Bacteria of dental caries in primary and permanent teeth in children and young adults

Abstract

Although Streptococcus mutans has been implicated as a major etiological agent of dental caries, our cross-sectional preliminary study indicated that 10% of subjects with rampant caries in permanent teeth do not have detectable levels of S. mutans. Our aims were to use molecular methods to detect all bacterial species associated with caries in primary and permanent teeth and to determine the bacterial profiles associated with different disease states. Plaque was collected from 39 healthy controls and from intact enamel and white-spot lesions, dentin lesions, and deep-dentin lesions in each of 51 subjects with severe caries. 16S rRNA genes were PCR amplified, cloned, and sequenced to determine species identities. In a reverse-capture checkerboard assay, 243 samples were analyzed for 110 prevalent bacterial species. A sequencing analysis of 1,285 16S rRNA clones detected 197 bacterial species/phylotypes, of which 50% were not cultivable. Twenty-two new phylotypes were identified. PROC MIXED tests revealed health- and disease-associated species. In subjects with S. mutans, additional species, e.g., species of the genera Atopobium, Propionibacterium, and Lactobacillus, were present at significantly higher levels than those of S. mutans. Lactobacillus spp., Bifidobacterium dentium, and low-pH non-S. mutans streptococci were predominant in subjects with no detectable S. mutans. Actinomyces spp. and non-S. mutans streptococci were predominant in white-spot lesions, while known acid producers were found at their highest levels later in disease. Bacterial profiles change with disease states and differ between primary and secondary dentitions. Bacterial species other than S. mutans, e.g., species of the genera Veillonella, Lactobacillus, Bifidobacterium, and Propionibacterium, low-pH non-S. mutans streptococci, Actinomyces spp., and Atopobium spp., likely play important roles in caries progression.

FIG. 1.

Phylogenetic relationships of species detected from dental caries in permanent teeth and from healthy controls. Subjects with (w Sm) and without (w/o Sm) detectable levels of S. mutans (Sm) were selected for clonal analysis. The distribution and levels of bacterial species/phylotypes among the five subjects and two healthy controls are shown by the columns of boxes to the right of the tree as either not detected (clear box), <15% of the total number of clones assayed (shaded box), or ≥15% of the total number of clones assayed (darkened box). Fifteen percent was chosen as the arbitrary level. GenBank accession numbers are provided. The marker bar represents a 5% difference in nucleotide sequences.

FIG. 2.

Mean levels of health-associated species or species groups in primary teeth determined by a reverse-capture checkerboard assay for 15 subjects with caries (green bars) and 14 healthy controls (white bars). *, P ≤ 0.05 for pairwise comparisons of the healthy group to other groups by PROC MIXED. P indicates the overall P value comparing all groups and sites by PROC MIXED. Strep., Streptococcus; Porphyrom., Porphyromonas; Eubact., Eubacterium; Capnocyto., Capnocytophaga; Fusobact., Fusobacterium; cl., clone.

FIG. 3.

Mean levels of health-associated species or species groups in permanent teeth determined by a reverse-capture checkerboard assay for 36 subjects with caries (green bars) and 25 healthy controls (white bars). *, P ≤ 0.05 for pairwise comparisons of the healthy group to other groups by PROC MIXED. P indicates the overall P value comparing all groups and sites by PROC MIXED. Strep., Streptococcus; Eubact., Eubacterium; Eubact. sabur., Eubacterium saburreum; Capnocyto., Capnocytophaga; Fusobact. nuc. ss poly., Fusobacterium nucleatum subsp. polymorphum; Actino, Actinomyces; cl., clone.

FIG. 4.

Mean levels of caries-associated species or species groups in primary teeth determined by a reverse-capture checkerboard assay for 15 subjects with caries (red bars) and 14 healthy controls (white bars). *, P ≤ 0.05 for pairwise comparisons of the healthy group to other groups by PROC MIXED. P indicates the overall P value comparing all groups and sites by PROC MIXED. Strep., Streptococcus; Actino, Actinomyces; cl., clone.

FIG. 5.

Mean levels of caries-associated species or species groups in permanent teeth determined by a reverse-capture checkerboard assay for 36 subjects with caries (red bars) and 25 healthy controls (white bars). *, P ≤ 0.05 for pairwise comparisons of the healthy group to other groups by PROC MIXED. P indicates the overall P value comparing all groups and sites by PROC MIXED. Strep., Streptococcus; genomo., genomospecies.