Oral dysbiosis induced by Porphyromonas gingivalis is strain-dependent in mice

Abstract

Background: Porphyromonas gingivalis strain W83, one of the most widely investigated, is considered virulent in the context of periodontitis. The recently isolated P. gingivalis TDC60 has been reported to be highly pathogenic, although it has not yet been investigated in a mouse periodontitis model by oral gavage. Aim: Our aim was to compare the virulence of both strains by evaluating their impact on alveolar bone loss and the composition of oral microbiota. Methods: We inoculated by oral gavage C57BL/6 mice with either one of the two P. gingivalis strains and compared to a sham-treated group, without antibiotics pre-treatment. The mandibular alveolar bone of treated mice and controls were assessed, one month after the final inoculation, by microCT measurements. Moreover, at this time, we characterized their oral microbiota by 16S rRNA gene sequencing. Results: While P. gingivalis W83 successfully initiated periodontitis, TDC60-treated mice only experienced moderate lesions. Furthermore, only W83-treated mice exhibited a specific distinct microbiota, with significantly lower richness and evenness than other samples, and decreased proportions of taxa usually found in healthy individuals. Conclusion: This association between alveolar bone loss and a major persistent shift of the oral microbiota gives insights into virulence discrepancies among these bacterial strains.

Keywords: P. gingivalis TDC60; Periodontitis; Porphyromonas gingivalis W83; alveolar bone loss; animal model; mice; oral microbiota.

Figure 1.

P. gingivalis specific 16S rRNA gene amplicons from DNA of oral swabs after electrophoresis on a 3% agarose gel. 1–2: sham-infected mice (controls); 3–4: W83-treated mice; 5–6: TDC60-treated mice; N: negative control; P: positive control (expected amplicon size: 161 bp). M: molecular weight marker (Quick-Load Low Molecular Weight DNA Ladder, NEB®).

Figure 2.

Alveolar bone loss in mice. Measurements of bone levels were made by comparing the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) (a, b) and the percentage of remaining bone (c, d) at two interdental sites: between the first and second mandibular molars (M1M2) and between the second and the third mandibular molars (M2M3). Root lengths (from the CEJ to the root apex) were also measured to assess the percentage of remaining alveolar bone: Percent remaining bone (%) = ([root length – CEJ-ABC]/root length) x 100. Representative images of micro-CT of control (e), TDC60-treated (f) and W83-treated (g) mice are presented. Control and P. gingivalis infected mice were 16 weeks old (n = 6 mice/group). Dunn’s Kruskal–Wallis multiple comparisons, statistically significant at: *p < 0.05; **p < 0.01.

Figure 3.

Alpha and beta diversity of the oral microbiota of mice. Bioinformatics processing of the microbiota samples from the oral cavity of control and P. gingivalis infected mice (n = 6 mice/group, 16 weeks old) allowed for Principal Coordinate Analysis (PCoA). The PCoA calculated with weighted UniFrac metric revealed clustering of W83-treated mice (a). Analysis of distances between samples showed significant differences between W83- and TDC60-treated mice, with both PERMANOVA and ANOSIM tests (**p < 0.01, b). Alpha diversity analysis showed significantly lower richness (S_obs, c) and lower evenness (Shannon-Weaver, d) in W83-treated mice; Dunn’s Kruskal–Wallis multiple comparisons, statistically significant at: *p < 0.05.

Figure 4.

Analysis of taxa relative abundances in the oral cavity according to the treatment of mice. The linear discriminant analysis with effect size (LEfSe) cladogram identified taxa that were differentially abundant across the groups (n = 6 mice/group; p < 0.05) (a). Taxa at the genus-level were then filtered (excluding those with a mean relative abundance < 1% in all groups), and plotted when the Kruskal–Wallis test returned significant result (p < 0.05) (b). Dunn’s Kruskal–Wallis multiple comparisons, statistically significant at: *p < 0.05; **p < 0.01.