Assessment of intraradicular bacterial composition by T-RFLP analysis

Objectives: The aim of this study was to assess the bacterial community structures associated with endodontic infections by use of the Terminal Restriction Length Polymorphism (T-RFLP) technique, and to investigate the correlation of the observed community profiles with particular clinical features. Methods: Intraradicular samples were aseptically collected from 25 teeth harbouring endodontic infections. Specimens were classified in 3 study groups, according to the following symptomatic features: asymptomatic (n= 7), tender (n= 5) and symptomatic (n= 13). The genomic DNA was extracted and amplified with universal 16S ribosomal DNA primers targeting most bacterial species. The forward primer was fluorescently labelled to allow quantitative detection of terminal fragments. The PCR products were digested with two tetrameric endonucleases (HhaI and MspI) and submitted to capillary electrophoresis in an automated DNA sequencer. Results: The total number of terminal restriction fragments (T-RFs) detected per subject varied from 8 to 34 (HhaI), and from 8 to 32 (MspI), with an overall of 123 (HhaI) and 122 (MspI) unique T-RFs (means 20.8 and 20.0, respectively). Approximately 50% of the T-RFs were detected in no more than 2 subjects, indicating a high variability in the bacterial assemblages. In silico prediction of T-RFs using a 16S rDNA database from oral bacteria revealed a high dominance of the species Tannerella forsythia, Selenomonas spp. oral clones JI021 and EZ011, and Veillonella spp. No statistically significant differences in T-RF richness among the study groups could be observed. Likewise, hierarchical and multivariate analyses did not display any observable clusters of T-RFLP profiles according to the predefined clinical parameters. Conclusion: Taken together, the results obtained from this study support the concept that the intraradicular bacterial community structures, in broad, do not play a relevant role in the establishment of symptomatic features of endodontic origin. Financial support: CAPES (BEX0567/06-0) and FAPESP (04/01674-6).