Changes in periodontal health status are associated with bacterial community shifts as assessed by quantitative 16S cloning and sequencing

Abstract

The gingival sulcus contains a complex ecosystem that includes many uncultivated bacteria. Understanding the dynamics of this ecosystem in transitions between health and disease is important in advancing our understanding of the bacterial etiology of periodontitis. The objective of this longitudinal study was to examine the stability of bacterial colonization in the gingival crevice and to explore the relationship between shifts in microbial composition and changes in periodontal health status using a comprehensive, quantitative, culture-independent approach. Subgingival plaque samples and periodontal data were collected from 24 subjects over 2 years. Baseline and 2-year plaque samples were analyzed using quantitative ribosomal 16S cloning and sequencing. Ten subjects remained periodontally healthy over 2 years, the periodontal health of seven subjects worsened, and seven subjects showed clinical improvement. Bacterial stability was greatest among healthy, clinically stable subjects and lowest for subjects whose periodontal status worsened (P = 0.01). Higher numbers of species lost or gained were also observed for subjects whose clinical status changed (P = 0.009). This provides evidence that a change in periodontal status is accompanied by shifts within the bacterial community. Based on these data, measures of microbial stability may be useful in clinical diagnosis and prognosis. Regarding individual species, increases in levels of the uncultivated phylotype Veillonella sp. oral clone X042, a gram-negative bacterium and the most common member of the subgingival bacterial community, were associated with periodontal health (P = 0.04), suggesting that this is an important beneficial species. Filifactor alocis, a gram-positive anaerobe, was found at higher levels in subjects with disease (P = 0.01).

FIG. 1.

Clinical stability of 24 subjects over 2 years. For the improved group, the shaded and open bars taken together indicate the number of sites with probe depths of >4 mm (deep sites) at baseline. Open bars represent decreases in the number of deep sites that occurred between the baseline and final sampling times. For the worse group, the shaded bars indicate initial clinical status and the solid bars represent increases in the number of deep sites that occurred between the baseline and final sampling times.

FIG. 2.

Relationship between periodontal status and microbial stability and diversity for 24 subjects over 2 years. (Top) Relationship between clinical status and microbial conservation. (Center) Relationship between the number of species lost or gained over 2 years and clinical status. (Bottom) Relationship between mean microbial diversity (number of species present in each subject over 2 years) and clinical status. The standard deviation (small lines), group mean (central line in diamond), and 95% confidence intervals (height of diamond) are shown for each group.

FIG. 3.

Mean levels at 2 years (top) and change in levels (mean level at 2 years minus mean level at baseline) (bottom) for species significantly associated with improvement or worsening of periodontitis. (Top) Percentage of total clones accounted for by each species for the stable healthy, improved, and worse groups at the final sampling time. (Bottom) Percentage of change between the initial and final sampling times for the same species and clinical groups. Species showing significant differences (P < 0.05) among the three clinical groups are indicated by an asterisk.