INTRODUCTION
Nearly 42% of periodontitis in the United States is attributable to tobacco smoking (
50), and numerous studies have reported a critical role for smoking in increasing the risk for developing extensive and severe forms of this disease (
3,
22,
26). With there being over 1 billion adult smokers worldwide (
55), smoking-related periodontitis presents a significant global public health issue. Although there is a large body of evidence on the clinical effects of smoking on the periodontium, little is known about the biological mechanisms by which it increases the risk for disease. Bacteria in dental plaque are the primary etiological agents of periodontal diseases, and elucidating the effects of smoking on this ecosystem is critical to understanding the role of smoking in the etiopathogenesis of periodontal diseases.
Studies using molecular approaches for bacterial identification and characterization have demonstrated that the subgingival microbial profile associated with periodontitis in smokers is diverse and distinct from that in nonsmokers (
44,
53,
57). Recent evidence also indicates that smoking cessation alters patterns of microbial recolonization following periodontal therapy, with a decrease in the levels of putative periodontal pathogens (
12,
18). Further, evidence from the nasopharyngeal ecosystem indicates that smoking alters the microbial signatures of these communities, with a decrease in the commensal population and a concomitant increase in pathogens (
7,
9). Together, these investigations suggest that the disease-associated subgingival biofilm in smokers is enriched for pathogenic bacteria. However, the effects of smoking on a health-compatible ecosystem have not been previously examined.
Bacteria colonize a tooth surface within a few minutes after its eruption into the oral cavity and form complex communities on the tooth surface and in the subgingival sulcus (
36,
45). Early colonization of tooth-associated ecosystems is a specific and selective process, and the development of a mature community is influenced by these early interbacterial as well as host-bacterium interactions (
28,
33,
42). In the gastrointestinal tract, it has been shown that early colonizers occupy this spatial niche in large numbers, thereby playing an important role in resisting colonization by pathogenic species. This phenomenon of niche saturation, whereby a few selected species occupy an ecological habitat and provide resistance to colonization of this niche by pathogenic organisms, along with metabolic synergism, plays a critical role in maintaining a healthy, stable community (
6). Examining the effect of smoking on these nascent communities thus is an important initial step in understanding the etiopathogenic role of smoking in periodontal diseases.
Therefore, the purpose of the present investigation was to compare bacterial acquisition and colonization in current smokers and those who have never smoked during marginal and subgingival plaque formation over 7 days.
RESULTS
Of the 23 subjects recruited, 8 withdrew from the study at various stages due to antibiotic use or inability to wear the stent for a prolonged period. Thus, 15 current smokers and 15 individuals who had never smoked completed the study, and samples from these subjects were included in the analysis. A power analysis revealed that the use of 15 subjects in each group will give 80 to 90% power with α = 0.05 to detect a difference of 1% between the 2 groups at any given time point (given a standard deviation [SD] of 1.4% based on our previous data [
31]). The demographic characteristics of the subjects are shown in
Table 1. There were no differences between the groups except for tobacco exposure, as measured by salivary cotinine levels (
P < 0.05, 2-sample
t test).
The clinical features of the two groups are shown in
Fig. 1. The plaque index increased significantly between day 1 and day 7 (
P < 0.05 by repeated-measures ANOVA); however, there were no differences in gingival or plaque index between the two groups for any of the 4 observation periods (
P > 0.05).
A total of 12,078 high-quality, chimera-depleted sequences were used in all analyses. Overall, these sequences represented the phyla
Firmicutes,
Proteobacteria,
Actinobacteria,
Bacteroidetes,
Spirochaetes, and
Synergistes, with
Firmicutes accounting for 83.4% of all sequences. These sequences represented 158 and 271 s-OTUs in the marginal biofilms and 216 and 241 s-OTUs in the subgingival biofilms of nonsmokers and current smokers, respectively. Smokers demonstrated greater bacterial diversity than nonsmokers during the early days of bacterial colonization in both marginal and subgingival plaques (
P < 0.05 by repeated-measures ANOVA) (
Fig. 2). Moreover, smokers also demonstrated a decrease in bacterial diversity in both the marginal and the subgingival habitats over 7 days of plaque development (
P < 0.05 by repeated-measures ANOVA). In nonsmokers, 71% of the community remained stable over the 7-day period, while in smokers, only 46% of the community was stable over the same period (data not shown). This was reflected by the statistically significant decrease in diversity in smokers, but not nonsmokers, from day 1 to day 7 in both habitats.
