Bacterial 16S sequence analysis of severe caries in young permanent teeth

Abstract

Previous studies have confirmed the association of the acid producers Streptococcus mutans and Lactobacillus spp. with childhood caries, but they also suggested these microorganisms are not sufficient to explain all cases of caries. In addition, health-associated bacterial community profiles are not well understood, including the importance of base production and acid catabolism in pH homeostasis. The bacterial community composition in health and in severe caries of the young permanent dentition was compared using Sanger sequencing of the ribosomal 16S rRNA genes. Lactobacillus species were dominant in severe caries, and levels rose significantly as caries progressed from initial to deep lesions. S. mutans was often observed at high levels in the early stages of caries but also in some healthy subjects and was not statistically significantly associated with caries progression in the overall model. Lactobacillus or S. mutans was found either at low levels or not present in several samples. Other potential acid producers observed at high levels in these subjects included strains of Selenomonas, Neisseria, and Streptococcus mitis. Propionibacterium FMA5 was significantly associated with caries progression but was not found at high levels. An overall loss of community diversity occurred as caries progressed, and species that significantly decreased included the Streptococcus mitis-S. pneumoniae-S. infantis group, Corynebacterium matruchotii, Streptococcus gordonii, Streptococcus cristatus, Capnocytophaga gingivalis, Eubacterium IR009, Campylobacter rectus, and Lachnospiraceae sp. C1. The relationship of acid-base metabolism to 16S rRNA gene-based species assignments appears to be complex, and metagenomic approaches that would allow functional profiling of entire genomes will be helpful in elucidating the microbial pathogenesis of caries.

FIG. 1.

Distribution of species by phylum for all samples. Spirochaetes, Deinococcus-Thermus, and Fusobacteria were detected but are not shown because each represented less than 1% of total clones.

FIG. 2.

Mean levels of the most common species as caries severity increased. From left to right within a species, bars indicate levels for healthy control, intact enamel, white spot, cavitated, and dentin samples. Species found at greater than 0.75% of total clones are shown, with 95% confidence intervals. The upper limits for the 95% confidence interval bars for the cavitated and dentin samples of the total Lactobacillus group, which are not shown, were 42.2% and 58.2%, respectively.

FIG. 3.

Changes in distributions of the most common genus groups as caries severity increased. S. mutans was considered separately because of its importance in caries. Estimates of the percent change per one level of severity were determined by PROC MIXED analysis. Genus groups found present at greater than 1% of total clones are shown, with 95% confidence intervals. P values were adjusted using a false discovery rate correction. *, corrected P < 0.05; **, corrected P < 0.01.

FIG. 4.

Decreasing species diversity as caries severity increased. The decrease in severity was modeled using PROC MIXED analysis and is shown as a dotted line (estimate, −0.13; P < 0.00002). Mean Shannon diversity indices at the species level are shown with 95% confidence intervals for each level of caries severity. Samples that were statistically different according to post hoc t tests and paired t tests are connected by light gray lines.

FIG. 5.

Levels of Streptococcus spp., Propionibacterium FMA5, Streptococcus mutans, and Lactobacillus spp. at each level of caries experience. The Streptococcus group includes levels of health-associated streptococci S. mitis-S. pneumoniae-S. infantis, S. oralis, S. cristatus, S. gordonii, and S. sanguinis. At each level in the caries group, subjects are presented in the same order, according to level of Lactobacillus in cavitated samples.

FIG. 6.

Hierarchical cluster analysis of Lactobacillus species present in all caries samples.