A protocol for developing reporting guidelines for laboratory studies in Endodontology.

Laboratory-based research studies are the most common form of research endeavour and make up the majority of manuscripts that are submitted for publication in the field of Endodontology. The scientific information derived from laboratory studies can be used to design a wide range of subsequent studies and clinical trials and may have translational potential to benefit clinical practice. Unfortunately, the majority of laboratory-based articles submitted for publication fail the peer-review step, because unacceptable flaws or substantial limitations are identified. Even when apparently well-conducted laboratory-based articles are peer-reviewed, they can often require substantial corrections prior to the publication. It is apparent that some authors and reviewers may lack the training and experience to have developed a systematic approach to evaluate the quality of laboratory studies. Occasionally, even accepted manuscripts contain limitations that may compromise interpretation of data. To help authors avoid manuscript rejection and correction pitfalls, and to aid editors/reviewers to evaluate manuscripts systematically, the purpose of this project is to establish and publish quality guidelines for authors to report laboratory studies in the field of Endodontology so that the highest standards are achieved. The new guidelines will be named-'Preferred Reporting Items for Laboratory studies in Endodontology' (PRILE). A steering committee was assembled by the project leads to develop the guidelines through a five-phase consensus process. The committee will identify new items as well as review and adapt items from existing guidelines. The items forming the draft guidelines will be reviewed and refined by a PRILE Delphi Group (PDG). The items will be evaluated by the PDG on a nine-point Likert scale for relevance and inclusion. The agreed items will then be discussed by a PRILE face-to-face consensus meeting group (PFCMG) formed by 20 individuals to further refine the guidelines. This will be subject to final approval by the steering committee. The approved PRILE guidelines will be disseminated through publication in relevant journals, presented at congresses/meetings, and be freely available on a dedicated website. Feedback and comments will be solicited from researchers, editors and peer reviewers, who are invited to contact the steering committee with comments to help them update the guidelines periodically.

Influence of solvent and a supplementary step with a finishing instrument on filling material removal from canals connected by an isthmus.

AIM: To evaluate the effectiveness of a solvent (eucalyptol) in improving filling material removal from canals connected by isthmuses, and the additional cleaning effect of a finishing instrument. METHODOLOGY: The mesial canals from 32 mandibular molars (Vertucci's type II morphology) were instrumented and filled with the single-cone technique using Reciproc R25 gutta-percha points (VDW, Munich, Germany) combined with Sealer 26 (Dentsply, Petrópolis, RJ, Brazil). Each root was then subjected to retreatment using the Mtwo instrument system (VDW), with or without a solvent (n = 16 per group). The volume of filling material in the canals was assessed by micro-computed tomographic (micro-CT) scans taken before and after retreatment. Canals with remnants of filling material received a supplementary procedure with the XP-endo Finisher R instrument (FKG Dentaire, La Chaux-de-Fonds, Switzerland), with or without eucalyptol, and another micro-CT scan was taken. All retreatment procedures were performed inside a cabinet under a controlled temperature (37 °C). Filling material removal was evaluated in the 5-mm apical canal system for the canal+isthmus space or the isthmus alone. Statistical analyses were performed to compare the removal of filling material with and without eucalyptol, and after a supplementary approach with XP-endo Finisher R. The level of significance was set at 5% for all statistical tests (P < 0.05). RESULTS: The amount of filling material removed from the canal+isthmus with Mtwo instruments was 83.2% when no solvent was used and 83.8% using the solvent (P > 0.05). When the isthmus area was evaluated separately, most specimens were associated with a reduction in the filling material, with no significant difference between the groups with or without using a solvent (P > 0.05). The supplementary step with XP-endo Finisher R significantly improved removal of filling material from both canal and isthmus area (P < 0.05), regardless of the use of a solvent (P > 0.05). CONCLUSION: The use of eucalyptol did not improve filling material removal from Vertucci's type II molar mesial canals and isthmuses. XP-endo Finisher R significantly enhanced removal of filling material from the canals and isthmuses.

Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis.

