Molecular Analysis of Infection in Advanced Caries

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DESCRIPTION: Extension of the carious lesion into the matrix of dentine undermines the physical and functional integrity of the tooth and provides an increasingly noxious insult to the underlying pulp tissue. The bacteria associated with this process are not well understood. Currently, there are no reliable means of identifying the status of the pulp tissue underlying a carious lesion and no enhanced therapeutic modalities are available. This seriously impairs the efficacy of current diagnostic and treatment regimes for this clinically important disease. Preliminary studies have used a universal bacterial amplicon based on 16S rDNA to define the major bacterial populations of carious dentine. This work was complemented by real-time polymerase chain reaction quantification of bacterial DMA to determine levels of bacterial species identified by phylogenetic analysis. A complex array of predominantly anaerobic bacteria was demonstrated with characteristic Lactobacillus and Prevotella spp. dominant. Further dissection of the microbial composition of the developing lesion will be enhanced by development of methods for enriching the display in population analysis of low abundance species of interest. The sequence information obtained will be used to design specific oligonucleotide probes for the determination of the spatial localization of organisms within the lesion by fluorescence in situ hybridization combined with confocal microscopy. Experimental analysis will extend to provide information on the mechanisms of bacterial invasion of the Dental pulp at high resolution using both extractive and in situ approaches and relate this to the histopathological responses of the pulps. Data obtained from this study will provide an objective basis for an understanding of the extension of the carious process with potential diagnostic value for predicting pulp response including pulpal death and endodontic infection.