The characteristics of the study subjects were shown in Additional file 1: Table S1. No difference in age, gender, race, and alcohol drink were found by smoking status. Smokers had slightly higher periodontal screening and recording (PSR) scores than nonsmokers.

By the Wilcoxon rank-sum test, smokers did not differ significantly from nonsmokers at any site on any measure of alpha diversity, beta diversity, or taxa-relative abundance with one exception—PD_whole tree diversity was lower in the smokers’ buccal mucosa (p = 0.05) (Fig. 1b–d, Additional file 2: Table S2). Based on the t test for the buccal mucosa, smokers had marginally lower observed species (p = 0.046), PD_whole tree (p = 0.032), and nearly significant lower Shannon index (p = 0.074). Thus, it appears that smokers had lower alpha diversity than that in the nonsmokers’ buccal mucosa based on both Wilcoxon rank-sum and t tests. We plotted the mean difference between smokers and nonsmokers for observed species and PD_whole tree (Additional file 1: Figure S1). For most sites, we can exclude large differences in means, but for sub and supragingival plaques, the confidence intervals are wide, reflecting small sample size. Thus, we cannot exclude the possibility of a substantial smoking effect at these two sites. Moreover, Additional file 2: Figure S1 indicates that alpha diversity in the buccal mucosa is lower in smokers as described above.

Regardless of the smoking status, microbiota among the oral sites differed greatly (Fig. 2, Additional file 2: Table S3). We found no difference in any microbial measurement by age, gender, race, alcohol consumption, and PSR score (data not shown).

Our study reported an effect of cigarette smoke on the oral microbiota in the buccal mucosa. Although our sample size is modest, it is remarkable that overall, we found no associations between microbial features and smoking status in other oral sites. Our results for the buccal mucosa should be examined in other studies since we tested multiple sites. Previous studies have shown inconsistent results regarding the effect of smoking on the oral microbiota. For example, an altered microbiota composition was noted in studies of 62 swabs from the tonsillar pillars [3], 200 subgingival samples [4], 30 marginal and subgingival plaque and gingival crevicular fluid samples [5], and oral wash samples comparing 13–41 current smokers with 77–194 never smokers in 4 groups [6] and 64 saliva samples [7] by sequencing 16S rRNA gene and in a study of 292 stimulated oral samples by Human Oral Microbe Identification Microarray (HOMIM) [8]. These studies, however, reported that different taxa were changed by smoking status. Some studies [9–11] reported no association between oral microbiota and smoking. Heterogeneity in smoking definitions, oral sites sampled, study population, and inclusion/exclusion of participants with related conditions (i.e., periodontitis), and in methods for collecting the specimens, likely account for some of the inconsistency with respect to smoking. In addition, our results suggested that smoking did not affect the oral microbiota with a large effective size. Large studies capable of exploring smaller magnitude effects of smoking on specific oral niches are needed to fully understand subtle smoking-induced alteration in microbiota.

Data for smoking effects on the nasal microbiota are sparser and even less clear. In contrast to our null results, swabs from the left and right nasopharynx in 62 subjects found significant differences in relative abundance of diverse taxa by smoking status, but 55% of these smoking-associated taxa were found only in the left or right nasopharynx, suggesting inconsistency [3].

Differences of microbiota across oral sites, regardless of smoking, point to distinct microbial niches. Consistent with our findings and with the Human Microbiome Project (HMP) [12], a recent study of 66 Chinese subjects showed significant differences in microbiota composition among the buccal mucosa, saliva, and dental plaque sites [13]. Different oral sites harbor a distinct microbiota, suggesting that the various oral surfaces might provide different receptors for bacterial adhesion, species interactions, and environmental conditions (e.g., oxygen level) for microbial survival and growth.

Ecological studies have suggested that more diverse and complex communities are generally more resistant and resilient to perturbations [14]. In the HMP, the oral microbiota, despite its heterogeneity across subsites, was more stable over time than the other body site evaluated [12]. As shown here, the oral cavity is a highly heterogeneous ecological system containing distinct microbial niches. In addition, it is well accepted that microbial organisms living on oral surfaces can switch from a free-living state to a sessile mode in biofilms with advantageous properties that include enhanced tolerance to many adverse conditions including antimicrobial agents [15]. Consistent with this view, a recent study of 66 healthy subjects with saliva samples at baseline, immediately and 1, 2, 4, and 12 months after antibiotics usage found that saliva microbial composition remained stable [16]. Likewise, our findings suggest that the microbial communities in the oral cavity might be resistant or resilient to disturbances such as cigarette smoking.

Strengths of the current study include extensive sampling of the oral cavity with a well-developed protocol, careful matching of case and control groups, a contrast of never with heavy smokers, state-of-the-art assays including quality control samples for batch effects, and rigorous statistical analysis. These methods should have identified significant associations with smoking status in the most sampled sites. Weaknesses include the modest sample size, especially for subgingival plaque samples due to sequencing failure. The study only allows us to detect the effect of smoking with a large effect size on the oral microbiota. Subtle effects or temporal effects of smoking could not be examined in this study.

Our study showed that cigarette smoking had a significant effect on the microbiota of the buccal mucosa, but not in other oral sites and nasal cavity. The oral cavity is heterogeneous with distinct communities across sites, which may contribute to its stability in the face of potential perturbing factors such as smoking. Larger studies are needed to further examine oral and nasal microbiota effects produced by behaviors such as cigarette smoking.