Principal-component analysis of unweighted UniFrac distances revealed significant clustering of bacterial sequences by tobacco exposure in both the subgingival and marginal communities (
P < 0.05 by distance-based ANOVA with permutation) (
Fig. 3A and B).
Smokers demonstrated significantly divergent patterns of bacterial acquisition and colonization over 7 days compared to nonsmokers. This difference was apparent in both the marginal and subgingival microbiomes. When averaged over the 7-day observation period, the genera
Streptococcus,
Veillonella,
Neisseria,
Abiotrophia, and
Selenomonas formed 70% of the subgingival community in nonsmokers, while in smokers,
Streptococcus,
Veillonella,
Selenomonas,
Campylobacter,
Pseudomonas,
Dialister,
Abiotrophia,
Neisseria, and
Prevotella accounted for this number (
Fig. 4). Moreover, during the first 7 days, smokers acquired several species/phylotypes belonging to the genera
Lactobacillus,
Fusobacterium,
Centipeda,
Pseudomonas,
Leptotrichia,
Synergistes,
Propionibacterium, and
Cardiobacterium, while these species were absent in nonsmokers. In marginal plaque (
Fig. 5), smokers demonstrated consistently greater 7-day average levels of
Streptococcus,
Haemophilus,
Kingella,
Selenomonas,
Lachnospira, and
Pseudomonas and lower levels of
Neisseria,
Actinomyces,
Rothia, and
Lautropia than nonsmokers (
P < 0.05 by repeated-measures ANOVA on transformed variable). Only smokers acquired the genera
Lactobacillus,
Treponema, and
Pseudomonas during this observation period.
Smokers demonstrated significantly higher levels of G-CSF, IL-1β, IL-2, IL-8, IL-9, IL-12, IFN-γ, RANTES, tumor necrosis factor alpha (TNF-α), and VEGF than nonsmokers during one or more days of plaque development (
P < 0.05 by repeated-measures ANOVA on normalized score) (
Table 2). VEGF and G-CSF were consistently higher in smokers over the whole observation period. Nonsmokers demonstrated significantly higher levels of IL-15, IL-10, and MCP-1 than current smokers (
P < 0.05).
Significantly opposing patterns of interactions were observed between subgingival levels of
Streptococcus and certain immune mediators among current smokers and nonsmokers (
Fig. 6). In both groups, these interactions were significant (
P < 0.05). In smokers, a positive correlation was observed between the levels of
Streptococcus and IL-2, G-CSF, VEGF, and RANTES, whereas nonsmokers showed a negative correlation. On the other hand, a positive interaction was observed between the levels of
Selenomonas and IL-2 and RANTES in nonsmokers but not in current smokers.
When these correlations were examined irrespective of smoking status, there was a negative correlation between levels of streptococci and the levels of these immune mediators, while a positive correlation was observed between IL-2, IL-9, RANTES, and Selenomonas.
DISCUSSION
In order to understand the temporal effects of smoking on oral bacterial colonization, the marginal and subgingival biofilms of 15 current smokers and 15 individuals who had never smoked were examined over 7 days of plaque development. Previous studies have demonstrated that the oral microbiome has several distinct microbial niches, e.g., the tooth surface, the subgingival sulcus, and oral mucosal surfaces (
48,
56). The subgingival sulcus provides both a shedding mucosal surface (the sulcular epithelium) and a nonshedding surface (the tooth). Therefore, a tooth-associated habitat and a mucosa-associated habitat were selected to obtain a representational characterization of the oral microbiome. Combining an open-ended molecular approach with a multiplex immunological assay and an adequately powered, longitudinal clinical study design revealed a critical role for smoking in altering colonization in a health-compatible oral microbiome. To the best of our knowledge, this is the first study to demonstrate such an influence.
