Endodontic infections have been traditionally studied by culture methods, but recent reports showing that over 50% of the oral microbiota is still uncultivable (B. J. Paster et al., J. Bacteriol. 183:3770-3783, 2001) raise the possibility that many endodontic pathogens remain unknown. This study intended to investigate the prevalence of several uncultivated oral phylotypes, as well as newly named species in primary or persistent endodontic infections associated with chronic periradicular diseases. Samples were taken from the root canals of 21 untreated teeth and 22 root-filled teeth, all of them with radiographic evidence of periradicular bone destruction. Genomic DNA was isolated directly from each sample, and 16S rRNA gene-based nested or heminested PCR assays were used to determine the presence of 13 species or phylotypes of bacteria. Speciesspecific primers had already been validated in the literature or were developed by aligning closely related 16S rRNA gene sequences. Species specificity for each primer pair was confirmed by running PCRs against a panel of several oral bacteria and by sequencing DNA from representative positive samples. All species or phylotypes were detected in at least one case of primary infections. The most prevalent species or phylotypes found in primary infections were Dialister invisus (81%), Synergistes oral clone BA121 (33%), and Olsenella uli (33%). Of the target bacteria, only these three species were detected in persistent infections. Detection of uncultivated phylotypes and newly named species in infected root canals suggests that there are previously unrecognized bacteria that may play a role in the pathogenesis of periradicular diseases.

Samples from infected root canals of 43 teeth with chronic apical periodontitis were analyzed for the presence and relative levels of 83 oral bacterial species and/or phylotypes using a reverse-capture checkerboard hybridization assay. Associations between the most frequently detected taxa were also recorded. The most prevalent taxa were Olsenella uli (74%), Eikenella corrodens (63%), Porphyromonas endodontalis (56%), Peptostreptococcus anaerobius (54%), and Bacteroidetes oral clone X083 (51%). When prevalence was considered only for bacteria present at levels >10 5 , Bacteroidetes clone X083 was the most frequently isolated bacterium (37%), followed by Parvimonas micra (28%), E. corrodens (23%), and Tannerella forsythia (19%). The number of target taxa per canal was directly proportional to the size of the apical periodontitis lesion, with lesions >10 mm in diameter harboring a mean number of approximately 20 taxa. Several positive associations for the most prevalent taxa were disclosed for the first time and may have important ecological and pathogenic implications. In addition to strengthening the association of several cultivable named species with chronic apical periodontitis, the present findings using a large-scale analysis allowed the inclusion of some newly named species and as-yet-uncultivated phylotypes in the set of candidate pathogens associated with this disease.Chronic apical periodontitis is arguably one of the most common forms of biofilm-induced diseases that affect humans (9). The disease develops after dental pulp necrosis and infection as a result of caries, trauma, or iatrogenic clinical procedures. The environmental conditions in the necrotic root canal are conducive to the establishment of a microbiota conspicuously dominated by anaerobic bacteria. Bacterial profiles of the endodontic microbiota vary from individual to individual (37); i.e., each individual harbors a unique microbiota in terms of species richness and abundance. This indicates that apical periodontitis has a heterogeneous etiology, where no single species can be considered to be the main endodontic pathogen, and multiple bacterial combinations can play a role in disease causation.Early studies of the microbiota associated with apical periodontitis were conducted using broad-range culture methods. Those studies were followed by a generation of studies employing molecular detection methods such as species-specific PCR and the original checkerboard DNA-DNA hybridization assay to target cultivable bacteria previously isolated from infected canals or from other oral diseased sites. These methods allowed the inclusion of some culture-difficult species in the set of candidate endodontic pathogens. The adoption of 16S rRNA gene clone library analysis allowed an even more comprehensive broad-range investigation of bacterial communities in endodontic infections. By this technique, not only cultivable species but also as-yet-uncultivated and uncharacterized bacteria can be identified. Studies using the 16S rRNA gene clone library ana...

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