The Responses of Dental Plaque Microcosms to Experimental Environmental Changes

Abstract

The overall objective of this study was to increase our understanding of the compositional changes in dental plaque biofilms during and after the application of antimicrobial agents. Dental plaque contains thousands of species, and is a causative factor in dental caries and periodontal disease. Culture techniques identify only a fraction of species present as many are currently unculturable, but the development of molecular techniques such as “checkerboard DNA:DNA hybridisation” (CKB) has allowed for more comprehensive evaluations of plaque species. In this thesis, checkerboard DNA:DNA hybridization was established and refined with a panel focussed on cariogenic microbes.

In a preliminary clinical study, plaque from five oral sites was collected from 24 five-to-six year-old Christchurch children, eight caries-free (decayed, missing or filled deciduous teeth (dmft) = 0), 16 with high caries (dmft ≥5) and analysed by CKB. There were a number of caries- and site-specific composition differences with high-caries children having high levels, at specific oral sites, of Candida albicans and Lactobacillus fermentum compared to caries free children along with various other bacteria not normally associated with caries. Bacteria present at higher levels in caries-free children included most of the anaerobes in the CKB panel, but also Streptococcus mutans and Lactobacillus acidophilus. There appears to be a complex site-specific bacterial relationship to dental caries with a number of species, two in particular (Haemophilus parainfluenzae and Selenomonas noxia), associated with either high levels of caries or a caries-free state depending on the oral site sampled. In laboratory antimicrobial investigations, dental plaque microcosms grown in an “artificial mouth” were treated with antimicrobials and analyzed by CKB to determine species profiles, with sampling immediately after treatment (day 11 or 15 of culture) then after subsequent regrowth for 7 or 15 days. The initial experiment investigated the application of the commercial mouthwashes Listerine, Plax, Savacol and Oral B to pre-formed (day 3) plaques. Subsequently, the effects of Listerine, chlorhexidine, and Savacol applied from shortly after inoculation (day 0) or to three-day-old pre-formed plaques were investigated. Plaque wet weights were determined daily to track changes in plaque biomass and CKB results were subjected to principal component analysis (PCA) and analysis of variance (ANOVA) to determine the significance of any changes observed. Oral B had no detected effect on growth. The treatment effects of Plax (triclosan) Savacol (containing chlorhexidine) were similar, with immediate growth suppression and minimal changes in the CKB-based biofilm species composition during treatment compared to the controls. Listerine allowed composition changes during treatment and the microcosms contained more viable organisms. Stringent antimicrobials that strongly inhibited plaque growth prevented changes in the plaque microbiota by CKB analysis, but resulted in greater changes than in the Listerine-treated plaques during regrowth. These plaques tended to contain higher levels of pathogens at the end of the experiment. Saliva donor effects complicated the statistical analysis of inter-experiment species changes as the species composition changes in the biofilms appeared to be affected by the composition of the saliva inoculum, which depended on the donor used.