The benefit of culture-independent methods to detect bacteria and fungi in re-infected root filled teeth: a pilot study

Abstract

Aim: To identify dominant microorganisms in root filled teeth with apical periodontitis by Pan-PCRs in comparison with a culture-dependent approach, focusing on fungal species profiling. Methodology: The root filling material (gutta-percha) removed from 42 teeth with periapical radiolucencies undergoing root canal retreatments was analysed by molecular genetics techniques. Real-Time Pan-PCRs were conducted for the diagnosis of predominant bacteria (targeting 16S rDNA) and fungi (targeting ITS1-2 region). Identification of microorganisms was performed by Sanger sequencing of the PCR products and BLAST analysis. Additionally, subgingival plaque samples were collected and cultured to review the composition of the microbial flora. The McNemar test and the repeated measures anova were used for statistical analyses (significance level was set at P < 0.05). Results: Overall, 42/42 plaque samples had bacterial growth, whereas 32/42 gutta-percha samples had bacterial growth with a dominance of Streptococcus spp. (12/42) and Enterococcus faecalis (9/42). The mean number of bacterial taxa per gutta-percha sample was 1.6 cultivatable taxa, significantly lower than in the plaque sample that had six taxa/sample (P < 0.001). Fungus-specific cultures were negative for gutta-percha samples, and only one plaque sample had growth of a fungus. In total, 36/42 plaque samples were positive in bacterial Pan-PCRs. In bacterial Pan-PCRs of 31/42 gutta-percha samples, dominant microorganisms were identified including Streptococcus spp. (5/42) and E. faecalis (4/42). Moreover, in 7/42 gutta-percha samples, DNA of bacteria which are difficult-to-cultivate in microbiology routine culture (Acinetobacter, Pyramidobacter, Bacteroidetes, Synergistes, Atopobium and Pseudoramibacter) was found. DNA of Candida spp. was detected in 5/42 root canals by fungal Pan-PCR (1/5) and genus-specific Candida-PCR (5/5). Conclusions: Pan-PCR assays remain appropriate as a broad-range approach for the detection of a dominant pathogen in gutta-percha samples which have less diverse microbial composition. The molecular genetic Pan-PCR approach has the advantage of detecting microorganisms that are as-yet-uncultivable or difficult-to-cultivate and should be therefore complement conventional microbiological diagnostics.