Candida albicans biofilms in denture wearers

Jackson, Sarah Louise (2013) Candida albicans biofilms in denture wearers. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The term denture stomatitis describes an inflammation of the oral mucosa in contact with the fitting surface (usually maxillary) of the denture. Although considered to have a multi-factorial aetiology, the incidence of denture stomatitis is strongly associated with poor denture hygiene and the presence of Candida albicans, in denture plaque. Dentures provided an ideal abiotic substratum for microbial attachment, retention and growth, providing hard, non-shedding of variable topography. Although a wide range of denture hygiene products and procedures are available for use, targeted activity towards Candida is not typically reported. In addition, denture cleansing protocols often use abrasive pastes and/or brushes which can alter denture topography and potentially increase the susceptibility to plaque accumulation and reduce cleanability. The aim of this work was to investigate interactions between denture surface topography and C.albicans, with a view to identifying factors that would enhance denture hygiene. Two sets of denture acrylic test surfaces were abraded in a linear direction; one using emery papers of increasing grit size, and the other by abrasion using toothbrush and dentifrices. All test surfaces were characterised using white light profilometry, enabling derivation of roughness parameters (Ra/Sa) and measurement of feature dimensions (width, depth). A method was developed that allowed biofilm growth from adherent blastospores or hyphae on these surfaces. Initial retention on the abraded surfaces increased with increasing roughness values, but there was no effect on topography on the resultant biofilm. Biofilms developed from hyphae had significantly higher biomass than those from blastospores, and also presented a more open network structure. After biofilm removal, surface roughness significantly affected retention of remaining cells: rougher surfaces retained more cells, and retention was increased by the presence of hyphae. The orientation of hyphae on the abraded surfaces did not seem to be effected by topographic features, nor by the proximity of other cells. This latter observation was confirmed using an optical tweezer method, which enabled the specific placement of individual cells, with subsequent monitoring of germ tube formation. The production of quorum sensing (QS) molecules was investigated as a factor influencing biofilm development. Using gas chromatography and mass spectrometry, volatile compounds were collected from planktonic and biofilms of C.albicans over time. Ethanol and the QS molecule farnesol were amongst the molecules identified, being produced at 4hour and 10hours respectively during the 24 hour incubation period. Farnesol was also shown to inhibit hyphal production by attached cells, when applied to the surfaces as a conditioning film, as well as in vapour form. A denture cleanser with improved anti-Candida activity was effective against blastospore and hyphal biofilms during extended 1 and 16 hour soak times. Using confocal scanning laser microscopy (CSLM) live-dead staining of Candida biofilm indicated that the denture cleanser inactivated cells throughout the biofilm, with increased effect and penetration over time, but no difference between hyphal and blastospore biofilms was observed. When mixed biofilms were generated with C.albicans and Streptococcus oralis, the presence of the bacteria appeared to increase the effect of the cleanser on Candida biofilm. It is proposed that the inactivation of the bacteria may have disrupted the biofilm and enhanced its susceptibility. However this requires further investigation. In order to reduce the accumulation of denture plaque and C.albicans, there is a need to limit substrate abrasion, reduce retention on the surface, prevent hyphal growth and ideally kill and remove denture plaque. Strategies towards these aims include targeted but gentle cleaning and the potential use of quorum sensing molecules.