Growth and molecular characterization of dental plaque microcosms

Aims: (i) To compare the effects of feeding protocols upon the composition and stability of dental plaque microcosms formed in constant-depth film fermenters (CDFF). (ii) To evaluate the utility of denaturing gradient gel electrophoresis (DGGE) and culture methodologies for the investigation of such models. Methods and Results: Microcosms were established anaerobically in the CDFFs from freshly collected saliva. These were fed either with artificial saliva alone (famine) or combined with discontinuous feeding (feast-famine). Culture and 16s rDNA sequencing indicated that supplemental feeding gave ca . 2 log increases in Lactobacillus rhamnosus and Prevotella buccae . Feast-famine microcosms were then further characterized by DGGE using primers specific for the V2-V3 region of eubacterial rDNA. These gave single major bands with pure cultures (eight species) and resolved all strains apart from Lact. rhamnosus and Actinomyces naeslundii . Whilst culture with selective media indicated a degree of stability and reproducibility between replicate microcosms, DGGE showed a considerable degree of variability that related to several putatively uncultured bacteria. Conclusions: Feast-famine regimes altered community composition. DGGE analyses identified putatively unculturable species and demonstrated variability between replicate fermenters. Significance and Impact of the Study: This study demonstrates the utility of DGGE for the analysis of dental plaque, especially with respect to unculturable bacteria. Results question the assumptions of reproducibility of plaque microcosms established in non-replicated CDFFs made on the basis of selective media. Feeding regimes, particularly those involving complex nutrients, will dramatically affect population dynamics.