Bacteriological Investigations of the Human Oral Microbiota in Health and Disease

Abstract

The human oral cavity harbours a diverse consortium of microoganisms which has a complex relationship with host health and disease. Dental caries for example, have mulitispecies aetiologies and analysis of the mechanisms which underlie the condition is complicated by the large portion of the oral microbiota which remain uncharacterised. In vivo studies can be problematical in terms of reproducibility, ethics and inter-individual variation; reproducible and representative in vitro models of the human oral cavity are therefore of considerable utility. This doctoral thesis presents a series of in vitro or ex vivo investigations of oral microbial ecology. In order to reproduce inter-individual variation within oral consortia, a gingival model was further validated for generating salivary microcosms. Plaques were analysed using differential viable counting, PCR-DGGE, combined with principal components analysis (PCA) and Confocal laser scanning microscopy (CLSM). Data indicated that inter-individual variation in human salivary composition could be replicated in modelled microcosms (Chapter 3). In Chapter 4, poloxamer hydrogel constructs were validated for the growth and analyses of oral biofilms. Biofilms were analysed using differential viable counting, PCR-DGGE combined with principal components analysis (PCA), Confocal laser scanning microscopy (CLSM) and pH was measured using a microelectrode. Data indicated that poloxamer can support oral bacterial growth in both single and multi species biofilms, maintain compositional stability and reproduce inter-individual variation and acidogenesis. In Chapter 5, the taxonomic composition of saliva, supragingival plaque and degraded dentine was investigated in 30 volunteers [10 healthy and 20 Type 2 diabetes mellitus (T2DM)], using culture, PCR-DGGE and cluster analysis combined with PCA. Unique PCR amplicons were sequenced for identity. Dental caries incidence was elevated in T2DM compared to healthy individuals but the oral bacterial composition in supragingival plaque between two groups was not significantly different. Salivary eubacterial profiles from T2DM patients with and without caries clustered separately. With respect to bacterial consortia associated with degraded dentine, Prevotella nigrescens, Neisseria mucosa and Streptococcus sanguinis were associated with diabetes. In order to investigate potential familial factors in oral disease aetiology, the oral microbiotas of members of 10 families [5 caries free (CF) and 5 severe early childhood caries (SECC) families] were characterised using differential viable counting, together with PCR-DGGE, cluster analysis and PCA (Chapter 6). There was a highly significant association between numbers of lactobacilli in saliva and plaque and caries experience in SECC patients. Additionally, the numbers of streptococci in dental plaque were significantly higher in SECC patients than in CF controls. Regression analysis showed that plaque index score correlated significantly with DMFT and with lactobacillus abundance in dental plaque in a highly predictive manner. However, eubacterial fingerprints did not cluster within or between CF and SECC groups and bacterial counts for the three sampling sites in SECC did not differ significantly. Numbers of anaerobes, facultative anaerobes and streptococci of parents and children were correlated. In terms of familial microbial similarities, DGGE concordance between children, their mothers and their siblings ranged between 43 percent to 73 percent, and 35 percent to 82 percent, respectively. Data indicated a possible aetiological role of lactobacilli and potentially provide novel means of prediction.

Date of Award	3 Jan 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Mcbain (Supervisor) & Ruth Ledder (Supervisor)