Dalwai, F.; (2008) In vitro modelling of bacterial population shifts in oral biofilms. Doctoral thesis , University of London.

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Abstract

In vitro models of dental plaque are a valuable tool for understanding the development of plaque-related diseases and assessing potential treatments for these diseases. The main focus of this study was the development of an in vitro model to characterise the changes in bacterial populations from dental plaque associated with health to one associated with gingivitis. By emulating environmental conditions in the oral cavity associated with gingivitis it was possible to see changes in the oral microbiota associated with gingivitis. Using traditional culture techniques the ascendancy of Actinomyces spp. at the expense of Streptococcus spp. was observed with the onset of gingivitis conditions, along with increased proportions of Gram-negative species. To assess the range of cultivable species present isolates, which had previously been cultured in the model, were identified by sequencing of the 16S rRNA gene. After the onset of gingivitis conditions a greater richness of species was identified. Examination of these communities with confocal microscopy and viability staining also revealed structural changes associated with environmental conditions emulating gingivitis. To assess the presence of species which were not frequently identified by culture, but previously shown to be associated with gingivitis, quantitative PCR (qPCR) was used to enumerate Prevotella spp., Fusobacterium spp. and Porphyromonas gingivalis. Prevotella and Fusobacterium spp. were found to be significant members of the microbial communities developed in the CDFF, with Prevotella spp. increasing significantly under conditions emulating gingivitis. Furthermore, the total bacterial counts enumerated by culture were underestimated by approximately 80% compared to the total counts obtained by qPCR. This model was ultimately used to assess the effectiveness of tetracycline, chlorhexidine and silver ion-releasing dental materials against the accumulation of plaque. All of these agents influenced the microbial composition, rather than total microbial numbers, with reduced levels of Actinomyces, Prevotella and Fusobacterium spp. This study has shown that in vitro models of microbial communities associated with health and disease are valuable tools for observing key factors in disease progression. When disease results from changes in the resident microflora the use of such models allows the influence of individual environmental factors to be assessed and also allows the effect of potential treatments on these communities to be examined.

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