Molecular characterization of subject-specific oral microflora during initial colonization of enamel

Abstract

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.

FIG. 1.

Confocal micrographs (238 by 238 μm) of 8-h plaque development on enamel chips in subject 1 (A) and subject 2 (B). Cells were simultaneously labeled with the kingdom-level bacterial probe EUB338 (red) and the genus-level Streptococcus probe STR405 (green). Panels show the overlay of the two images. Notice the difference in biomass between the two samples but the similar proportion of Streptococcus spp. (yellow, colocalization of the two probes). Bar, 40 μm.

FIG. 2.

Collector's curves of observed and estimated (ACE and Chao1) phylotype richness as a function of the number of clones. These curves depict the change in richness values as the number of clones in the library increased and are used to assess the sufficiency of the sampling effort for each specific library. A sufficient number of clones was sequenced in all libraries except in the 8-h library from subject 1, in which the gap between the observed and estimated phylotype curves had not closed when sampling stopped.

FIG. 3.

∫-LIBSHUFF comparisons of 16S rRNA gene sequence libraries from 4-h and 8-h biofilms in subjects 1, 2, and 3. Significant P values after correction for an experiment-wide type 1 error rate of 5% appear in boldface type and correspond to those values of <0.0017. Libraries are distinct if both X versus Y and Y versus X are statistically significant. If X versus Y is significant but Y versus X is not, then Y is a subset of X. If X versus Y is not significant but Y versus X is significant, then X is a subset of Y. Comparisons of the pooled combination of 4-h and 8-h samples of each subject with the pooled combination of each other subject are not presented; they resulted in significant values (P = 0.0000) for all comparisons (subject 1 versus subject 2, subject 2 versus subject 3, and subject 1 versus subject 3).

FIG. 4.

Maximum-likelihood phylogenetic trees of the 97 phylotypes and related database sequences of 16S rRNA genes (shown in boldface type with associated EMBL accession numbers). Novel phylotypes are underlined. Symbol colors and shapes, associated with each phylotype, represent the subject (red represents subject 1, green represents subject 2, and blue represents subject 3) and the time period at which the sequence was isolated (left half circle, isolated at 4 h; right half circle, isolated at 8 h; full circle, isolated at 4 h and 8 h). (A) The 69 phylotypes that share close identity with published gram-positive species in the EMBL database library. (B) The 29 phylotypes that share close identity with published gram-negative species in the EMBL database library. The scale bar represents one substitution for every 10 nucleotides. The species used as the outgroup was Thermus thermophilus (EMBL accession number AJ251938).

FIG. 4.

Maximum-likelihood phylogenetic trees of the 97 phylotypes and related database sequences of 16S rRNA genes (shown in boldface type with associated EMBL accession numbers). Novel phylotypes are underlined. Symbol colors and shapes, associated with each phylotype, represent the subject (red represents subject 1, green represents subject 2, and blue represents subject 3) and the time period at which the sequence was isolated (left half circle, isolated at 4 h; right half circle, isolated at 8 h; full circle, isolated at 4 h and 8 h). (A) The 69 phylotypes that share close identity with published gram-positive species in the EMBL database library. (B) The 29 phylotypes that share close identity with published gram-negative species in the EMBL database library. The scale bar represents one substitution for every 10 nucleotides. The species used as the outgroup was Thermus thermophilus (EMBL accession number AJ251938).

FIG. 5.

Confocal micrographs of typical multigeneric clusters of cells found on enamel chips at 4 h (A and B) and 8 h (C and D) of plaque development. Cells were simultaneously labeled with all-bacterium-specific EUB338 probe (red) and either the Streptococcus-specific STR405 probe (A and B) (green) or the Prevotella-specific PRV392 probe (C and D) (green). (A and B) Unidentified bacterial cells (EUB338 reactive; red) juxtaposed with Streptococcus cells (EUB338 and STR405 reactive; red + green = yellow). (C and D) Unidentified bacterial cells (EUB338 reactive; red) in association with Prevotella cells (EUB338 and PRV392 reactive; red +green = yellow). Scale bar for all images, 5 μm.