Quantitative analysis of diverse Lactobacillus species present in advanced dental caries

Abstract

Our previous analysis of 65 advanced dental caries lesions by traditional culture techniques indicated that lactobacilli were numerous in the advancing front of the progressive lesion. Production of organic acids by lactobacilli is considered to be important in causing decalcification of the dentinal matrix. The present study was undertaken to define more precisely the diversity of lactobacilli found in this environment and to quantify the major species and phylotypes relative to total load of lactobacilli by real-time PCR. Pooled DNA was amplified by PCR with Lactobacillus genus-specific primers for subsequent cloning, sequencing, and phylogenetic analysis. Based on 16S ribosomal DNA sequence comparisons, 18 different phylotypes of lactobacilli were detected, including strong representation of both novel and gastrointestinal phylotypes. Specific PCR primers were designed for nine prominent species, including Lactobacillus gasseri, L. ultunensis, L. salivarius, L. rhamnosus, L. casei, L. crispatus, L. delbrueckii, L. fermentum, and L. gallinarum. More than three different species were identified as being present in most of the dentine samples, confirming the widespread distribution and numerical importance of various Lactobacillus spp. in carious dentine. Quantification by real-time PCR revealed various proportions of the nine species colonizing carious dentine, with higher mean loads of L. gasseri and L. ultunensis than of the other prevalent species. The findings provide a basis for further characterization of the pathogenicity of Lactobacillus spp. in the context of extension of the carious lesion.

FIG. 1.

Neighbor-joining tree of the Lactobacillus species and phylotypes identified from 58 carious dentine samples, based on 100 sequences of the Lactobacillus-specific 16S rRNA amplicon (≈400 bp) and closely related amplicon regions from Lactobacillus 16S rRNA gene sequences in GenBank (accession numbers in parentheses). Sequences were aligned with clustalw, and the distance matrix was calculated with the Jukes-Cantor algorithm. Shaded boxes adjacent to the representative sequence correspond to the number of clones of the different Lactobacillus spp. or phylotypes identified. Lactobacillus spp. or phylotypes quantified by real-time PCR in the carious dentine samples are marked by the species-specific primer pairs used. Novel phylotypes (*) were defined to differ by >2% in sequence comparisons of their 16S rDNA amplicons from their closest related species. Bootstrap values (>50) near the nodes are represented as percentages of 100 replicates. The phylogenetic tree was rooted with the appropriate amplicon region of the 16S rRNA gene sequence of E. coli K-12 strain MG1655. The scale bar represents the genetic distance.

FIG. 2.

Graphic representation of the DNA loads of the nine predominant Lactobacillus species or phylotypes in carious dentine determined by real-time PCR. Forty samples with the highest total Lactobacillus loads are shown. The difference in the total Lactobacillus load with the cumulative total of the nine species in each sample (referred to as unknown) was attributed to the presence of other, less-prevalent Lactobacillus spp. that were not quantified by real-time PCR. Patient samples are indicated on the x axis.