Comparison of Subgingival and Buccal Mucosa Microbiome in Chronic and Aggressive Periodontitis: A Pilot Study

Abstract

Periodontal microorganisms not only colonize subgingival pockets, but also are detected on various mucous membranes in patients with periodontitis. The object of this pilot study was, using the next-generation sequencing of 16S RNA gene, to characterize the microbiota in two oral habitats (buccal mucosas and subgingival pockets) in patients with different forms of periodontitis. Thirty-two buccal swab samples and 113 subgingival samples were obtained from eleven subjects with chronic periodontitis (ChP), twelve subjects with aggressive periodontitis (AgP), and nine periodontally healthy individuals (HP). Using Miseq Sequencing of 16S rRNA gene, we found that the subgingival and buccal mucosa microbiome of ChP and AgP patients both differed from HP. Meanwhile, Veillonella, Treponema, Filifactor, Fretibacterium, Peptostreptococcaceae_[XI][G-6], Peptostreptococcaceae_[XI][G-5], Bacteroidetes_[G-5], Bacteroidetes_[G-3], Peptostreptococcaceae_[XI][G-4], Peptostreptococcaceae_[XI][G-2] significantly increased both in buccal and subgingival plaque samples in periodontitis subjects (ChP and AgP) compared with HP. Moreover, the results based on the Unweighted UniFrac distance showed that buccal and subgingival plaque samples from the same individuals show higher community divergence than same habitats from different subject samples. This study demonstrated that the microbiome of buccal mucosa can be influenced by periodontitis. However, subgingival and buccal mucosa microbiome seem to be characterized by species-specific colonization patterns. This pilot study provides a glimpse at the changes of subgingival and buccal mucosa associated with periodontitis from a holistic view. Further studies should be taken to illuminate the interplay between these detected changes and periodontitis development.

Keywords: 16S sequencing; aggressive periodontitis; buccal mucosa microbiome; chronic periodontitis; subgingival microbiome.

Figure 1

Relative abundance of bacterial composition on phylum level of buccal (shown as B) and subgingival plaque (shown as Sub) samples in health group (HP), chronic periodontitis group (ChP), and aggressive periodontitis group (AgP).

Figure 2

Relative abundance of bacterial composition on genus level of buccal (shown as B) and subgingival plaque (shown as Sub) samples in health group (HP), chronic periodontitis group (ChP), and aggressive periodontitis group (AgP).

Figure 3

Bacterial community alpha-diversity as calculated by ACE and Shannon index of buccal (shown as B) and subgingival plaque (shown as Sub) samples in health group (HP), chronic periodontitis group (ChP), and aggressive periodontitis group (AgP). The error bars indicate mean with standard error.

Figure 4

Venn diagram of the number of different OTUs common/unique to the buccal (shown as B) and subgingival plaque (shown as Sub) samples in health group (HP), chronic periodontitis group (ChP), and aggressive periodontitis group (AgP). The overlapping areas represent the number of OTUs shared by the counterpart samples. (A) A total of 685 kind of OTUs were detected in buccal samples of three groups. Three hundred nineteen kind of OTUs were shared by the HP_B, AgP_B, and ChP_B groups, while 18, 75, and 81 kind of OTUs were unique to the respective groups. (B) A total of 875 kind of OTUs were detected in the subgingival plaque samples of three groups. Five hundred fourteen kind of OTUs were shared by the HP_Sub, AgP_Sub, and ChP_Sub groups, and 7, 111, and 30 kind of OTUs were unique to the respective groups.

Figure 5

β-diversity of buccal (Left) and subgingival plaque (Right) samples in three groups. Principal coordinates analysis (PCoA) of unweighted UniFrac distance were performed based on the OTU abundances.

Figure 6

The ternary plots analysis of buccal (Left) and subgingival plaque (Right) samples on the OTU level which based on three main status: the intraoral environment of periodontal healthy status (HP), and the intraoral environment of periodontitis (ChP/AgP). The dots represented as OTU, the size of the dots represented the OTU abundances and the color represented as different phylum. The OTUs whose abundance <0.01 in all samples were classified into others.

Figure 7

The Unweighted UniFrac distances were calculated and compared between groups to evaluate the similarity of all samples. The distances between subgingival plaque from the same (Intra Sub-Sub) or different individuals (Inter Sub-Sub), the distances between buccal bacterial samples and subgingival plaque from the same individuals (Intra Sub-B) and the distances between buccal bacterial samples from different individuals (Inter B-B) were presented in three groups. ^***p < 0.001. The error bars indicate mean with standard error.

Figure 8

Comparisons of microbiota that presented significantly different contents in both buccal (up) and subgingival plaque (down) samples of periodontitis groups (AgP/ChP) shown the same trend of differences when compared with HP group at genus level. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.