Clonal analysis of the microbiota of severe early childhood caries

Abstract

Background/aims: Severe early childhood caries is a microbial infection that severely compromises the dentition of young children. The aim of this study was to characterize the microbiota of severe early childhood caries.

Methods: Dental plaque samples from 2- to 6-year-old children were analyzed using 16S rRNA gene cloning and sequencing, and by specific PCR amplification for Streptococcus mutans and Bifidobacteriaceae species.

Results: Children with severe caries (n = 39) had more dental plaque and gingival inflammation than caries-free children (n = 41). Analysis of phylotypes from operational taxonomic unit analysis of 16S rRNA clonal metalibraries from severe caries and caries-free children indicated that while libraries differed significantly (p < 0.0001), there was increased diversity than detected in this clonal analysis. Using the Human Oral Microbiome Database, 139 different taxa were identified. Within the limits of this study, caries-associated taxa included Granulicatella elegans (p < 0.01) and Veillonella sp. HOT-780 (p < 0.01). The species associated with caries-free children included Capnocytophaga gingivalis (p < 0.01), Abiotrophia defectiva (p < 0.01), Lachnospiraceae sp. HOT-100 (p < 0.05), Streptococcus sanguinis (p < 0.05) and Streptococcus cristatus (p < 0.05). By specific PCR, S. mutans (p < 0.005) and Bifidobacteriaceae spp. (p < 0.0001) were significantly associated with severe caries.

Conclusion: Clonal analysis of 80 children identified a diverse microbiota that differed between severe caries and caries-free children, but the association of S. mutans with caries was from specific PCR analysis, not from clonal analysis, of samples.

Fig. 1

Phylogenetic trees of species and phylotypes identified from clones compared with the HOMD. The trees were created from full 16S rRNA reference sequences although it is recognized that use of 500-bp data from study clones could have created a different, but less accurate, treeing topography. Taxa in trees include identifications at >0.1 mean percent positive taxa by subject. A total of 139 taxa were identified. Numbers of children with taxa detected are in the table to the right of each tree. Species or phylotypes that differed in detection frequencies are noted at ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, by χ² analysis, or Fishers exact test. a Phylogenetic tree of 51 taxa in Proteobacteria, Bacteroidetes and Actinobacteria detected in severe caries and caries-free children, with positions of Clostridia and Bacillus as phylogenetic references. The tree includes 28 taxa of Proteobacteria, 15 taxa of Bacteroidetes, 1 Treponema species, 5 Actinobacteria and 2 Leptotrichia taxa. b Phylogenetic tree of 49 taxa in the Clostridia branch of Firmicutes, which includes the Gram-negative Veillonella and Selenomonas genera, detected in severe ECC and caries-free children. This group comprises several named and unnamed taxa, including the Centipeda species. c Phylogenetic tree of 38 taxa in the Bacillus branch of Firmicutes detected in severe caries and caries-free children. The tree includes 29 Streptococcus species, comprising 10 unnamed species, 2 Gemella, 2 Granulicatella and 4 Lactobacillus species.

Fig. 1

Phylogenetic trees of species and phylotypes identified from clones compared with the HOMD. The trees were created from full 16S rRNA reference sequences although it is recognized that use of 500-bp data from study clones could have created a different, but less accurate, treeing topography. Taxa in trees include identifications at >0.1 mean percent positive taxa by subject. A total of 139 taxa were identified. Numbers of children with taxa detected are in the table to the right of each tree. Species or phylotypes that differed in detection frequencies are noted at ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, by χ² analysis, or Fishers exact test. a Phylogenetic tree of 51 taxa in Proteobacteria, Bacteroidetes and Actinobacteria detected in severe caries and caries-free children, with positions of Clostridia and Bacillus as phylogenetic references. The tree includes 28 taxa of Proteobacteria, 15 taxa of Bacteroidetes, 1 Treponema species, 5 Actinobacteria and 2 Leptotrichia taxa. b Phylogenetic tree of 49 taxa in the Clostridia branch of Firmicutes, which includes the Gram-negative Veillonella and Selenomonas genera, detected in severe ECC and caries-free children. This group comprises several named and unnamed taxa, including the Centipeda species. c Phylogenetic tree of 38 taxa in the Bacillus branch of Firmicutes detected in severe caries and caries-free children. The tree includes 29 Streptococcus species, comprising 10 unnamed species, 2 Gemella, 2 Granulicatella and 4 Lactobacillus species.

Fig. 1

Phylogenetic trees of species and phylotypes identified from clones compared with the HOMD. The trees were created from full 16S rRNA reference sequences although it is recognized that use of 500-bp data from study clones could have created a different, but less accurate, treeing topography. Taxa in trees include identifications at >0.1 mean percent positive taxa by subject. A total of 139 taxa were identified. Numbers of children with taxa detected are in the table to the right of each tree. Species or phylotypes that differed in detection frequencies are noted at ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, by χ² analysis, or Fishers exact test. a Phylogenetic tree of 51 taxa in Proteobacteria, Bacteroidetes and Actinobacteria detected in severe caries and caries-free children, with positions of Clostridia and Bacillus as phylogenetic references. The tree includes 28 taxa of Proteobacteria, 15 taxa of Bacteroidetes, 1 Treponema species, 5 Actinobacteria and 2 Leptotrichia taxa. b Phylogenetic tree of 49 taxa in the Clostridia branch of Firmicutes, which includes the Gram-negative Veillonella and Selenomonas genera, detected in severe ECC and caries-free children. This group comprises several named and unnamed taxa, including the Centipeda species. c Phylogenetic tree of 38 taxa in the Bacillus branch of Firmicutes detected in severe caries and caries-free children. The tree includes 29 Streptococcus species, comprising 10 unnamed species, 2 Gemella, 2 Granulicatella and 4 Lactobacillus species.

Fig. 2

The most frequently detected species from severe caries and caries-free children. The scale represents the square root of data to enhance the visualization of taxa less frequently detected or in lower proportions. a Detection frequencies of taxa by child in severe caries and caries-free children. Taxa are ordered in decreasing order of detection in severe ECC children. Taxa that differed in detection frequencies are noted at ∗ p ≤ 0.05, ∗∗ p ≤ 0.01 by χ² analysis. b Mean proportions of taxa detected in severe caries and caries-free children. Taxa are given in decreasing order of mean clone levels in severe ECC children. Taxa that differed in detection frequencies are noted at ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001 by Mann Whitney U test. Error bars = standard error of the mean.

Fig. 3

Detection frequencies by child of S. mutans and Bifidobacteriaceae species assayed by species-specific PCR. Streptococcus mutans (p < 0.002) and Bifidobacteriaceae species (p < 0.0001) were strongly associated with severe early childhood caries.