Supplementary sampling of obturation materials enhances microbial analysis of endodontic treatment failures: a proof of principle study

Root canal treatment failures often correlate with persistent biomaterial-associated endodontic infections. The aim of the present study was to assess the impact of endodontic obturation material sampling from root canals with posttreatment apical periodontitis on improving standard study protocols. Samples from previously filled root canals and their corresponding endodontic filling materials were obtained from five root-filled teeth with posttreatment periradicular lesions. After cultivation, the isolated microorganisms were quantified and biochemically identified. Moreover, clone libraries were constructed after the amplification of bacterial 16S ribosomal DNA (rDNA) from the same samples. DNA from selected clones was sequenced to identify microbial species. Transmission electron microscopy (TEM) aided visualization of the detected bacteria. Overall, 22 taxa of the phyla Firmicutes, Actinobacteria, and Bacteroidetes were detected in both obturation and root canal samples by culture-dependent and culture-independent methods. Root canal fillings sheltered 17 species (3.30-7.50 x 10(3) CFU/ml). Of these, nine were detected solely in the retrieved obturation materials. The reinfected root canals harbored 13 taxa (3.48-7.36 x 10(3) CFU/ml). Obligate and facultative anaerobic bacteria prevailed. The number of different species ranged from 1 to 5 within a single sample. Fungi were not detected. Bacteria can colonize both root canals and endodontic fillings in vivo. Integrating the sampling of obturation materials with standard root canal sample collection offers a clearer insight into the actual microbial flora of reinfected root canals and improves the study protocols of secondary/persistent endodontic infections.