The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation

Abstract

The goals of this study were to better understand the ecology of oral subgingival communities in health and periodontitis and elucidate the relationship between inflammation and the subgingival microbiome. Accordingly, we used 454-pyrosequencing of 16S rRNA gene libraries and quantitative PCR to characterize the subgingival microbiome of 22 subjects with chronic periodontitis. Each subject was sampled at two sites with similar periodontal destruction but differing in the presence of bleeding, a clinical indicator of increased inflammation. Communities in periodontitis were also compared with those from 10 healthy individuals. In periodontitis, presence of bleeding was not associated with different α-diversity or with a distinct microbiome, however, bleeding sites showed higher total bacterial load. In contrast, communities in health and periodontitis largely differed, with higher diversity and biomass in periodontitis. Shifts in community structure from health to periodontitis resembled ecological succession, with emergence of newly dominant taxa in periodontitis without replacement of primary health-associated species. That is, periodontitis communities had higher proportions of Spirochetes, Synergistetes, Firmicutes and Chloroflexi, among other taxa, while the proportions of Actinobacteria, particularly Actinomyces, were higher in health. Total Actinomyces load, however, remained constant from health to periodontitis. Moreover, an association existed between biomass and community structure in periodontitis, with the proportion of specific taxa correlating with bacterial load. Our study provides a global-scale framework for the ecological events in subgingival communities that underline the development of periodontitis. The association, in periodontitis, between inflammation, community biomass and community structure and their role in disease progression warrant further investigation.

Figure 1

Periodontal destruction, but not increased periodontal inflammation, is associated with greater α-diversity. (a) shows community richness (observed and estimated) for subjects with periodontitis (P) in sites with bleeding (PB) and those without bleeding (PnB) and for communities from healthy subjects (H). In (b) evenness of communities was compared, while (c) shows comparisons of diversity. *indicates a P<0.05. To compare P and H, communities from bleeding and non-bleeding sites were combined. α-diversity metrics were calculated after subsampling to obtain equal number of sequences per library.

Figure 2

Communities from periodontitis cluster apart from those in health, while bleeding does not drive community clustering. Graphs are principal coordinate analysis plots based on community membership metrics [(a) is based on unweighted UNIFRAC and (b) on the Jaccard Index] or community structure metrics [(c) is based on weighted UNIFRAC, while (d) is based on the θ_YC distance]. Communities from healthy subjects appear in green. For the periodontitis group, communities from sites with BoP appear in red, while communities from sites without BoP appear in blue.

Figure 3

The core subgingival microbiome in health and periodontitis. OTUs that are part of the core microbiome were first defined as those present in at least 50% of subjects in both the healthy and periodontitis groups (gray), in at least 50% of healthy subjects (green) or in at least 50% of subjects with periodontitis (red). Prevalence and relative abundance data were used for further filtering. OTUs in gray represent the core subgingval OTUs, present at equal prevalence and relative abundance in health and periodontitis. OTUs in green represent the core health-associated species, appearing at increased prevalence and relative abundance in health compared to disease. OTUs in red represent the core periodontitis-associated species, present at increased prevalence and relative abundance in periodontitis compared to health. Inner circles labeled with 1, contain highly prevalent and highly abundant OTUs, that is, present in at least 2/3 of samples from each group (core, health or periodontitis) and numerically dominant with a mean relative abundance of ⩾2% of total sequences. Middle circles labeled with 2 contain OTUs highly prevalent but present in low abundance, that is present in at least 2/3 of samples from the specific group but with a mean relative abundance of <2% of total sequences. Outer circles labeled with 3 contain OTUs moderately prevalent and present in low abundance, that is OTUs present in 1/2 to 2/3 of samples from each group and with a mean relative abundance of <2% of total sequences.

Figure 4

Bacterial load is higher in periodontitis than in health, is also higher in bleeding than in non-bleeding sites and correlates with the relative abundance of specific taxa within periodontitis samples. (a) shows bacterial load measurements in periodontitis (P) in sites with bleeding (PB) or without bleeding (PnB) and in health (H). *indicates a P<0.05. To compare P and H, communities from bleeding and non-bleeding sites were combined. (b) shows taxa from periodontitis communities that showed a significant negative correlation between their relative abundance and bacterial load, while (c) shows taxa from periodontitis communities with a significant positive correlation between their relative abundance and bacterial load. Relative abundance (%) and bacterial load (number of 16S rRNA copies) data were transformed (see methods).