane phospho-lipidsunsaturationandergosterolcontenthasbeenreported recently.10,20_{Thereducedergosterolcontentofamphotericin}

B resistant strain11 _{could have been saturated with}

phos-pholipids incorporated byAA. Thereforeincreased amount ofmembranephospholipidsenablesAmBRtoproducemore PGE2thanitsparentstrain.

Table3–PGE2level(pg/ml)estimatedundertheinfluenceofarachidonicacid(AA).

Strains PGE2level(pg/ml)indifferentconditions

YNB+AAa _{YNB+AA+1/2MICFLZ YNB+AA+1/2MICTER}

C.albicans 1387±15.0 2157.3±26.1(p<0.0001) 1655.3±14.1(p<0.0001) AmBR 1878±11.0 1935±16.5(p<0.005) 2114±12.2(p<0.0001) C.glabrata 1123.0±15.5 1174.3±9.2(p<0.05) 1374.3±16.5(p<0.0001) C.parapsilosis 1189.3±13.2 1944±14.5(p<0.0001) 1942.3±2.5(p<0.0001) C.tropicalis 1846±15.3 1966.6±14.0(p<0.005) 1858±9.1(p<0.1) a _PGE

2levelinYNB+AAwasusedasbasalconcentrationforcomparison.

In this experiment the results show that prostaglandin production is enhanced when AA was supplemented withsubinhibitory concentrationoffluconazole, C.albicans exhibited 2157.3±26.1pg/ml PGE2 followed by C. tropicalis

(1966.6±14.0pg/ml),C.parapsilosis(1944±14.5pg/ml),AmBR (1935±16.5pg/ml)and C.glabrata (1174.3±9.2pg/ml) (Fig. 3 and Table 3). PGE2 productionin the presence ofAA with

subinhibitoryconcentration(1/2MIC)ofterbinafinewasmore pronounced (p<0.0005; Fig. 3 and Table 3) against AmBR (2114±12.2pg/ml) and C. glabrata (1374.3±16.5pg/ml). The present results open a new dimension in prostaglandin researchasCandidanon-albicansandC.albicansdrug-resistant strain(AmBR)exhibitedanotabledifferenceinproductionof PGE2individually.

ThevariationinPGE2productioninbiofilmsofthedifferent

teststrainscouldbeindicativeoftheirpersistenceinhost tis-sues.ThelevelofPGE2inallteststrains,includingAmBR,was

0 500 1000 1500 2000 2500

YNB AA FLZ TER FLZ+AATER+AA

PG E 2 c o n c e n tr at io n (pg /m l) C.albicans C.glabrata C.parapsilosis C.tropicalis AmBR

Fig.3–PGE2levelestimationofC.albicansand non-albicansbiofilms.

ThelevelofPGE2producedbyCandidaalbicansand non-albicansbiofilmindifferentmediatreatmentwas estimatedafter48hincubation.SynthesisinYNBmedium wasusedascontrol,furthersupplementedwith

arachidonicacid(AA),1/2MICoffluconazole(FLZ)and terbinafine(TER)individually.ResultexhibitedthatPGE2 formationwasincreasedinthepresenceof1/2MIC antifungalsanditismorepronouncedinC.tropicalisand AmBRagainstTER.ThelevelofPGE2inallteststrains includingAmBR,wasfoundtobeenhancedundermedium ofYNBsupplementedwithAA,1/2MICFLZ(p<0.0005)and AA,1/2MICTER(p<0.0005).PGE2levelwasestimatedby ELISAmethod(CaymanKit).ResultsuggestedthatAA enhancedPGE2productioninthepresenceofsubinhibitory concentrationofantifungals(1/2MIC).

foundtobeenhancedundermediumofYNBwithAA+1/2MIC fluconazoleandwithAA+1/2MICterbinafine(p<0.0005,Fig.3). ThiscanbeexplainedbythefactthatAAincreases suscep-tibility ofC.albicansaswellasCandidanon-albicansspecies againstantifungalagentsand mayaffectthe prostaglandin productionaswell.Thus,itisevidentfromtheresultsthat theexposuretoantifungalagentsenhancestheproductionsof PGE2toagreaterextentinbiofilmswhichmayberesponsible

fordevelopmentofresistanceagainstantifungalagents.

Conclusion

The main general conclusion from this study is that the effectofsubinhibitoryconcentrationsofantifungalagentson biofilmformationisdependentonthetypeofantifungalagent and fungal strain used. Fluconazole and terbinafine when tested incombinationwithAAcausedleastbiofilm forma-tionincaseofC.parapsilosisfollowedbyAmBRandC.glabrata. However,thiseffectwasmorepronouncedwithterbinafine in terms of antifungal activity as well as reduced biofilm formation. Our results furthersuggest that AA could have beenincorporatedintothephospholipidsofyeasts, influenc-ingthesaturationlevelandfluidityofyeastcellmembranes. Further,the enhancedprostaglandin productioninCandida non-albicansanddrugresistantC.albicansascomparedtoC. albicansopensanewareaofresearchtoinvestigatetherole of prostaglandin inantifungal resistancedevelopment and pathogenesis.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

WethanktheDirectorofCDRIandheadoftheFermentation Technology Division, CSIR-CDRI,Lucknow,India for provid-ing facilities.WealsothankICMR,NewDelhiforgivingme fellowship duringthiswork CSIR-CDRICommunicationNo; 8541.

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