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Sample Abstract

Longitudinal Analysis of Subgingival Bacterial Stability Using 16S Cloning and Sequencing

Introduction: Recent investigations have revealed that much of the subgingival flora is composed of uncultivated and previously unknown bacteria, and little is known about the composition and stability of the predominant species. Open-ended investigations such as cloning and sequencing the ribosomal 16S DNA provide a method to study this natural history. Objective: The purpose of this investigation was to examine the stability of the predominant subgingival bacterial species over a two-year period using 16S cloning and sequencing. Materials and methods: 25 subjects over 40 years of age with no history of systemic disease, smoking or recent antibiotic use were selected. Subgingival plaque samples were collected on paper points at a two-year interval. Probe depths, bleeding on probing, and plaque and gingival indices were recorded at the time of sampling. DNA was isolated directly from each sample and 16S rRNA genes were amplified by PCR using universal primers and cloned into E.coli. Colonies were screened for inserts of the expected molecular size by PCR. The amplicons were sequenced to identify bacterial species. 100 clones from each sample were sequenced, allowing detection of species that comprised 3% or more of the total flora with a 95% confidence. Results: At initial presentation, subjects were either periodontally healthy (n=12), or showed signs of slight (n=8) to moderate (n=5) disease. 12 subjects remained periodontally stable over the two-year period, while 6 subjects showed an improvement in periodontal health, and 7 showed evidence of disease progression. The mean microbial stability of the periodontally stable group was 55.5% (±3.6) and that of the unstable group was 38.3% (±3.4). This difference was statistically significant (P=0.002, t-test). Summary and Conclusions: The major subgingival bacterial flora appears to be stable in periodontally stable individuals. Shifts in microbial composition are associated with changes in periodontal health status.