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dc.contributor.advisorSissons, Christopher Hal
dc.contributor.advisorWong, Lisa
dc.contributor.advisorAnderson, Sally
dc.contributor.advisorHolmes, Ann
dc.contributor.advisorCannon, Richard
dc.contributor.advisorDelahunt, Brett
dc.contributor.authorWeerasekera, Manjula Manoji
dc.date.available2012-03-14T20:09:30Z
dc.date.copyright2011
dc.identifier.citationWeerasekera, M. M. (2011). Role and dynamics of yeast species in oral biofilms (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/2142en
dc.identifier.urihttp://hdl.handle.net/10523/2142
dc.description.abstractOral biofilms are complex microbial ecosystems. In humans there are more than 900 oral microbial species, most with unknown properties or roles in disease. Candida albicans has cariogenic properties but its role in dental caries and in oral biofilms is still unclear. The incidence of oral candidosis and the involvement of non-albicans Candida species, particularly Candida dubliniensis, has recently increased due to HIV infection and immunosuppressive chemotherapy. Oral yeast identification in clinical laboratories mainly relies on culture analysis using CHROMagarTM Candida medium which yields uncertain results when differentiating C. albicans from C. dubliniensis. More reliable molecular methods for the identification of oral yeasts are needed both clinically, and for understanding oral ecosystems. Aims: (1) To develop PCR-DGGE to presumptively identify oral yeast species, in particular to differentiate C. albicans from C. dubliniensis, and to compare its performance with CHROMagar for identification of oral yeasts. (2) To investigate yeast prevalence and diversity in saliva and oral plaque microcosms cultured from different individuals, with and without exposure to three sucrose pulses daily. (3) To identify characteristic populations of bacterial species by DGGE eubacterial fingerprinting of saliva and oral microcosm plaques derived from different individuals, and to determine if any particular population is ecologically associated with the presence and prevalence of Candida species. Methods: PCR-DGGE and CHROMagar were evaluated for the presumptive identification of yeast species in saliva samples (n=25) and microcosm plaques (with confirmation by DNA sequencing). A range of yeast species (11 Candida species, 4 non-Candida species and 20 C. albicans isolates) were used to optimise PCR-DGGE. Previously published primer sets targeting the large subunit rDNA gene (25S–28S) (denoted primer sets N and U, respectively), and small subunit rDNA gene (18S) (primer set E) were used. Microcosm plaque biofilms were cultured from 24 individuals with and without 10% sucrose pulsing (6 minutes every 8 hour) for 11 days in a “Multiplaque Artificial Mouth”. Eubacteria were fingerprinted by PCR-DGGE. Results: Primer set N was highly discriminatory between yeast species and showed 100% specificity in the differentiation of C. dubliniensis from C. albicans. Primer set U often produced multiple bands, and could be used to distinguish six groups of C. albicans strains. Primer set E gave poor discrimination. PCR-DGGE of saliva samples from 25 donors identified yeasts, which were not discriminated by CHROMagar, and were confirmed by sequencing. C. albicans was the predominant yeast (carriage rate 56%) followed by C. dubliniensis (16%). In microcosm plaques, sucrose pulsing selectively promoted the growth of C. albicans and not non-albicans yeast species. No association was found between C. albicans and bacterial DGGE fingerprints of plaque microcosms. Yeasts and bacteria from different people responded differently to sucrose pulsing. Saliva bacterial clusters identified by PCR-DGGE were maintained to a significant degree during plaque microcosm development. Conclusion: PCR-DGGE using primer set N is a specific, sensitive, economical and reproducible technique: to presumptively identify yeast species in the oral cavity; to directly differentiate important multiple yeast species in clinical specimens; and to facilitate oral microbial ecological studies.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectYeast
dc.subjectPCR-DGGE
dc.subjectoral biofilms
dc.titleRole and dynamics of yeast species in oral biofilms
dc.typeThesis
dc.date.updated2011-12-09T09:20:46Z
thesis.degree.disciplineMolecular Medicine and Pathology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanyes
otago.openaccessAbstract Only
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