A “zero-plaque” model was used in the present study to examine the compositional and inflammatory changes that occur in the biofilm during undisturbed plaque formation. Traditionally, longitudinal evaluations of plaque formation have been limited by disruption of the biofilm when plaque samples are collected at specific intervals. Previous investigations have sought to overcome this by sampling different teeth at different time points (
43). However, sampling different teeth at each time point may not be representational of the changes that occur in the mouth, since it is known that tooth morphology and position play a role in determining the composition of the biofilm (
47). Therefore, in the present investigation, the sampling sites were professionally cleaned to remove the biofilm following sampling at each time point. By resetting the “sampling clock” after each visit, undisturbed plaque samples were collected at 1, 2, 4, and 7 days. Samples collected immediately following oral prophylaxis did not demonstrate the presence of bacterial DNA (data not shown), indicating that
de novo colonization occurred following each visit.
Over 12,000 near-full-length, high-quality sequences were analyzed, allowing high-resolution phylogenetic comparisons to be made. Sanger sequencing was used as the method of choice for this open-ended investigation, because even though deep-sequencing methodologies provide a greater depth of coverage, it has been demonstrated that short-read “tag” sequences do not provide the same stringency of phylogenetic classification as longer sequence reads (
27).
UniFrac distances were used to quantify the similarities between each pair of samples. This methodology uses 16S rRNA gene sequences from each community to create a common phylogenetic tree, from which branch lengths for each community are computed. Thus, a lower UniFrac value indicates that the lineages that make up both communities are closely related, while higher values indicate a greater phylogenetic difference. Principal-coordinate analysis of UniFrac distances was used to examine the contributions of smoking to the compositions of the marginal and subgingival plaque communities. Our data revealed a high level of partitioning between current smokers and nonsmokers irrespective of the age of the biofilm, suggesting that tobacco exposure influences bacterial composition during all stages of plaque development. The data also indicated the need for further studies examining the long-term effects of smoking on the oral microbiome.
Bacterial colonization of the plaque biofilm occurs in a specific temporal sequence, leading to the formation of a health-compatible climax community (
29). A central characteristic of a healthy, stable community is niche occupation or niche saturation, whereby a few selected species occupy an ecological habitat and provide resistance to colonization of this niche by pathogenic organisms (
6). Recent evidence indicates that increased species diversity within an ecosystem drives further speciation (
17). Smokers demonstrated a greater diversity than nonsmokers during initial colonization of both marginal and subgingival plaques. These microbial communities also demonstrated lower stability over the 7-day period than those of nonsmokers. The predominant genera found in nonsmokers (
Streptococcus,
Neisseria, and
Veillonella) were previously reported to be abundant members of health-associated subgingival and supragingival communities (
1,
32). Also, the levels of these genera did not change significantly during the 7-day observation period. Taking the findings together, it appears that periodontal health is associated with large numbers of a few species that stably saturate this niche. This phenomenon of niche saturation did not occur to the same extent in smokers, who were colonized by several types of genera within 24 h, many of which have been associated with disease (
30,
46). The communities in smokers also demonstrated greater fluctuation than those in nonsmokers. These characteristics are similar to those of the community associated with periodontitis in smokers (
24,
44,
52,
53). Our data suggest that an initial lack of niche saturation lowers colonization resistance and increases the susceptibility of this ecosystem to future colonization by pathogenic organisms. Further studies are needed to examine the long-term effects of smoking on subgingival and marginal biofilms.