AIM: To evaluate the association between the presence of selected bacterial species/groups in the apical root canal and expression of mediators of soft and bone tissue destruction in apical periodontitis lesions. Relationships between bacteria and some other features of apical periodontitis were also investigated. METHODOLOGY: Seventeen freshly extracted teeth with pulp necrosis and apical periodontitis were included. The apical root segment was sectioned and cryopulverized; DNA was extracted and evaluated for the presence of 9 bacterial species/groups using real-time polymerase chain reaction. Lesions were processed for histopathological and immunohistochemical analyses, which targeted matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), receptor activator of NFκB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). Associations of the target bacteria with expression of these mediators, presence of symptoms, lesion size and histopathological diagnosis were evaluated. Data were analysed using the chi-square, Fisher's exact, Mann-Whitney and Pearson tests. P values lower than 0.05 were considered significant. RESULTS: All pulverized apical root samples were positive for bacteria. The most prevalent taxa were Actinobacteria (53%), Streptococcus species (35%), Fusobacterium species and Parvimonas micra (18%). The target mediators exhibited a high mean expression in the lesions (MMP-2: 82%; MMP-9: 73%; RANK: 78%; RANKL; 81%; OPG; 83%). Mean RANKL:OPG ratio was significantly higher in granulomas than cysts (P < 0.05, Mann-Whitney test). Actinobacteria were associated with granulomas, higher MMP-2 expression, lower OPG expression, and higher RANKL:OPG ratio (P < 0.05 for all, Fisher's exact test or Mann-Whitney test). No other significant associations were found. CONCLUSION: Actinobacteria may play an important role in the active phase of soft and bone tissue destruction in apical periodontitis.

Bacteremia after supragingival scaling and dental extraction: Culture and molecular analyses.

OBJECTIVE: To study the incidence and magnitude of bacteremia after dental extraction and supragingival scaling. SUBJECTS AND METHODS: Blood samples were taken before and 5 and 30 min after dental extraction and supragingival scaling from individuals at high (n = 44) or negligible risk (n = 51) for infective endocarditis. The former received prophylactic antibiotic therapy. Samples were subjected to aerobic and anaerobic culture and quantitative real-time polymerase chain reaction to determine the incidence of bacteremia and total bacterial levels. RESULTS: Patients who did not receive prophylactic antibiotic therapy had a higher incidence of positive blood cultures (30% 5 min after extraction) than patients who received prophylactic antibiotic therapy (0% 5 min after extraction; p < .01). Molecular analysis did not reveal significant differences in the incidence or magnitude of bacteremia between the two patient groups either 5 or 30 min after each of the procedures evaluated. Extraction was associated with higher incidence of bacteremia than supragingival scaling by blood culture (p = .03) and molecular analysis (p = .05). CONCLUSIONS: Molecular methods revealed that dental extraction and supragingival scaling were associated with similar incidence of bacteremia in groups receiving or not prophylactic antibiotic therapy. However, blood culture revealed that antibiotic therapy reduced viable cultivable bacteria in the bloodstream in the extraction group.

What happens to unprepared root canal walls: a correlative analysis using micro-computed tomography and histology/scanning electron microscopy.

AIM: To microscopically examine the cleanliness of root canal walls that remained unprepared as revealed by micro-CT. METHODOLOGY: The root canals of 10 freshly extracted mandibular premolars with necrotic pulps and apical periodontitis along with the mesiobuccal canals of 11 mandibular molars with vital pulps were prepared using Reciproc instruments R40 and R25, respectively, and 2.5% sodium hypochlorite irrigation. Specimens were scanned in micro-CT before and after preparation, and the unprepared areas were identified. The outer root surface corresponding to the untouched areas was marked on each root third to guide further analysis using histological (for teeth with vital pulps) and scanning electron microscopic (SEM; for necrotic teeth) examination. In the teeth with vital pulps, the root canal area occupied by tissue remnants was calculated. In SEM analysis of teeth with necrotic pulps, scores were attributed for the amount of debris on the untouched areas. RESULTS: The proportion of unprepared areas in the mesiobuccal molar canals was 18.1% and 9.6% over the full canal length and apical canal, respectively. In premolars, corresponding figures were 34.6% and 17.6%, respectively. Histological analysis of canals with vital pulps revealed tissue remnants over the untouched walls almost exclusively in the apical canal. SEM analysis of the canals with necrotic pulps revealed debris along the untouched walls in all root canal thirds. CONCLUSION: The areas that remain untouched by Reciproc instruments used with 2.5% NaOCl irrigation as revealed by micro-CT analysis were usually covered with debris, in the form of pulp tissue remnants, bacteria and dentine chips, especially in the apical root canal.

Micro-CT evaluation of the efficacy of hard-tissue removal from the root canal and isthmus area by positive and negative pressure irrigation systems.

