Analysis of Active Bacteria Persisting after Chemomechanical Procedures: An RNA- and DNA-based Molecular Study

IntroductionBecause active cells present higher abundance of ribosomal RNA (rRNA) than rDNA (rRNA genes), data obtained with rDNA-based quantitative polymerase chain reaction (qPCR) and rRNA-based qPCR (RT-qPCR) were correlated to search for active bacteria after chemomechanical procedures (CMP). In addition, the ability of both assays to detect bacteria in endodontic samples was evaluated.MethodsSamples were taken from 40 teeth with primary endodontic infections before (S1) and after CMP (S2). DNA and cDNA (synthetized from RNA) were used as templates for qPCR using universal primers for bacteria and species-specific primers for Bacteroidaceae sp. HOT-272, Cutibacterium acnes, Selenomonas spp., and Enterococcus faecalis.ResultsAfter CMP, there was a drastic reduction in the number of total bacteria, Selenomonas spp., and E. faecalis, whereas no significant difference was observed for the levels of Bacteroidaceae sp. HOT-272 and C. acnes. The concentration of rRNA copies in S2 samples was significantly higher than the corresponding levels of rDNA for assays targeting total bacteria, Bacteroidaceae sp. HOT-272, and C. acnes (P < .05), indicating persistence of active bacteria. The rDNA-based qPCR presented low sensitivity and high specificity when compared with RT-qPCR. For most assays, samples positive for rDNA were also positive for rRNA (positive predictive value = 100%).ConclusionsCMP was effective in reducing levels but not the metabolic activity of total bacteria. Bacteroidaceae sp. HOT-272 and C. acnes were active members of the persistent community. Although less sensitive than RT-qPCR, most rDNA-based qPCR assays had a low risk of providing false-positive results in postinstrumentation samples.     

In Vivo Quantitative Evaluation of Live and Dead Bacteria in Root Canal Infection by Using Propidium Monoazide with Real-Time PCR

IntroductionFor selective detection of viable bacteria with molecular methods, propidium monoazide (PMA) treatment has been successfully applied to a wide range of bacteria. The purpose of this study was to compare the quantity of live cells with the total amounts of both live and dead cells before and after chemomechanical preparation by using PMA in combination with real-time polymerase chain reaction (qPCR).MethodsTwenty-one teeth with pulp necrosis and a periapical lesion were included. Bacterial sampling of the root canals was performed before (S1) and after (S2) chemomechanical root canal treatment. Each sample was separated into 2 different tubes. PMA was added to one of the tubes, and the other was left untreated. Then, DNA extraction and qPCR were performed. To evaluate the validity of the PMA treatment, the defined mixtures containing different ratios of live and dead cell suspensions of Enterococcus faecalis were either subjected to PMA treatment or not subjected to PMA treatment, followed by qPCR quantification.ResultsA paired t test showed a highly significant difference in the mean threshold cycle values between S1 with and without PMA (P = .0002), and this difference (0.89) was similar to that (0.96) obtained from the samples consisting of 80% live cell suspension and 20% dead cell suspension of E. faecalis. The threshold cycle values between the S2 samples with and without PMA were also significantly different (P = .0134), and this difference (0.37) was similar to that obtained from the 100% live cell suspension of E. faecalis (0.42).ConclusionsPMA in conjunction with qPCR appeared to be useful in analyzing the primary infections of root canals because there were significant amounts of dead bacteria in the root canals. Although the use of PMA treatment in post-preparation samples significantly reduced the detection of dead bacteria, this difference was still small, so further studies should be carried out to confirm the necessity of PMA treatment.     

The Apical Root Canal System of Teeth with Posttreatment Apical Periodontitis: Correlating Microbiologic,Tomographic, and Histopathologic Findings

IntroductionThis study evaluated the microbiological conditions of the apical root canal system of teeth with posttreatment apical periodontitis and correlated them with observations from cone-beam computed tomographic (CBCT) imaging, micro–computed tomographic (micro-CT) imaging, and histopathology.MethodsRoot apices were obtained from 36 root canal–treated teeth subjected to periradicular surgery. CBCT examination was available before surgery. The apical root specimens were scanned in a micro-CT device and then cryopulverized. The powder was subjected to DNA extraction for real-time polymerase chain reaction quantification of total bacteria, Streptococcus species, members of the phylum Actinobacteria, and Enterococcus faecalis. Microbiological findings were evaluated for associations with CBCT, micro-CT, and histopathologic data. An association between lesion size and the proportion of unfilled apical canal system volume was also assessed.ResultsAll cryopulverized specimens were positive for total bacteria. Actinobacteria and streptococci occurred in 35 and 33 specimens, respectively, and were usually dominant in the community. Actinobacteria counts were 2.23 times higher in granulomas than in cysts. Streptococci were significantly more present in small lesion cases. E. faecalis was detected in only 7 samples, always as a dominant community member. The association of total bacteria, streptococci, and Actinobacteria counts with the unfilled canal volume was significant in the univariate analyses but not confirmed in the adjusted analyses. Large lesions were significantly associated with a higher volume of unfilled apical canals.ConclusionsBacterial infection occurred in all root apices, with high prevalence and dominance of Actinobacteria and streptococci. The volume of the unfilled apical canal system was significantly associated with the lesion size and possibly with bacterial counts. Findings illustrate the need to thoroughly disinfect and fill the apical root canal of infected teeth during endodontic therapy.