Beyond Streptococcus mutans: dental caries onset linked to multiple species by 16S rRNA community analysis

Abstract

Dental caries in very young children may be severe, result in serious infection, and require general anesthesia for treatment. Dental caries results from a shift within the biofilm community specific to the tooth surface, and acidogenic species are responsible for caries. Streptococcus mutans, the most common acid producer in caries, is not always present and occurs as part of a complex microbial community. Understanding the degree to which multiple acidogenic species provide functional redundancy and resilience to caries-associated communities will be important for developing biologic interventions. In addition, microbial community interactions in health and caries pathogenesis are not well understood. The purpose of this study was to investigate bacterial community profiles associated with the onset of caries in the primary dentition. In a combination cross-sectional and longitudinal design, bacterial community profiles at progressive stages of caries and over time were examined and compared to those of health. 16S rRNA gene sequencing was used for bacterial community analysis. Streptococcus mutans was the dominant species in many, but not all, subjects with caries. Elevated levels of S. salivarius, S. sobrinus, and S. parasanguinis were also associated with caries, especially in subjects with no or low levels of S. mutans, suggesting these species are alternative pathogens, and that multiple species may need to be targeted for interventions. Veillonella, which metabolizes lactate, was associated with caries and was highly correlated with total acid producing species. Among children without previous history of caries, Veillonella, but not S. mutans or other acid-producing species, predicted future caries. Bacterial community diversity was reduced in caries as compared to health, as many species appeared to occur at lower levels or be lost as caries advanced, including the Streptococcus mitis group, Neisseria, and Streptococcus sanguinis. This may have implications for bacterial community resilience and the restoration of oral health.

Figure 1. Relative levels of bacterial taxa by advancing stage of caries.

Graphs at the level of phylum, genus and species are shown. Taxa are sorted by magnitude of change with stage of caries (linear mixed effects model estimates), so that taxa associated with health sort at the bottom and taxa associated with caries are shown at the top. “*” indicates taxa with p<0.05 and “**” indicates taxa with p<0.01 after the false discovery rate correction was applied. Only genera found at greater than 0.1% of total clones and species found at greater than 0.2% of total clones are shown, and only those taxa significantly associated with caries or health are shown in the species-level graph.

Figure 2. Correlation between relative levels of Veillonella and acidogenic streptococci in white spot lesions.

The total % abundance of S. mutans, S. sobrinus, and S. vestibularis/salivarius combined is plotted against the abundance of the Veillonella atypica/dispar/parvula group expressed as a fraction of the remaining community. The result of a linear regression is shown as a line with the indicated parameters.

Figure 3. Heatmap and cluster analysis of baseline samples from white spot lesions.

Abundances of those bacterial species significantly associated with caries are shown, except for Veillonella which was ubiquitous and therefore omitted. The samples (one from each of 36 subjects) are arranged by hierarchical clustering using the average method and Bray-Curtis dissimilarity. Abundance as percentage of the total community is indicated by the color scale. The bar along the left side indicates longitudinal caries activity.

Figure 4. Decreasing species diversity was observed with increasing caries severity both within and among subjects.

Mean Shannon Diversity Indices with 95% confidence intervals are shown. The upper panel shows diversity within subjects for stage of caries at baseline. Diversity was modeled using a linear mixed effects model (SAS PROC MIXED), and is shown as a dashed line (estimate =  −0.26). Post hoc comparisons between sample types were significant, except between white spot and cavitated lesions. The lower panel shows species diversity comparisons among subjects by their baseline and longitudinal caries status for samples collected from noncarious enamel (the only type of sample available from all groups) using ANOVA. Significant post hoc comparisons are indicated by blue lines.

Figure 5. Within-subject differences in plots of a non-metric multidimensional scaling (NMDS) ordination based on Bray-Curtis Dissimilarity.

Baseline samples from only the subset of subjects whose caries status remained constant over time (caries subjects that continued to develop caries and healthy subjects that remained healthy) was included, and within-subject differences by stage of caries were observed. A single sample from each stage of caries is included for each subject, and each point represents a single sample. The top panel shows the NMDS plot, with the centroid for each stage of caries marked. The metaMDS algorithm used puts the largest dimension of change along the horizontal axis. The p-value is for the overall ANOSIM model. The points in lower panels are sized by abundance for the most common species significantly associated with caries and health, and p-values are for the linear mixed effects model estimates. Empty plot symbols represent samples where that species was not detected.

Figure 6. Sample sizes at baseline and outcomes at longitudinal follow-up.

Figure 7. Candidate microbial risk and protective factors.

Candidate microbial risk and protective factors are listed for the onset of caries in subjects that were caries free at baseline and the progression of caries in subjects that had caries at baseline.

Figure 8. Among-subject differences visualized by non-metric multidimensional scaling (NMDS) ordination based on Bray-Curtis Dissimilarity.

Among-subject differences by longitudinal caries activity were observed. The plots represent baseline community profiles for healthy subjects and subjects with caries. The baseline patient class and subsequent disease activity are color-coded. Samples from healthy enamel were the only stage available from all subjects and so were used here. The top panel shows the NMDS plot, with the centroid for each clinical group marked. The p-value is for the overall ANOSIM model. The points in lower panels are sized by abundance for the most common species significantly associated with caries and health, and p-values are for the linear mixed effects model estimates.