AIM: To evaluate the removal of accumulated hard-tissue debris (AHTD) from the root canal system of mandibular molars by positive and negative pressure irrigation systems, using micro-CT imaging analysis. METHODOLOGY: Mandibular molars with a single canal in the distal root and 2 canals connected by an isthmus in the mesial root were matched based on similar morphological dimensions using micro-CT evaluation and assigned to 2 experimental groups (n = 20 mesial and 10 distal canals), according to the irrigation protocol: apical positive (conventional irrigation) or negative (EndoVac system) pressure. Changes in root canal volume and surface area as well as percentage of uninstrumented canal wall surface and accumulated hard-tissue debris (AHTD) after canal preparation were compared statistically using the independent sample t-test and Mann-Whitney U-test, with the significance level set at 5%. RESULTS: Volume, surface area and percentage of static voxels in either mesial or distal root canal systems were not significantly different between groups before or after root canal preparation (P > 0.05). After preparation, AHTD was not observed in the distal canal of both groups. However, in the mesial root canal system, the conventional irrigation group was associated with a significantly higher median percentage of AHTD (11.48%; IQR: 5.9-22.6; range: 1.86-41.98) than the EndoVac group (3.40%; IQR: 1.5-7.3; range: 0.82-12.84) (P < 0.05). CONCLUSIONS: Neither irrigation protocol succeeded in rendering the mesial canal system free of AHTD; however, apical negative pressure irrigation resulted in lower levels of AHTD than conventional irrigation.

Sealing ability of two root-end filling materials in a bacterial nutrient leakage model.

AIM: To compare in vitro the sealing ability of root-end fillings with mineral trioxide aggregate (MTA) and EndoSequence BioCeramic Root Repair Material-Fast Set (BC-RRM) Putty using a novel bacterial nutrient leakage model, which provides information on whether or not intracanal bacteria are receiving nutrients from serum via leakage channels. METHODOLOGY: Sixty single-rooted decoronated mandibular incisors with instrumented root canals were subjected to root-end resection and ultrasonic preparation. The root specimens were mounted in the experimental apparatus, and the root-end cavities filled with the test materials. The positive control group used warm Gutta-percha and no sealer. In the negative controls, the entire resected surface was covered with varnish. After sterilization in ethylene oxide, the root canal was inoculated with 1.5 × 10(5) washed cells of Enterococcus faecalis. The apparatus was filled with foetal bovine serum, leaving only the apical root immersed. After 30-day incubation, samples were taken from the canal, cultured and the colony-forming units (CFUs) counted. Statistical analysis was performed using the Mann-Whitney test for quantitative and the Fisher exact test for qualitative data. RESULTS: In the MTA group, 10 of 20 (50%) specimens still had detectable viable bacteria in the canals (mean, 8.97 × 10(3)  CFUs). In the BC-RRM Putty group, 5 of 18 (28%) specimens were positive for bacterial growth (mean, 2.88 × 10(4)  CFUs). There was no significant difference when comparing the quantitative or presence/absence data from the MTA and BC-RRM Putty groups. Positive and negative controls yielded the expected results. CONCLUSIONS: MTA and BC-RRM Putty had similar sealing ability. The experimental model was effective in determining whether or not residual intracanal bacteria could survive by receiving nutrients from outside.

Causes and management of post-treatment apical periodontitis.

Endodontic treatment failure is usually characterised by the presence of post-treatment apical periodontitis, which may be persistent, emergent or recurrent. The major aetiology of post-treatment disease is persistent intraradicular infection, but in some cases a secondary intraradicular infection due to coronal leakage or an extraradicular infection may be the cause of failure. Understanding the causes of endodontic treatment failure is of paramount importance for the proper management of this condition. Teeth with post-treatment apical periodontitis can be managed by either nonsurgical endodontic retreatment or periradicular surgery, both of which have very high chances of restoring the health of the periradicular tissues and maintaining the tooth function in the oral cavity. This review article focuses on the aetiological factors of post-treatment apical periodontitis and discusses the indications and basics of the procedures for optimal clinical management of this condition.

Clinical antibacterial effectiveness of the self-adjusting file system.

AIM: To evaluate in vivo the antibacterial effectiveness of the self-adjusting file (SAF) using molecular methods. METHODOLOGY: Root canals from single-rooted teeth with apical periodontitis were instrumented using the SAF system under continuous irrigation with 2.5% NaOCl. DNA extracts from samples taken before and after instrumentation were subjected to quantitative analysis of total bacteria counts and levels of streptococci by quantitative real-time polymerase chain reaction (qPCR). The reverse-capture checkerboard assay was also used to identify 28 bacterial taxa before (S1) and after (S2) SAF instrumentation. SAF was also compared with a conventional hand nickel-titanium instrumentation technique for total bacterial reduction. Data from qPCR were analysed statistically within groups using the Wilcoxon matched pairs test and between groups using the Mann-Whitney U-test and the Fisher's exact test, with significance level set at P < 0.05. RESULTS: Self-adjusting file significantly reduced the total bacterial counts from a mean number of 1.96 × 10(7) cells to 1.34 × 10(4) cells (P < 0.001). Quantitatively, the 99.9% reduction in total bacterial counts associated with the SAF system was significantly superior to the 95.1% reduction obtained by hand instrumentation (P < 0.001). Qualitatively, SAF resulted in significantly more cases with negative PCR results for bacteria (54.5%) than hand instrumentation (4.5%) (P < 0.001). The SAF system succeeded in significantly reducing the streptococcal levels, but four cases still harboured these bacteria in S2. Checkerboard analysis revealed that not only streptococci but also some anaerobic and even as-yet-uncultivated bacteria may resist the effects of chemomechanical procedures. CONCLUSION: The SAF instrumentation system was highly effective in reducing bacterial populations from infected root canals and performed significantly better than hand instrumentation. However, because half of the samples still had detectable bacteria after preparation with SAF, supplementary disinfection is still required to maximize bacterial elimination.

