Subgingival dysbiosis in smoker and non‑smoker patients with chronic periodontitis

Abstract

Periodontitis is one of the most common oral inflammatory diseases, and results in connective tissue degradation and gradual tooth loss. It manifests with formation of periodontal pockets, in which anaerobic and Gram‑negative bacteria proliferate rapidly. Consequently, alteration of the subgingival microbiota is considered the primary etiologic agent of periodontitis. Previous studies have reported that smokers are at increased risk of periodontal disease, in both prevalence and severity, indicating that smoking is a risk factor for the onset and progression of the pathology. In the present study, 16S rRNA sequencing was employed to assess the subgingival microbiota in 6 smoker patients with chronic periodontitis, 6 non‑smoker patients with chronic periodontitis and 8 healthy controls. The results demonstrated significant alterations in the microbial structure of periodontitis patients. High relative abundance of Parvimonans, Desulfubulbus, Paludibacter, Haemophilus, and Sphaerochaeta genera characterized subgingival microbiota of periodontitis patients, both smokers and non‑smokers. Due to the high precision and sensitivity of the 16S rRNA sequencing method, analysis for low‑abundant genera (including Pedobacter, Granulicatella, Paracoccus, Atopobium, Bifidobacterium, Coprococcus, Oridobacteriu, Peptococcus, Oscillospira and Akkermansia) was feasible, and revealed novel phylotypes associated with periodontitis. Of note, a major microbial community alteration was evident in smoker patients, suggesting an association between smoking and severity of subgingival dysbiosis. The present study confirmed that chronic periodontitis is a polymicrobial disease where changes in the equilibrium of subgingival microbiota contribute to severity of pathology.

Figure 1.

Comparison of subgingival microbial diversity among PCS, PCnoS and CTRL groups. Diversity within bacterial communities is demonstrated by (A) the number of observed species and (B) the Shannon Index. Results are presented as the mean ± standard deviation. *P<0.05 vs. CTRL, by Tukey's test for multiple comparisons. (C) Diversity among bacterial communities is displayed in the principal coordinate analysis plot, based on unweighted Unifrac distances (34,559 sequences/sample), with the amount of variance along each axis in brackets. CTRL, healthy controls; PCS, smokers with periodontitis; PCnoS, non-smokers with periodontitis.

Figure 2.

Subgingival microbiota composition in PCS, PCnoS and CTRL groups. (A) The percentage distribution of bacterial phyla identified with relative abundance >1%. The mean ± standard error of the mean are plotted. Significant differences are indicated by *P<0.05. (B) Pie charts representing dominant genera with relative abundance >1% of the total reads. The mean values of the major genera are presented for each study group. (C) Number of key and shared phylotypes in CTRLvsPCS, CTRLvsPCnoS and PCSvsPCnoS comparisons as determined by LEfSe algorithm analysis (LDA>3 with alpha <0.05). PCS, smokers with periodontitis; PCnoS, non-smokers with periodontitis; CTRL, healthy controls; U, unknown.

Figure 3.

Predicted functional changes in the subgingival microbiome. Significantly different gene functions between (A) CTRL and PCS, (B) CTRL and PCnoS and (C) PCS and PCnoS groups. Differentially abundant gene functions derived from level 3 of the Kyoto Encyclopedia of Genes and Genomes database, were predicted using PICRUSt, and statistically significant pathways were identified using LDA effect size analysis (LDA score >2). PCS, smokers with periodontitis; CTRL, healthy controls; LDA, linear discriminative analysis.