Smokers were colonizers by pathogens within 24 hours of biofilm development, and these organisms persisted during the observation period. While the implications of acquiring periodontal pathogens in the biofilm are obvious, these individuals also acquired systemic pathogens such as
Haemophilus and
Pseudomonas. Members of both of these genera are prolific biofilm formers and have been associated with refractory and antibiotic-resistant disease (
15,
19). It is known that smokers are at a higher risk for developing invasive antibiotic-resistant infections (
4,
7,
9,
16,
23,
37), and the results of the present study suggest that smoking increases susceptibility to disease by promoting early acquisition and colonization of biofilm-forming pathogens.
Periodontal disease occurs due to an exaggerated host response to a bacterial trigger. Although it is known that smokers exhibit higher levels of certain proinflammatory cytokines in disease (
3,
20), the baseline immune response of these high-risk individuals to normal bacterial colonization is not known. Examining a panel of immune mediators known to be important in the etiology of periodontal disease allowed for the quantification of the effects of smoking on the host response to early bacterial colonization. The levels of immune mediators exhibited a large interindividual variation, as reported by other investigators (
54). A variance-stabilizing transformation was created to overcome these large interindividual differences. This has been previously validated on serum levels of immune mediators (
5,
10). In the present study, onset of clinical disease occurred earlier in smokers than in nonsmokers, suggesting a prematurely amplified immune response to bacterial colonization. This clinical picture was corroborated by the immunological findings, with smokers demonstrating significantly higher levels of proinflammatory immune mediators (e.g., IL-1β, IL-2, IL-8, G-CSF, etc.) than nonsmokers from the first day. It is possible that this early elevation in immune mediator levels may persist during biofilm maturation, thereby increasing susceptibility to periodontitis in smokers; this warrants further investigation using long-term studies on plaque development and maturation.
Several opposing patterns of host-bacterium coassociations were observed in smokers and nonsmokers. The most robust of these associations were between streptococci and IL-2, RANTES, VEGF, and G-CSF and between the selenomonads and IL-2 and RANTES. Smokers not only demonstrated greater subgingival levels of streptococci than nonsmokers but also displayed a strong, positive correlation between a highly proinflammatory response and these bacterial levels. In contrast, nonsmokers showed an equally strong but negative correlation between bacterial levels and a proinflammatory response. Streptococci are early commensal colonizers of the subgingival and supragingival habitats (
14,
29) and are abundant members of these communities in health (
1,
31,
32,
58). Our data suggest that in nonsmokers, these commensals play an immunomodulatory role in the developing biofilm, primarily by attenuating the proinflammatory response. Our results are in agreement with emerging evidence demonstrating that streptococci exert a powerful anti-inflammatory effect on oral mucosal cells (
11,
25). Our data also suggest that this immunomodulation is reversed in smokers. A strong correlation was also observed between the genus
Selenomonas and the proinflammatory response in nonsmokers but not in smokers, providing further evidence of an altered host-bacterium interaction in these high-risk individuals.
Selenomonas spp. are putative periodontal pathogens that have been associated with both gingivitis and periodontitis and are abundant members of these disease-associated communities (
30,
40,
46,
49). Thus, the data support a critical role for smoking in modulating host-bacterium interactions in the oral cavity. However, it is not clear from this study whether smoking exerts this effect by decreasing the host response to bacteria, by altering the immunomodulatory behavior of the commensals, or by a combination of both. Further studies using
in vitro or
ex vivo methods are required to elucidate this mechanism.
In summary, smokers demonstrated a highly diverse, relatively unstable initial colonization of both marginal and subgingival biofilms, with lower niche saturation than that seen in nonsmokers. Several pathogens are acquired within the first 24 hours of biofilm formation in smokers and continue to persist over the observation period, suggesting that smoking alters bacterial acquisition and colonization in oral biofilms in favor of periodontopathogens. Smokers demonstrated a highly proinflammatory response to this early colonization, which persisted over 7 days. Opposing patterns of coassociations between predominant genera and proinflammatory cytokines were observed between current smokers and nonsmokers, providing evidence of altered host-bacterium interactions. Thus, the present investigation suggests a critical role for smoking in altering the oral microbiome in health, and further studies are required to examine the long-term effects of smoking on this host-associated ecosystem.