Drift of the Subgingival Periodontal Microbiome during Chronic Periodontitis in Type 2 Diabetes Mellitus Patients

Abstract

Since periodontitis and type 2 diabetes mellitus are complex diseases, a thorough understanding of their pathogenesis requires knowing the relationship of these pathologies with other disorders and environmental factors. In this study, the representability of the subgingival periodontal microbiome of 46 subjects was studied by 16S rRNA gene sequencing and shotgun sequencing of pooled samples. We examined 15 patients with chronic periodontitis (CP), 15 patients with chronic periodontitis associated with type 2 diabetes mellitus (CPT2DM), and 16 healthy subjects (Control). The severity of generalized chronic periodontitis in both periodontitis groups of patients (CP and CPT2DM) was moderate (stage II). The male to female ratios were approximately equal in each group (22 males and 24 females); the average age of the subjects was 53.9 ± 7.3 and 54.3 ± 7.2 years, respectively. The presence of overweight patients (Body Mass Index (BMI) 30-34.9 kg/m²) and patients with class 1-2 obesity (BMI 35-45.9 kg/m²) was significantly higher in the CPT2DM group than in patients having only chronic periodontitis or in the Control group. However, there was no statistically significant difference in all clinical indices between the CP and CPT2DM groups. An analysis of the metagenomic data revealed that the alpha diversity in the CPT2DM group was increased compared to that in the CP and Control groups. The microbiome biomarkers associated with experimental groups were evaluated. In both groups of patients with periodontitis, the relative abundance of Porphyromonadaceae was increased compared to that in the Control group. The CPT2DM group was characterized by a lower relative abundance of Streptococcaceae/Pasteurellaceae and a higher abundance of Leptotrichiaceae compared to those in the CP and Control groups. Furthermore, the CP and CPT2DM groups differed in terms of the relative abundance of Veillonellaceae (which was decreased in the CPT2DM group compared to CP) and Neisseriaceae (which was increased in the CPT2DM group compared to CP). In addition, differences in bacterial content were identified by a combination of shotgun sequencing of pooled samples and genome-resolved metagenomics. The results indicate that there are subgingival microbiome-specific features in patients with chronic periodontitis associated with type 2 diabetes mellitus.

Keywords: 16S rRNA gene sequencing; metagenomics; oral microbiome; periodontitis; type 2 diabetes mellitus.

Figure 1

Schematic visualization of the experimental design. CP: chronic periodontitis. CPT2DM: chronic periodontitis with type 2 diabetes mellitus.

Figure 2

Alpha diversity distribution between the study groups. (A) Chao1 index. (B) Shannon index. (C) Simpson index. The data were analyzed by the Wilcoxon rank-sum test with FDR (false discovery rate) correction for multiple testing. Median, interquartile range and standard deviation are indicated.

Figure 3

The major bacterial genera present in the subgingival periodontal microbiota of the control and CP/CPT2DM patient groups. (A) Columns correspond to the samples; the group is denoted with a top color bar. Hierarchical clustering was performed using the Euclidean distance and complete linkage. Logarithmic transformation of read counts was performed using pseudo counts. The top eight families in terms of relative abundance were selected for further analysis. (B) Non-metric multidimensional scaling (NMDS) biplot of taxonomic profiles (family level) of patients’ plaque samples using 16S rRNA gene sequencing and Aitchison distance (MDS1 and MDS2; coordinates scaled to standard deviation unit and centered to the mean). Taxonomic profiles at family level referring to the samples from control subjects, CP, and CPT2DM patients, are shown in red, blue, and green, respectively. (C) The CoDa dendrogram shows an ecological model of differences between the experimental groups. Decomposition of total variance by balances between groups of genera is shown using vertical bars (red bars denote the Control group; blue, the CP group; and green, the CPT2DM group). The mean balances are shown using anchoring points of vertical bars. The red area denotes the “healthy” state balance; the blue area denotes the “disease” state balance. CP: chronic periodontitis. CPT2DM: chronic periodontitis with type 2 diabetes mellitus. CoDa: compositional data analysis.

Figure 4

The results of the Songbird analysis. The X-axis denotes the effect size; the Y-axis denotes the bacterial families. The statistically significant decrease is shown in red; the statistically significant increase is shown in blue.

Figure 5

Phylogenetic analysis of the metagenome-assembled genomes. The phylogenetic tree is based on 43 marker proteins obtained from 26 MAG sequences and related eHOMD genomes [44]. The MAG groups are shown in different colors (red for the Control group, blue and green for CP and CPT2DM, respectively). The colored areas also denote the bacterial phyla. MAG: metagenome-assembled genome. eHOMD: expanded Human Oral Microbiome Database.