Characterizing oral microbial communities across dentition states and colonization niches

Abstract

Methods: The present study aimed to identify patterns and processes in acquisition of oral bacteria and to characterize the microbiota of different dentition states and habitats. Mucosal, salivary, supragingival, and subgingival biofilm samples were collected from orally and systemically healthy children and mother-child dyads in predentate, primary, mixed, and permanent dentitions. 16S rRNA gene sequences were compared to the Human Oral Microbiome Database (HOMD). Functional potential was inferred using PICRUSt.

Results: Unweighted and weighted UniFrac distances were significantly smaller between each mother-predentate dyad than infant-unrelated female dyads. Predentate children shared a median of 85% of species-level operational taxonomic units (s-OTUs) and 100% of core s-OTUs with their mothers. Maternal smoking, but not gender, mode of delivery, feeding habits, or type of food discriminated between predentate microbial profiles. The primary dentition demonstrated expanded community membership, structure, and function when compared to the predentate stage, as well as significantly lower similarity between mother-child dyads. The primary dentition also included 85% of predentate core s-OTUs. Subsequent dentitions exhibited over 90% similarity to the primary dentition in phylogenetic and functional structure. Species from the predentate mucosa as well as new microbial assemblages were identified in the primary supragingival and subgingival microbiomes. All individuals shared 65% of species between supragingival and subgingival habitats; however, the salivary microbiome exhibited less than 35% similarity to either habitat.

Conclusions: Within the limitations of a cross-sectional study design, we identified two definitive stages in oral bacterial colonization: an early predentate imprinting and a second wave with the eruption of primary teeth. Bacterial acquisition in the oral microbiome is influenced by the maternal microbiome. Personalization begins with the eruption of primary teeth; however, this is limited to phylogeny; functionally, individuals exhibit few differences, suggesting that microbial assembly may follow a defined schematic that is driven by the functional requirements of the ecosystem. This early microbiome forms the foundation upon which newer communities develop as more colonization niches emerge, and expansion of biodiversity is attributable to both introduction of new species and increase in abundance of predentate organisms.

Keywords: 16S; Acquisition; Bacteria; DNA; Pyrosequencing; Saliva; Subgingival; Supragingival.

Fig. 1

Bacterial acquisition in the predentate microbiome. Species-level operational taxonomic units (s-OTUs) that were identified in the predentate mucosa of 47 infants are shown in a. Bars indicate presence of species and are not sized by their abundances. Species that were part of the core predentate mucosal microbiome are indicated in orange font. Orange bars indicate s-OTUs that were shared by each child and their biological mother (based on 11 mother-child dyad samples). Principal component analysis of unweighted UniFrac distances colored by delivery mode (b) and feeding mode (c) are shown. The inferred functional potential encoded in the core predentate mucosal microbiome is shown in d. The pie slices are sized by relative abundances of genes encoding for each function. The similarity between each mother-child, infant-infant, and infant-unrelated adult dyad is shown in e. The metrics are unweighted UniFrac distances, weighted Unifrac, and Bray-Curtis similarity. Groups that are bracketed are significantly different from each other (p < 0.05, Dunn’s test). Principal component analysis of unweighted UniFrac distances of the predentate microbiome is shown in f. The samples are colored by maternal smoking (self reported). The relative abundances (expressed as a percent) of species that were significantly different between predentate infants of smokers and nonsmokers is shown in g

Fig. 2

Phylogenetic shifts in the core salivary microbiome during various dentition states. a The salivary core microbiome (present in at least 75% of individuals in the group) of children in predentate, primary, mixed, and adult dentitions, as well as those of unrelated adults. Each bar represents presence of species. Data for a is presented in Additional file 4: Table S2. Shannon diversity index is shown in Fig. b and equitability in Fig. c. The distribution of species by gram staining characteristics and oxygen requirements in each dentition state is shown in Fig. d. Probable gram staining characteristics and oxygen requirements were attributed to uncultivated species based on phylogenetic relatedness to the closest cultivated species. In all the figures, groups that share the same symbol are significantly different from each other (p < 0.05, Dunn’s test). The similarity between children in different dentition states and their biological mother is shown in e. Groups that are bracketed are significantly different from each other (p < 0.05, Dunn’s test)

Fig. 3

Phylogenetic characteristics of the supragingival microbiome. a A stacked bar chart of supragingival s-OTUs by dentition (phylogenetic tree constructed using the webserver iTOL.embl.de). Data for a is presented in Additional file 7: Table S3. The length of each bar represents the normalized mean relative abundance of a species. Data for this graph (b) shows the core supragingival microbiome in the three dentate stages. The bars represent species that were detected in 75% or more individuals in a group. The distribution of s-OTUs by gram staining characteristics and oxygen requirements in each dentition are shown in c, and Shannon diversity and equitability in d and e respectively. Probable gram staining characteristics and oxygen requirements were attributed to uncultivated species based on phylogenetic relatedness to the closest cultivated species. In all the figures, groups that share the same symbol are significantly different from each other (p < 0.05, Dunn’s test)

Fig. 4

Phylogenetic characteristics of the subgingival microbiome. Figure 4A is a stacked bar chart representing the normalized mean relative abundances of subgingival s-OTUs by dentition (phylogenetic tree constructed using the webserver iTOL.embl.de). Data for Fig. 4a is presented in Additional File 9-supplemental table S5. b shows the core subgingival microbiome in the three dentate stages. The bars represent species that were detected in 75% or more individuals in a group. The distribution of s-OTUs by gram staining characteristics and oxygen requirements in each dentition are shown in panel c, and Shannon diversity and equitability in panels d and e respectively. Probable gram staining characteristics and oxygen requirements were attributed to uncultivated species based on phylogenetic relatedness to the closest cultivated species. Panel f shows the number of species shared between the supragingival and subgingival environments in all dentition states. In all the figures, groups that share the same symbol are significantly different from each other (p < 0.05, Dunn’s test)