Metagenomic Analysis of Dental Plaque on Pit and Fissure Sites With and Without Caries Among Adolescents

Abstract

Caries is one of the most prevalent infectious diseases worldwide and is driven by the dysbiosis of dental biofilms adhering to tooth surfaces. The pits and fissured surfaces are the most susceptible sites of caries. However, information on the taxonomic composition and functional characteristics of the plaque microbiota in the pit and fissure sites is very limited. This study aimed to use metagenomic sequencing analyses to investigate the relationship between the plaque microbiome in the pit and fissure site and caries in adolescents. A total of 20 adolescents with active pit and fissure surface caries were involved as well as 20 age-matched, caries-free teenagers for control tests. Plaque samples were collected from the pit and fissure site and were subjected to metagenomic analyses, in which the microbial communities were investigated. Our results showed that the microbiota diversity was similar between those two groups. At the species level, the relative abundances of A. gerencseriae, P. acidifaciens, P. multisaccharivorax, S. oralis, S. mutans, and P. denticolens were higher in the caries-active group. N. elongata, C. hominis, and A. johnsonii were relatively more abundant in the caries-free groups. Functional analysis suggested that the metabolic pathway was the most abundant pathway, and the functional traits of the level 2 pathways included amino acid metabolism, metabolism of cofactors, and vitamins and carbohydrate metabolism. Our results also revealed that the caries group displayed several alterations in metabolic pathways, including enriched functions in carbohydrate digestion and absorption. This study suggested that in addition to the specific anatomical structures of the pit and fissured surfaces, the fundamental differences in the plaque microbiome may also be related to the susceptibility of pit and fissure caries.

Keywords: biofilms; bioinformatics; biomarkers; dental caries; microbiome.

Figure 1

Relative abundances of plaque microbial in caries active and caries free groups. (A–C) Relative abundances of phyla, genera, and species from the top 20 annotated microbial taxa, respectively, are shown in a bar plot. (D) Significant differences in relative abundance of plaque microbial taxa between the caries active and caries-free groups, as identified by linear discriminant analysis (LDA) effect size analysis (LEFSe) (DUNN test, p < 0.05).

Figure 2

The diversity analysis between groups: PCoA (A) and Non-Metric Multi-Dimensional (B) analysis based on Bray-Curtis distances according to the abundance tables of microbial taxa at the genus level. Circles in different colors represent different groups.

Figure 3

Venn diagram at the species level. The overlap region A represents the microbiome shared in all samples among caries-active and caries-free groups. Region B represented the unique taxa in caries-free controls and Region C represented the unique taxa in caries-active group.

Figure 4

Comparing the functional characteristics of the microbiome caries active group and caries free controls. (A) The number of genes annotated in the Kyoto Encylopedia of Genes from the 40 samples. (B) The comparison of functional KEGG between the two groups at the level 1 pathway, (C) level 2 pathway, (D) level 3 pathway, and (E) ortholog level. The horizontal axis represents the relative abundance of annotated genes. The histograms show the top 20 annotated genes predicted in the metabolic pathways.

Figure 5

Linear discriminant analysis (LDA) effect size analysis (LEFSe) was used to identify level 3 pathways (A) and KOs (B) with significant differences in relative abundance between the caries active and caries-free groups.

Figure 6

Species contributions to key functions. For each identified KO, HUManN2 was used to calculate the species contribution. In the figure are species contributions to three features correlated to dental caries.

Figure 7

Networks in dental microbial communities under caries-active and caries free status are shown, with each microbial species and cooccurrence relationship indicated by a node and an edge, respectively. A connection (line between dots) indicates a strong (Spearman’s p >0.6) and significant (p < 0.05) correlation. The size of each node is proportional to the relative abundance. Lines between nodes indicate positive correlations (red) or negative correlations (blue). The top thress abundant genera are indicated in color.