Species-Level Salivary Microbial Indicators of Well-Resolved Periodontitis: A Preliminary Investigation

Abstract

Objective: To profile the salivary microbiomes of a Hong Kong Chinese cohort at a species-level resolution and determine species that discriminated clinically resolved periodontitis from periodontally healthy cases. Methods: Salivary microbiomes of 35 Hong Kong Chinese subjects' under routine supportive dental care were analyzed. All subjects had been treated for any dental caries or periodontal disease with all restorative treatment completed at least 1 year ago and had ≤3 residual pockets. They were categorized based on a past diagnosis of chronic periodontitis into "healthy" (H) or "periodontitis" (P) categories. Unstimulated whole saliva was collected, genomic DNA was isolated, and high throughput Illumina MiSeq sequencing of 16S rRNA (V3-V4) gene amplicons was performed. The sequences were assigned taxonomy at the species level by using a BLASTN based algorithm that used a combined reference database of HOMD RefSeqV14.51, HOMD RefSeqExtended V1.1 and GreenGeneGold. Species-level OTUs were subjected to downstream analysis in QIIME and R. For P and H group comparisons, community diversity measures were compared, differentially abundant species were determined using DESeq2, and disease indicator species were determined using multi-level pattern analysis within the R package "indicspecies." Results: P subjects were significantly older than H subjects (p = 0.003) but not significantly different in their BOP scores (p = 0.82). No significant differences were noted in alpha diversity measures after adjusting for age, gender, and BOP or in the beta diversity estimates. Four species; Treponema sp. oral taxon 237, TM7 sp. Oral Taxon A56, Prevotella sp. oral taxon 314, Prevotella sp. oral taxon 304, and Capnocytophaga leadbetteri were significantly more abundant in P than in the H group. Indicator species analysis showed 7 significant indicators species of P group. Fusobacterium sp oral taxon 370 was the sole positive indicator of P group (positive predictive value = 0.9, p = 0.04). Significant indicators of the H category were Leptotrichia buccalis, Corynebacterium matruchotii, Leptotrichia hofstadii, and Streptococcus intermedius. Conclusion: This exploratory study showed salivary microbial species could discriminate treated, well-maintained chronic periodontitis from healthy controls with similar gingival inflammation levels. The findings suggest that certain salivary microbiome features may identify periodontitis-susceptible individuals despite clinical disease resolution.

Keywords: 16S rRNA gene sequencing; oral microbiome; periodontal diseases; saliva; salivary microbiota.

Figure 1

Schematic diagram representing the species–level open reference taxonomy assignment for 16S rRNA Reads.

Figure 2

Phylogenetic relationship of 21 species shared by 80% of the subjects. Phylotypes assigned to single HOMD entity are shown. Five phylotypes assigned to multiple species are not shown (Granulicatella multispecies spp49 2, Streptococcus, multispecies spp1 2, Streptococcus multispecies spp24 14, Streptococcus multispecies spp32 2).

Figure 3

(A) Boxplots of alpha diversity measures in P and H groups. (B) Rarefaction plot of Shannon alpha diversity in P and H groups. P, blue color; H, red color.

Figure 4

Principal coordinate ordination plots based on the weighted Unifrac beta diversity distance. P, blue color; H, red color. No clustering of P and H samples was evident.

Figure 5

Phylogenetic relationship of Indicator (H indicators in green, P indicators in red) and DA species (in orange). Bars represent mean relative abundances.