The Subgingival Microbiome of Periodontal Pockets With Different Probing Depths in Chronic and Aggressive Periodontitis: A Pilot Study

Abstract

Periodontitis is a kind of infectious disease initiated by colonization of subgingival periodontal pathogens, which cause destruction of tooth-supporting tissues, and is a predominant threat to oral health as the most common cause of loss of teeth. The aim of this pilot study was to characterize the subgingival bacterial biodiversity of periodontal pockets with different probing depths in patients with different forms of periodontitis. Twenty-one subgingival plaque samples were collected from three patients with chronic periodontitis (ChP), three patients with aggressive periodontitis (AgP) and three periodontally healthy subjects (PH). Each patient with periodontitis was sampled at three sites, at different probing depths (PDs, one each at 4 mm, 5-6 mm, and ≥ 7 mm). Using 16S rRNA gene high-throughput sequencing and bioinformatic analysis, we found that subgingival communities in health and periodontitis samples largely differed. Meanwhile, Acholeplasma, Fretibacterium, Porphyromonas, Peptococcus, Treponema_2, Defluviitaleaceae_UCG_011, Filifactor, and Mycoplasma increased with the deepening of the pockets in ChP, whilst only Corynebacterium was negatively associated with PD. In AgP, Corynebacterium and Klebsiella were positively associated with PD, while Serratia, Pseudoramibacter, Defluviitaleaceae_UCG_011, and Desulfobulbus were negatively associated with PD. And among these two groups, Corynebacterium shifted differently. Moreover, in subgingival plaque, the unweighted UniFrac distances between samples from pockets with different PD in the same patients were significantly lower than those from pockets within the same PD category from different patients. This study demonstrated the shift of the subgingival microbiome in individual teeth sites during disease development. Within the limitation of the relative small sample size, this pilot study shed new light on the dynamic relationship between the extent of periodontal destruction and the subgingival microbiome.

Keywords: 16S rDNA; aggressive periodontitis; chronic periodontitis; high-throughput nucleotide sequencing; subgingival microbiome.

Figure 1

Microbiota alpha-diversity as calculated by ACE and Shannon index. The ACE index values were significantly lower in subgingival plaque samples of the health group (PH) than the chronic periodontitis group (ChP) and aggressive periodontitis group (AgP). A tendency toward an increase in the Shannon index was observed in samples of ChP/AgP compared to plaque from PH; however, the difference was not significant. ^*p ≤ 0.05; ^***p ≤ 0.001.

Figure 2

Relative abundance of bacterial composition at phylum level in health group (PH), chronic periodontitis group (ChP) and aggressive periodontitis group (AgP).

Figure 3

The distribution of the major genera in the microbiomes of periodontal health and disease. The y-axis shows the top 30 most abundant genera which constitute 87.06~91.63% of each bacterial microbiota. The relative abundance of each genus is indicated by the circle area.

Figure 4

Principal component analysis at the OTU level at 97% identity. Each sample is represented by a dot. Green dots represent the samples of the health group (PH). Blue dots represent the samples of the chronic periodontitis group (ChP). Red dots represent the samples of aggressive periodontitis group (AgP). The shape of the dots represents the samples from different persons (except PH). PC1 explained 54.2% of the variation observed, and PC2 explained 9.4% of the variation. ChP and AgP clustered together more closely, while PH was more dispersive, suggesting that the bacterial structures in diseased groups were more similar.

Figure 5

The Unweighted Unifrac distance between samples. A, distance between samples from the same AgP patients; A_PD4, distance between samples of PD = 4 mm sites from different AgP patients; A_PD5, distance between samples of PD = 5–6 mm sites from different AgP patients; A_PD7, distance between samples of PD ≥ 7 mm sites from different AgP patients; AA, distance between samples from different AgP patients. C, distance between samples from the same ChP patients; C_PD4, distance between samples of PD = 4 mm sites from different ChP patients; C_PD5, distance between samples of PD = 5–6 mm sites from different ChP patients; C_PD7, distance between samples of PD ≥ 7 mm sites from different ChP patients; CC, distance between samples from different ChP patients. HH, distance between samples from different healthy subjects.

Figure 6

Heatmap presenting the distribution of the genera which showed significant differences in relative abundance between PH, ChP, and AgP (Log2 transformed counts). Samples groups are represented in the x-axis. Genera are listed in the right y-axis, and hierarchical clusters in the left y-axis. High genera counts are represented in red and low genera counts are represented in green.

Figure 7

Scatter diagram of bacteria whose relative abundance were correlated significantly with the changing periodontal probing depths (PDs) in chronic periodontitis group (ChP).

Figure 8

Scatter diagram of bacteria whose relative abundance were correlated significantly with the changing periodontal probing depths (PDs) in aggressive periodontitis group (AgP).