Oral microbiome development during childhood: an ecological succession influenced by postnatal factors and associated with tooth decay

Abstract

Information on how the oral microbiome develops during early childhood and how external factors influence this ecological process is scarce. We used high-throughput sequencing to characterize bacterial composition in saliva samples collected at 3, 6, 12, 24 months and 7 years of age in 90 longitudinally followed children, for whom clinical, dietary and health data were collected. Bacterial composition patterns changed through time, starting with "early colonizers", including Streptococcus and Veillonella; other bacterial genera such as Neisseria settled after 1 or 2 years of age. Dental caries development was associated with diverging microbial composition through time. Streptococcus cristatus appeared to be associated with increased risk of developing tooth decay and its role as potential biomarker of the disease should be studied with species-specific probes. Infants born by C-section had initially skewed bacterial content compared with vaginally delivered infants, but this was recovered with age. Shorter breastfeeding habits and antibiotic treatment during the first 2 years of age were associated with a distinct bacterial composition at later age. The findings presented describe oral microbiota development as an ecological succession where altered colonization pattern during the first year of life may have long-term consequences for child´s oral and systemic health.

Fig. 1

Species richness and diversity of total microbiota in infant saliva samples. Bacterial richness and diversity (here presented by Chao1 and Shannon indices), obtained at different time points from birth to 7 years of age, were determined by 16S rRNA Illumina sequencing and OTU clustering at 97% sequence identity. a shows species richness and diversity in infants delivered vaginally (VD) or by caesarean section (C-section) at 3 months (N_VD = 62; N_CS = 11), 6 months (N_VD = 72; N_CS = 11), 12 months (N_VD = 59; N_CS = 10), 24 months (N_VD = 56; N_CS = 10) and 7 years of age (N_VD = 68; N_CS = 12). b represents species diversity and richness in infants breastfed for 12 months (BF) and in infants breastfed for less than 6 months of age (nBF). Analyzed samples were collected at 12 months (N_nBF = 52; N_BF = 17), 24 months (N_nBF = 50; N_BF = 16), and 7 years of age (N_nBF = 59; N_BF = 21). c shows species richness and diversity in children developing caries (CA) and children staying caries-free (CF) during the first 9 years of life. Saliva samples were collected at 3 months (N_CF = 40; N_CA = 26), 6 months (N_CF = 43; N_CA = 31), 12 months (N_CF = 37; N_CA = 26), 24 months (N_CF = 35; N_CA = 24) and 7 years of age (N_CF = 45; N_CA = 30). Data are presented with means and standard errors. (*p < 0.05; Mann–Whitney U-test)

Fig. 2

Salivary microbiota patterns through children’s development. Constrained correspondence analysis (CCA), here used to emphasize variations in microbiota species-level profiles, show the relationship between groups in total microbiota composition at different time points. The percentage of variation explained by constrained correspondence components is indicated on the axes. a Microbial pattern differences in saliva from infants delivered vaginally (VD) or by caesarean section (CS), p = 0.0016, at 3 months (N_VD = 62; N_CS = 11), 6 months (N_VD = 72; N_CS = 11), 12 months (N_VD = 59; N_CS = 10), 24 months (N_VD = 56; N_CS = 10) and 7 years of age (N_VD = 68; N_CS = 12). b Showing microbial composition pattern differences in infants who were breastfed for 12 months (BF) and in infants breastfed (nBF) for less than 6 months (p = 0.0017). The numbers of children were: 12 months (N_nBF = 52; N_BF = 17), 24 months (N_nBF = 50; N_BF = 16) and 7 years of age (N_nBF = 59; N_BF = 21). c Microbial composition patterns in children developing caries (CA) and children staying caries-free (CF) during the first 9 years of life (p = 0.0018). Saliva samples were collected at 3 months (N_CF = 40; N_CA = 26), 6 months (N_CF = 43; N_CA = 31), 12 months (N_CF = 37; N_CA = 26), 24 months (N_CF = 35; N_CA = 24) and 7 years of age (N_CF = 45; N_CA = 30). Numbers accompanying the variables (delivery mode, breastfeeding and caries onset) are representing the time points plotted. p-Values for CCA plots were determined by Adonis analysis (a nonparametric statistical method, R package Vegan) and significant values indicate that the factor provided can explain part of the total variability

Fig. 3

Microbiota composition of dominant bacterial genera in children being or not being breastfed until 12 months of age. a Genera classified as early colonizers. b Genera considered to “constantly increase” are already present at 3–6 months of age at >1% frequency, and are increasing in relative proportion with time. c Bacterial genera considered as “Late colonizers”, are defined as those present at 3–6 months of age below 1% relative abundance, which undergo a significant increase after 12 months of age. Plots are showing the relative abundance of dominant bacterial genera, as determined by Illumina sequencing of 16S rRNA gene, in saliva samples collected at 3 months, 6 months, 12 months (N_nBF = 52; N_BF = 17), 24 months (N_nBF = 50; N_BF = 16) and 7 years (N_nBF = 59; N_BF = 21) of age. Asterisks indicate cases where p < 0.05 by both Mann–Whitney U-test and Wilcoxon analysis

Fig. 4

Accumulative antibiotics effect on salivary microbiota development. a shows salivary microbiota patterns in children treated with antibiotics during the first 2 years of life and children not taking any antibiotics during the first 2 years of life; p = 0.017. p values for CCA plots were determined by Adonis analysis (a nonparametric statistical method, R package Vegan) and indicate that the factor provided can explain part of the total variability. b represents the influence of antibiotics intake on bacterial species distribution at 24 months and 7 years of age, here represented with a Venn’s diagram. The numbers indicate unique species found in children not treated with antibiotics (purple) and children treated with antibiotics (grey) and OTUs differentially distributed are stated in the tables. OTUs presented were filtered according to sequence length (>300 bp) and identity (>97% nucleotide similarity). Analyzed sample sizes were: 3 months (N_NO = 28; N_YES = 11), 6 months (N_NO = 33; N_YES = 13), 12 months (N_NO = 29; N_YES = 11), 24 months (N_NO = 26; N_YES = 10) and 7 years (N_NO = 35; N_YES = 11); NO = no antibiotics intake for the first 2 years of age; YES = antibiotic consumption during the first 2 years of age

Fig. 5

Relative abundance of most prevalent Streptococci species found in saliva samples of children staying caries-free and children developing caries during the first 9 years of age. Plots represent average relative abundance of Streptococci through time. Taxonomy assignments were performed with RDP classifier at 100% nucleotide identity. All data are presented as means with standard errors. Asterisks indicate cases where p < 0.05 by both Mann–Whitney U-test and Wilcoxon analysis