Establishment and Development of Oral Microflora in 12-24 Month-Old Toddlers Monitored by High-Throughput Sequencing

Abstract

A cohort study was conducted to evaluate oral microbial diversity among toddlers aged 12-24 months, and to describe the dynamic processes of colonization, development, and stabilization of the oral microflora during tooth eruption using high-throughput sequencing technology. A total of 20 healthy toddlers aged 12 months were included at baseline and followed up through 18-24 months. Clinical oral examinations of dental caries status and visible plaque index were carried out at three follow-up time points. Pooled supragingival plaque biofilm samples were also collected at ages 12, 18, and 24 months. Plaque biofilm DNA was extracted and analyzed by MiSeq sequencing. A total of 18 toddlers completed three follow-ups. At 12 months of age, all the toddlers only had eruption of the anterior teeth, without dental caries. At ages 18 and 24 months, one and two toddlers showed two and three teeth with carious white spots, respectively. Depth, Good's coverage, and sample size of sequencing were reasonable. The dominant bacterial genera in the oral cavity of 12-month-old toddlers were Capnocytophaga, Neisseria, Streptococcus, Kingella, and Leptotrichia; the oral microflora composition was relatively stable by 18 months of age and included unclassified Enterobacteriaceae, Selenomonas, Prevotella, Leptotrichia, and Veillonella as the dominant genera; unclassified Enterobacteriaceae, Streptococcus, Neisseria, Leptotrichia, and Selenomonas were the dominant genera by 24 months. There were significant differences among microbial compositions in the oral cavities of 12, 18, and 24-month-old toddlers, with relatively small differences observed between the 18 and 24 months samples. In conclusion, oral microbial community of toddlers showed a trend of dynamic development. Significant differences in oral microbial diversity among toddlers aged 12-24 months were observed, while the microbial diversity differences among toddlers aged 18-24 months tended to be more similar. The findings indicated that the oral microbial community gradually matures and tends to stabilize with the growth and development of toddlers.

Keywords: Streptococcus; early childhood caries; high-throughput sequencing; oral microbial diversity; toddlers.

Figure 1

A Venn diagram showing shared and unique OTUs at 97% identity among the four groups.

Figure 2

PCA based on the weighted UniFrac distances at the OUT level at 97% identity. Each sample is represented by a dot. PC1 explained 75.72% of the variation observed, and PC2 explained 7.12% of the variation. The samples formed well-separated clusters corresponding to the three groups, suggesting that the bacterial structures between groups were different.

Figure 3

Classification and compositional ratios of the samples obtained the three time points sequenced at the taxonomic level.

Figure 4

The linear discriminant analysis effect size (LEfSe). The colored nodes from the inner to outer circles represent the top abundant taxa from phylum to genus level. The yellow nodes represent taxa showing no significant difference between groups, while nodes with other colors represent taxa showing significant difference between groups.

Figure 5

Redundancy analysis (RDA). Bioplot of RDA of the microbiota between 12 and 18 m (A), 18 and 24 m (B) samples, responding the OTUs with at least 13 (A) and 15% (B) of the variability of the oral microbiota are indicated by blue arrows. Bioplot of RDA of the microbiota relative to the early dental caries (C), responding the OTUs with at least 16% of the variability of the oral microbiota.

Figure 6

Heat-map of the 50 relative abundant key bacteria genera. The color of the spot corresponds to the normalized relative abundances of the genera.