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. 2002 Mar;40(3):1001-9.
doi: 10.1128/JCM.40.3.1001-1009.2002.

Molecular analysis of bacterial species associated with childhood caries

Mitzi R Becker  1 , Bruce J Paster, Eugene J Leys, Melvin L Moeschberger, Sarah G Kenyon, Jamie L Galvin, Susan K Boches, Floyd E Dewhirst, Ann L Griffen
Affiliations

Molecular analysis of bacterial species associated with childhood caries

Mitzi R Becker et al. J Clin Microbiol. 2002 Mar.

Abstract

Although substantial epidemiologic evidence links Streptococcus mutans to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species: S. sanguinis was associated with health and, in order of decreasing cell numbers, Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum were associated with caries. These data suggest that A. gerencseriae and other Actinomyces species may play an important role in caries initiation and that a novel Bifidobacterium may be a major pathogen in deep caries. Further investigation could lead to the identification of _targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.

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Figures

FIG. 1.
FIG. 1.
Reverse capture checkerboard hybridization blot for 11 subjects with caries and 15 healthy control subjects (without caries). For the caries samples, lanes a represent intact enamel, lanes b represent white spot lesions, lanes c represent cavitated lesions, and lanes d represent carious dentin. Species-specific probes were applied in horizontal rows, and samples or standards were then hybridized in vertical lanes. Probe specificities are listed on the right. The last three columns contained DNA isolated from laboratory strains at a concentration of 108 cells per ml. The first lane of standards contained S. salivarius, S. oralis, S. sobrinus, S. gordonii, S. parasanguinis, A. gerencseriae, A. odontolyticus, S. mucilaginosus, and Veillonella parvula. The second lane contained S. sanguinis, S. mitis, S. mutans, S. intermedius, A. israelii, A. naeslundii, and L. fermentum. The third lane contained S. pneumoniae, S. anginosus, S. constellatus, S. mitis biovar II, A. naeslundii serotype III, R. dentocariosa, and B. dentium.
FIG. 2.
FIG. 2.
Mean levels of 23 species or species groups as determined by a reverse capture checkerboard assay for 30 healthy subjects and 30 subjects with caries. Species are ordered by decreasing quantity detected, and scales have been adjusted for each species. Levels in healthy subjects and at intact enamel sites in subjects with caries are shown as white bars. They were compared by the Wilcoxon rank sum test; P values are shown for significant differences above the first white bar. Levels at caries sites were compared to those found at intact enamel sites in the same subject by the Wilcoxon signed rank test. Levels that were significantly different are shown as black bars with P values, and those that were not are shown as gray bars. For all tests, the Bonferroni correction for multiple comparisons was used, adjusting alpha levels to 0.002. S. H6, Streptococcus group H6.
FIG. 3.
FIG. 3.
Phylogenetic tree showing the relationships of species and phylotypes detected by clonal analysis. Novel phylotypes are designated “Caries clone.” S. mitis and S. oralis as well as V. parvula and V. dispar could not be differentiated based on sequence comparisons and are grouped together. GenBank accession numbers are listed. The marker bar represents a 5% difference in nucleotide sequences.
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