Introduction
VolatilesproducedbyCandida SppJamesRedfern1,SarahJackson1,JoannaVerran1SchoolofHealthcareScience,ManchesterMetropolitanUniversity
GaschromatographyofC.albicansInordertoinvestigatetheMVOCsreleasedbyC.albicansbiofilms,gaschromatographycoupledwithmassspectrometryandsolidphasemicroextraction(SPMEGC-MS)wasused.Thecolumntemperaturewasmaintainedat70°Cfor10minutes,thenincreasedto270°Catarateof10°Cperminute,andheldforthreeminutes.Theinjectoranddetectortemperaturesweresetto270°C.Theinjectorwassplitlesswithaflowratesetto100mlperminute,resultinginaruntimeof33minutes.Thismethodwasdevelopedfromapreviousmethodusedtoidentifyvolatilecompoundsproducedbyfungalisolates(Bingleyetal.,2012).Blastosporeandhyphalbiofilmswereexaminedat24,48and72hoursforthevolatilecompoundsproduced.
Methods
Candidaalbicans isafunguscapableofcausingdenturestomatitis,themostcommonformoforalcandidiasis,presentinupto60%ofdenturewearers,affectingpredominantlypatientsover65yearsold(Daniluketal,2006).Symptomsincludemucosalbleeding,swelling,burningandotherpainfulsensations,halitosis,unpleasanttasteanddrynessinthemouth(Dorocka-Bobkowskaetal,2010).Likemostotherfungi,C.albicans iscapableofproducingmicrobialvolatileorganiccompounds(MVOCs),withover150describedintheliteratureItispossibletocreatebiosensorswherebyMVOCsinteractwithanelectrode(similarinconstructiontothewell-establishedglucosesensorwherethesurfaceconsistsofgraphiteparticles,apolymerbinderandotheradditives),changingsurfacepropertiesallowingamountofcompoundtobeinstantlydetected.Tothisend,uniqueprofilesofMVOCforCandidagrowthondenturematerialswillberequiredforthedevelopmentofasensorcapableofdetectingcolonizationofdentures.
Figure1-
Blastosporeandhyphalbiofilmswereexaminedat24,48and72hoursforthevolatilecompoundsproduced.At24,48and72hourstherewaslittledifferencebetweenthevolatilecompoundsdetectedfromblastosporebiofilmsandthosedetectedfromhyphalbiofilms.At24hours,bothbiofilmtypesproducedethanolandnerolidol(Figure3).At48and72hoursbothbiofilmtypescontinuedtoyieldpeaksforethanolandinadditiontothispeakrepresentingfarnesoltypemoleculeswereidentified.
Alemetal.,2006,reportedtheidentificationoftyrosolfromearlystageC.albicans biofilmsandplanktoniccultures.Tyrosolwasnotisolatedfromplanktonicorbiofilmculturesinthisworkwhichmaybearesultofanumberoffactors.Firstly,biofilmsanalysedinthisworkwereonlymeasuredfordetectablecompoundsat24and48hoursofgrowth.Alemetal.,detectedtyrosolafter10
Resultsanddiscussion
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Figure1- BiofilmsweregrownfromblastosporeofC.albicans(left)andC.albicanswithinducedhyphalgrowth(right)on1cm²piecesofgauze.Biofilmsweregrownfor24(top),48(middle)and72(bottom)hours.
Figure2- VolatilecompoundsandpotentialquorumsensingmoleculesdetectedfromA)24hourblastosporebiofilmsofC.albicansgrownongauze,B)48hourblastosporebiofilmsofC.albicansgrownongauze,C)72hourblastosporebiofilmsofC.albicansgrownongauze,D)24hourhyphal-inducedbiofilmsofC.albicansgrownongauze,E)24hourhyphal-inducedbiofilmsofC.albicans,F)72hourhyphal-inducedbiofilmsofC.albicansgrownongauze
24hours 48hours 72hoursBlastospore Hyphal Blastospore Hyphal Blastospore Hyphal
Carbondioxide 4 4 48 4 4Ethanol 90 90 90 90 90 90Aceticacid 38 49 49 49 72hours 861pentanol 831butanol 86 90 90 86 90 90Nerolidol 87 91 90 78alphafarnesene 89Farnesol 406,10dodecatrien-3-ol 64 906-Octen-1-ol 40betabisabolene 76 90dodecadien1-ol 54
Figure3- Volatilecompoundsandpotentialquorumsensingmoleculesasanalysedusinggaschromatographyanddatabasereferencingofchromatogram
hoursofgrowthinplanktonicculturesandin1-6hourbiofilmsandnotedthatfarnesoldominatedinlaterstagesofbiofilmgrowth.Inthisstudyanytyrosolpresentandtyrosolproductionbycellsat24and48hoursmayhavebeenlowincomparisontotheamountoffarnesoland thereforenotdetected.
OverallTheSPMEGC-MSmethodusedinthisstudywassuccessfulintheidentificationofvolatileandpotentialquorumsensingmoleculesproducedbyC.albicans.FurtherworkwillclarifyareproducibleGCMSmethodologytoworktowardstheidentificationofauniqueMVOCprofile, whengrowingbiofilmsofdenture(andotherhealthcareassociated)materials.
ReferencesAlem, M. A., Oteef, M. D., Flowers, T. H. and Douglas, L. J. (2006) 'Production of tyrosol by Candida albicans biofilms and its role in quorum sensing and biofilm development.' Eukaryot Cell., 5(10), Oct, pp. 1770-1779.Bingley, G.D., Verran, J., Munro, L.J., Banks, C.E., 2012. Identification of microbial volatile organic compounds (MVOCs) emitted from fungal isolates found on cinematographic film. Analytical Methods 4, 1265-1271.Dorocka-Bobkowska, B., Zozulinska-Ziolkiewicz, D., Wierusz-Wysocka, B., Hedzelek, W., Szumala-Kakol, A., Budtz-Jˆrgensen, E., 2010. Candida-associated denture stomatitis in type 2 diabetes mellitus. Diabetes Research and Clinical Practice 90, 81-86. Daniluk, T., Tokajuk, G., Stokowska, W., Fiedoruk, K., Sciepuk, M., Zaremba, M.L., Rozkiewicz, D., Cylwik-Rokicka, D., Kedra, B.A., Anielska, I., Górska, M., Kedra, B.R., 2006. Occurrence rate of oral Candida albicans in denture wearer patients. Advances in medical sciences 51 Suppl 1, 77-80.
