Comparison of conventional and molecular identification of endodontic isolates

Although a number of different bacteria have been associated with primary and secondary endodontic lesions, the actual causative agents are not known with certainty. Endodontic lesions are known to contain uncultured micro-organisms which can be studied by molecular methods. In addition, bacteria which are difficult to identify by traditional diagnostic methods are often isolated. Objective: The aim of this study was to identify the bacteria associated with root canal infection by both conventional microbiological identification and 16SrRNA sequencing. Methods: Samples were taken via paper points and cultured on a range of appropriate media and atmospheric conditions facilitating the isolation strict anaerobes, facultative and microaerophilic organisms. The bacteria present were identified by two methods; firstly, standard diagnostic methods based on staining and biochemical reactions; secondly, sequencing of the 16S rRNA gene. Sequences obtained were compared to established 16S  rRNA gene databases for identification. Results: Of the samples collected, 26% were obtained from primary root canal treatment and the remaining 74% from previously root canal filled teeth. In total, 45 bacterial isolates were obtained and the number of isolates per sample ranged from 1 to 6, with an average of 3. E.faecalis was detected in the secondary root canal treatment cases only. The 2 identification methods concurred for 42 of the 45 isolates. In the remaining 3 cases, the sequencing data indicated that these may represented previously uncharacterised bacteria. It will interesting to study these possibly novel bacteria by both phenotypic methods and further sequence analysis. Conclusion: Both the traditional biochemical and molecular approaches to the identification of the bacteria present in infected root canals proved to be valid. Currently, the molecular approach is more expensive but is less dependent on specialist microbiology skills and can therefore be applied in a wider research context.