Sequence of bacterial colonization during periodontal health and disease

Introduction: Although the role of subgingival plaque biofilm in the etiology of periodontal diseases is well established, very little is known about the constituents of a health compatible biofilm. Open-ended molecular approaches allow us to comprehensively examine the changes that occur in this microbial community during plaque formation and maturation, including colonization by as-yet-uncultivated and previously unknown bacteria. Objective: The purpose of this study was to examine the sequence of bacterial colonization in the subgingival biofilm over 21 days, using cloning and sequencing of 16S rRNA genes for bacterial identification. Methods: 15 subjects over 18 years of age with no history of systemic disease, smoking or recent antibiotic use were recruited. Stents were fabricated to protect 6 teeth in 2 quadrants during brushing. Subgingival plaque samples were collected using curettes and paper points at baseline, 1,2,4,7,14 and 21 days. Probe depths, plaque and gingival indices were also recorded. DNA was isolated from each sample and 16S rRNA genes were amplified by PCR using universal primers and cloned into E.coli. The amplicons were sequenced to identify bacterial species. 50 clones from each sample were sequenced, allowing detection of species that comprised 3% or more of the total flora with a 95% confidence. Results: 75-95% of the subgingival biofilm in health is predominantly colonized by species belonging to Veillonella, Abiotrophia, Streptococcus and Neisseria. The microbial profile shows a statistically significant shift (p<0.005, repeated measures ANOVA) on day 14 with decrease in the levels of health-associated species and a concomitant increase in species belonging to the genera Selenomonas, Dialister, Megasphaera and Eubacterium. Conclusions: The major subgingival bacterial flora appears to be comprised of a small group of species in periodontal health. Shifts in microbial composition are associated with changes in periodontal health status.

Supported by Philips Oral Healthcare grant.