Supragingival Plaque Microbiome Ecology and Functional Potential in the Context of Health and Disease

Abstract

To address the question of how microbial diversity and function in the oral cavities of children relates to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. Caries phenotypes contained statistically significant enrichments in specific genome abundances and distinct community composition profiles, including strain-level changes. Metabolic pathways that are statistically associated with caries include several sugar-associated phosphotransferase systems, antimicrobial resistance, and metal transport. Numerous closely related previously uncharacterized microbes had substantial variation in central metabolism, including the loss of biosynthetic pathways resulting in auxotrophy, changing the ecological role. We also describe the first complete Gracilibacteria genomes from the human microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology, and our results provide the information needed for next-generation diagnostic tools and therapeutics for caries.IMPORTANCE Oral health has substantial economic importance, with over $100 billion spent on dental care in the United States annually. The microbiome plays a critical role in oral health, yet remains poorly classified. To address the question of how microbial diversity and function in the oral cavities of children relate to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. This unveiled several new previously uncharacterized but ubiquitous microbial lineages in the oral microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology, and our results provide the information needed for next-generation diagnostic tools and therapeutics for caries.

Keywords: Streptococcus; metabolism; metagenomics; microbial ecology; oral microbiology.

FIG 1

Core supragingival microbiome composition in the context of health and disease. Microbial community abundance profiles and enrichment in phenotype-specific cohorts. Statistical significance (P < 0.001, ***; P < 0.01, **; and P < 0.05, *). Error bars represent SEM. (A) Mean of pairwise log₂ fold changes between phenotype subsets. (Top) Caries-positive versus caries-negative individuals with red indicating enrichment of taxa in the caries-positive cohort. (Bottom) Subjects with caries that has progressed to the dentin layer versus enamel-only caries with blue denoting taxa enriched in the enamel. (B) Relative abundance of TPM values for each genera of de novo assembly grouped by caries-positive and caries-negative cohorts. Actinomyces = 0.36, Alloprevotella = 0.0515, Campylobacter = 0.0124, “Candidatus Saccharimonas” = 0.107, Capnocytophaga = 0.000875, Cardiobacterium = 0.0621, Catonella = 0.000264, Corynebacterium = 0.0552, Granulicatella = 0.00255, Kingella = 0.252, Lachnoanaerobaculum = 0.0791, Lautropia = 0.279, Leptotrichia = 0.0829, Moraxella = 0.215, Neisseria = 0.455, Porphyromonas = 0.136, Prevotella = 0.12, Riemerella = 0.0621, Streptococcus = 0.000901, Tannerella = 0.0061, unclassified Bacteroidetes = 0.0111, unclassified Gracilibacteria = 0.0316, Veillonella = 0.471. (C) Kernel density estimation of Shannon entropy alpha diversity distributions for caries-positive (red) and caries-negative (gray) subjects calculated from normalized core supragingival community composition. Vertical lines indicate the mode of the kernel density estimate distributions for each cohort. Statistical significance (P = 0.0109).

FIG 2

Streptococcus community composition in the context of health and disease. Streptococcus community abundance analysis and enrichment in phenotype-specific cohorts. Statistical significance (P < 0.001, ***; P < 0.01, **; and P < 0.05, *). Error bars represent SEM. (A) Mean of pairwise log₂ fold changes between phenotype subsets. (Top) Caries-positive versus caries-negative individuals with red indicating enrichment of taxa in the caries-positive cohort. (Bottom) Subjects with caries that has progressed to the dentin layer versus enamel-only caries with blue denoting taxa enriched in the enamel. Pseudocount of 1e−4 applied to entire-count matrix for log transformation. (B) Relative abundance of MIDAS counts for each Streptococcus species grouped by caries-positive (red) and caries-negative (gray) subjects. (C) Fully connected undirected networks for diseased (right) and healthy (left) groups separately. Edge weights represent topological overlap measures, nodes colored by PageRank centrality, and the Fruchterman-Reingold force-directed algorithm for the network layout. (D) Kernel density estimation of Shannon entropy alpha diversity distributions for caries-positive (red) and caries-negative (gray) subjects calculated from normalized Streptococcus community composition. Vertical lines indicate the mode of the kernel density estimate distributions for each cohort. Statistical significance (P = 0.008).

FIG 3

Core supragingival microbial cooccurrence network topology. Fully connected undirected cooccurrence network from normalized abundance profiles. (Dendrogram) Clustering of taxa using topological overlap dissimilarity and ward linkage with PageRank centrality (top row) and statistical enrichment in healthy or diseased cohorts. Statistical significance (P < 0.05; red, enriched in diseased; blue, enriched in healthy).

FIG 4

KEGG module significance to phenotype. Venn diagram of numbers of significant KEGG modules identified by unanchored and anchored approaches (P < 0.05).

FIG 5

Metagenome assembled genomes recovered in assembly. Multiple MAGs of individual species and their functional differences. (A) t-SNE embeddings of center-log-ratio-transformed 5-mer profiles for each contig. Gold contigs have higher C and E coefficients in the ACE model distinguishing environmental acquisition while teal contigs indicate high A coefficients of heritable organisms. (B) Differences in functional potential of A. rava strains where one has a high heritability coefficient while the other has high environmentally associated coefficients according to the ACE model.

FIG 6

Supragingival microbial dark matter MAGs. TM7 and Gracilibacteria cooccurrence profiles and GC content distributions. (A) Spearman’s correlation for (left) TM7 and (right) Gracilibacteria abundance profiles against all other MAG abundance profiles. (B) GC content distributions for all contigs in corresponding MAGs from taxonomy groups III and IV.