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Comparative Study
. 2019 Jan 14;19(1):13.
doi: 10.1186/s12903-018-0693-1.

Comparison of the salivary and dentinal microbiome of children with severe-early childhood caries to the salivary microbiome of caries-free children

Eimear Hurley  1   2 , Maurice P J Barrett  1   3 , Martin Kinirons  2 , Helen Whelton  4 , C Anthony Ryan  5 , Catherine Stanton  6 , Hugh M B Harris  1   3 , Paul W O'Toole  7   8
Affiliations
Comparative Study

Comparison of the salivary and dentinal microbiome of children with severe-early childhood caries to the salivary microbiome of caries-free children

Eimear Hurley et al. BMC Oral Health. .

Abstract

Background: The main objectives of this study were to describe and compare the microbiota of 1) deep dentinal lesions of deciduous teeth of children affected with severe early childhood caries (S-ECC) and 2) the unstimulated saliva of these children and 3) the unstimulated saliva of caries-free children, and to compare microbiota compositional differences and diversity of taxa in these sampled sites.

Methods: Children with S-ECC and without S-ECC were recruited. The saliva of all children with and without S-ECC was sampled along with the deep dentinal microbiota from children affected by S-ECC. The salivary microbiota of children affected by S-ECC (n = 68) was compared to that of caries-free children (n = 70), by Illumina MiSeq sequencing of 16S rRNA amplicons. Finally, the caries microbiota of deep dentinal lesions of those children with S-ECC was investigated.

Results: Using two beta diversity metrics (Bray Curtis dissimilarity and UniFrac distance), the caries microbiota was found to be distinct from that of either of the saliva groups (caries-free & caries-active) when bacterial abundance was taken into account. However, when the comparison was made by measuring only presence and absence of bacterial taxa, all three microbiota types separated. While the alpha diversity of the caries microbiota was lowest, the diversity difference between the caries samples and saliva samples was statistically significant (p < 0.001). The major phyla of the caries active dentinal microbiota were Firmicutes (median abundance value 33.5%) and Bacteroidetes (23.2%), with Neisseria (10.3%) being the most abundant genus, followed by Prevotella (10%). The caries-active salivary microbiota was dominated by Proteobacteria (median abundance value 38.2%) and Bacteroidetes (27.8%) with the most abundant genus being Neisseria (16.3%), followed by Porphyromonas (9.5%). Caries microbiota samples were characterized by high relative abundance of Streptococcus mutans, Prevotella spp., Bifidobacterium and Scardovia spp.

Conclusions: Distinct differences between the caries microbiota and saliva microbiota were identified, with separation of both salivary groups (caries-active and caries-free) whereby rare taxa were highlighted. While the caries microbiota was less diverse than the salivary microbiota, the presence of these rare taxa could be the difference between health and disease in these children.

Keywords: Children; Dentine; Early childhood caries; Microbiota; Saliva.

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Conflict of interest statement

Ethics approval and consent to participate

Ethical approval was obtained from the Cork Teaching Hospitals Clinical Research Ethics Committee for the recruitment and sampling of these Cohorts of children. Ethical approval reference: ECM 3 (cc) 01/07/14. Informed and written consent was obtained from the parent or guardian of each child whom participated in this study.

Consent for publication

Not applicable

Competing interests

The authors have no financial relationships relevant to this article to disclose. The authors have no conflicts of interest to disclose.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
PCoA (principle co-ordinates) plots showing relatedness by two established metrics, Bray Curtis dissimilarity, and UniFrac distances, while unweighted UniFrac illustrates separation between the three groups (CAC, CAS & CFS). a. Plot of principle co-ordinates using Bray-Curtis dissimilarity. Points are coloured according to group and ellipses describe the distribution of points for each group. Percentage variation explained: PCA 1 (22.3%) and PCA 2 (7.7%). b. Plot of principle co-ordinates using weighted unifrac distance. Points are coloured according to group and ellipses describe the distribution of points for each group. Percentage variation explained: PCA 1 (45%) and PCA 2 (11.7%). c. Plot of principle co-ordinates using un-weighted unifrac distance. Points are coloured according to group and ellipses describe the distribution of points for each group. Percentage variation explained: PCA 1 (16.6%) and PCA 2 (4.9%)
Fig. 2
Fig. 2
Pairwise alpha diversity comparisons of saliva and caries microbiota. a. Boxplot of chao1 diversity in the three groups. Outliers are represented by black points. Significant differences between groups are shown by arrows and the following notation: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). b. Boxplot of observed species in the three groups. Outliers are represented by black points. Significant differences between groups are shown by arrows and the following notation: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). c. Boxplot of Phylogenetic diversity in the three groups. Outliers are represented by black points. Significant differences between groups are shown by arrows and the following notation: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). d. Boxplot of Shannon diversity in the three groups. Outliers are represented by black points. Significant differences between groups are shown by arrows and the following notation: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***)
Fig. 3
Fig. 3
Hierarchical clustering of microbiota data at bacterial family level. Abundances are colour-coded according to the colour key on the top left with grey representing a value of zero. Euclidean distance and complete linkage were used to cluster the rows and columns of the heatmap. The colour bar on top of the heatmap corresponds to sample type: CAC red, CAS green and CFS blue. All taxa present at less than 1% in all three groups are excluded from the heatmap
Fig. 4
Fig. 4
Hierarchical clustering of microbiota data at bacterial genus level. Abundances are colour-coded according to the colour key on the top left with grey representing a value of zero. Euclidean distance and complete linkage were used to cluster the rows and columns of the heatmap. The colour bar on top of the heatmap is coloured according to sample type: CAC red, CAS green and CFS blue. All taxa present at less than 1% in all three groups are excluded from the heatmap
Fig. 5
Fig. 5
Broad and fine detail compositional differences at Genus, phylum and species level. a. Microbiota composition at phylum level. Percentages for each taxon represent the median abundance values for the sample types. b. Barplot of percentage abundance at genus level. Percentages for each taxon represent the median values for the groups. c. Barplot of percentage abundance at species level. Percentages for each taxon represent the median values for the groups
Fig. 6
Fig. 6
Hierarchical clustering of microbiota data at bacterial species level. Abundances are colour-coded according to the colour key on the top left with grey representing a value of zero. Euclidean distance and complete linkage were used to cluster the rows and columns of the heatmap. The colour bar on top of the heatmap is coloured according to sample type: CAC red, CAS green and CFS blue. All taxa present with at least one species with a median value ≥0.5% in all three groups are included
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