Exploring the salivary microbiome of children stratified by the oral hygiene index

Abstract

Poor oral hygiene often leads to chronic diseases such as periodontitis and dental caries resulting in substantial economic costs and diminished quality of life in not only adults but also in children. In this study, the salivary microbiome was characterized in a group of children stratified by the Simplified Oral Hygiene Index (OHI-S). Illumina MiSeq high-throughput sequencing based on the 16S rRNA was utilized to analyze 90 salivary samples (24 Good, 31 Moderate and 35 Poor oral hygiene) from a cohort of Thai children. A total of 38,521 OTUs (Operational Taxonomic Units) with a 97% similarity were characterized in all of the salivary samples. Twenty taxonomic groups (Seventeen genera, two families and one class; Streptococcus, Veillonella, Gemellaceae, Prevotella, Rothia, Porphyromonas, Granulicatella, Actinomyces, TM-7-3, Leptotrichia, Haemophilus, Selenomonas, Neisseria, Megasphaera, Capnocytophaga, Oribacterium, Abiotrophia, Lachnospiraceae, Peptostreptococcus, and Atopobium) were found in all subjects and constituted 94.5-96.5% of the microbiome. Of these twenty genera, the proportion of Streptococcus decreased while Veillonella increased with poor oral hygiene status (P < 0.05). Furthermore, an unassigned species of Veillonella, Veillonella dispar and Veillonella parvula tended to be elevated in the Poor oral hygiene group. This is the first study demonstrating an important association between increase of Veillonella and poor oral hygiene status in children. However, further studies are required to identify the majority of Veillonella at species level in salivary microbiome of the Poor oral hygiene group.

Fig 1. Principal coordinate analysis (PCoA) plot showing the similarity relationships among bacterial community samples from 90 Thai children divided to the three oral hygiene groups using weighted UniFrac distance metric.

The green (triangles), blue (squares), and red (circles) indicate the Good, Moderate, and Poor oral hygiene groups, respectively. (a) The two components explained 36.78 and 11.60% of the variance, respectively. (b) The two components explained 36.78 and 5.85% of the variance, respectively. (c) Two components explained 5.85 and 11.60% of the variance, respectively. (d), (e) 3D PCoA plots were visualized by the EMPEROR.

Fig 2. Weighted UniFrac clustering tree of the 90 Thai children individuals.

This clustering tree shows the result of weighted UniFrac PCoA plots and visualized the phylogenetic relationship among each salivary microbiome. Each branch represents an individual salivary sample. Samples having similar salivary microbiome compositions are more clustered as highlighted. Green represents Good, blue Moderate and red Poor hygiene branches. Bar length is proportional to phylogenetic distance.

Fig 3. Mean number of operational taxonomic units (OTUs) and Shannon Index in the salivary microbiome of the 90 Thai children divided to the three groups.

The significant differences were evaluated by the Kruskal-Wallis H-test post hoc Mann-Whitney U-test with Bonferroni correction (P < 0.05). The error bar indicated 95% confidence intervals.

Fig 4. Relative abundances of 20 bacterial genera including upper-taxa among three oral hygiene groups.

C, f, and g indicated the class, family and genus, respectively. Some OTUs were not assignable to a genus since the sequence was absent from the database. The significant differences were evaluated using the Kruskal-Wallis H-test post hoc Mann-Whitney U-test with Bonferroni correction (P < 0.05). The error bars indicated 95% confidence intervals.

Fig 5. Heatmap displaying species of Streptococcus and Veillonella distribution patterns in the three oral hygiene groups.

To show the distribution of abundant OTUs, the relative abundances of OTU data were normalized to have a mean of 0 and standard deviation of 1 (z-score normalization) at the species level using cluster 3.0. The distribution of the OTUs represented as the color intensity of each grid (blue, low abundance; red, high abundance). All assigned species of genus Streptococcus and Veillonella in this study were ordered across the three oral hygiene groups. S indicates the species. “Other” indicates that no more than half of the sequences in each OTU were obtained from the taxonomic information of that genus. Some OTUs were not assignable to a species since the sequence was absent from the database.