Tobacco smoking affects bacterial acquisition and colonization in oral biofilms

Abstract

Recent evidence suggests that smoking affects the composition of the disease-associated subgingival biofilm, yet little is known about its effects during the formation of this biofilm. The present investigation was undertaken to examine the contributions of smoking to the composition and proinflammatory characteristics of the biofilm during de novo plaque formation. Marginal and subgingival plaque and gingival crevicular fluid samples were collected from 15 current smokers and from 15 individuals who had never smoked (nonsmokers) following 1, 2, 4, and 7 days of undisturbed plaque formation. 16S rRNA gene cloning and sequencing were used for bacterial identification, and multiplex bead-based flow cytometry was used to quantify the levels of 27 immune mediators. Smokers demonstrated a highly diverse, relatively unstable initial colonization of both marginal and subgingival biofilms, with lower niche saturation than that seen in nonsmokers. Periodontal pathogens belonging to the genera Fusobacterium, Cardiobacterium, Synergistes, and Selenomonas, as well as respiratory pathogens belonging to the genera Haemophilus and Pseudomonas, colonized the early biofilms of smokers and continued to persist over the observation period, suggesting that smoking favors early acquisition and colonization of pathogens in oral biofilms. Smokers also demonstrated an early proinflammatory response to this colonization, which persisted over 7 days. Further, a positive correlation between proinflammatory cytokine levels and commensal bacteria was observed in smokers but not in nonsmokers. Taken together, the data suggest that smoking influences both the composition of the nascent biofilm and the host response to this colonization.

Fig. 1.

Gingival and plaque indices of 15 current smokers and 15 nonsmokers over 7 days of plaque accumulation. Both groups demonstrated an increase in plaque and gingival indices over the observation period.

Fig. 2.

Shannon diversity index over 7 days in current smokers and nonsmokers. Results for subgingival biofilm are shown in the upper panel and those for marginal biofilm in the lower panel. Smokers demonstrated a decrease in diversity over 7 days (#, P < 0.05 by repeated-measures ANOVA) and showed a greater diversity than nonsmokers (*, P < 0.05; **, P < 0.01).

Fig. 3.

Clustering of microbial communities using principal-component analysis (PCoA) of unweighted UniFrac distances. (A) Subgingival communities; (B) marginal communities. Both communities demonstrated a significant clustering based on tobacco exposure.

Fig. 4.

Distribution by genus of clones in the subgingival biofilms of current smokers and nonsmokers over 7 days. Genera accounting for >0.5% of total clones are shown. Significant differences in the levels of several genera were observed between the two groups (by repeated-measures ANOVA on transformed variable, *, P < 0.05; **, P < 0.01).

Fig. 5.

Distribution by genus of clones in the marginal biofilms of current smokers and nonsmokers over 7 days. Genera accounting for >0.5% of total clones are shown. Significant differences in the levels of several genera were observed between the two groups (by repeated-measures ANOVA on transformed variable, *, P < 0.05; **, P < 0.01).

Fig. 6.

Best linear fits for coassociation between subgingival genera and inflammatory cytokines in current smokers and nonsmokers. P values highlighted in gray correspond to the null hypothesis that the two slopes are the same. P values corresponding to the null hypothesis that each slope is zero are also shown (*, P < 0.05; **, P < 0.01).