Reduction in bacterial counts in infected root canals after rotary or hand nickel-titanium instrumentation--a clinical study.

AIM: To compare the antibacterial efficacy of two instrumentation techniques, one using hand nickel-titanium (NiTi) instruments and the other using rotary NiTi instruments, in root canals of teeth with apical periodontitis. METHODOLOGY: Root canals from single-rooted teeth were instrumented using either hand NiTi instruments in the alternated rotation motion technique or rotary BioRaCe instruments. The irrigant used in both groups was 2.5% NaOCl. DNA extracts from samples taken before and after instrumentation were subjected to quantitative analysis by real-time polymerase chain reaction (qPCR). Qualitative analysis was also performed using presence/absence data from culture and qPCR assays. RESULTS: Bacteria were detected in all S1 samples by both methods. In culture analysis, 45% and 35% of the canals were still positive for bacterial presence after hand and rotary NiTi instrumentation, respectively (P > 0.05). Rotary NiTi instrumentation resulted in significantly fewer qPCR-positive cases (60%) than hand NiTi instrumentation (95%) (P = 0.01). Intergroup comparison of quantitative data showed no significant difference between the two techniques. CONCLUSION: There was no significant difference in bacterial reduction in infected canals after instrumentation using hand or rotary NiTi instruments. In terms of incidence of positive results for bacteria, culture also showed no significant differences between the groups, but the rotary NiTi instrumentation resulted in more negative results in the more sensitive qPCR analysis.

Clinical antimicrobial efficacy of NiTi rotary instrumentation with NaOCl irrigation, final rinse with chlorhexidine and interappointment medication: a molecular study.

AIM: To evaluate clinically the antibacterial effects of root canal treatment procedures using molecular microbiology analyses. METHODOLOGY: Samples were taken from 14 necrotic root canals of teeth with apical periodontitis before (S1) and after instrumentation with NaOCl irrigation (S2), a final rinse with chlorhexidine (CHX) (S3) and then one-week interappointment medication with calcium hydroxide/CHX paste (S4). The parameters examined included the following: incidence of positive broad-range PCR results for bacterial presence; impact on bacterial community structures evaluated by PCR-Denaturing Gradient Gel Electrophoresis (DGGE); quantitative bacterial reduction determined by real-time PCR; and identification of bacterial persisters by cloning and sequencing. Data from the different tests were subjected to statistical analyses and diversity indicator calculations. RESULTS: All S1 samples were positive for bacteria in all tests. Treatment procedures promoted a decrease in microbial diversity and significantly reduced the incidence of positive results and the bacterial counts (P < 0.05). In general, each subsequent treatment step improved disinfection. No specific taxon or community pattern was associated with post-treatment samples. CONCLUSION: Supplementary steps consisting of a final rinse with CHX followed by calcium hydroxide interappointment medication promoted further decrease in the bacterial bioburden to levels significantly below those achieved by the chemomechanical procedures alone. Because the long-term outcome of root canal treatment is dependent upon maximal bacterial reduction, the present results are of clinical relevance.

Quantitative molecular and culture analyses of bacterial elimination in oval-shaped root canals by a single-file instrumentation technique.

AIM: Bacterial reduction in oval-shaped root canals by a single-instrument technique was compared ex vivo with a conventional nickel-titanium rotary technique. Data obtained from two quantification methods, quantitative real-time polymerase chain reaction (qPCR) and culture, were also compared. METHODOLOGY: Oval-shaped canals of extracted teeth contaminated with Enterococcus faecalis were instrumented using either a single Reciproc instrument or the BioRaCe instrument series. Bacteriological samples were taken before (S1) and after instrumentation (S2). Bacterial quantification was performed using qPCR and culture. RESULTS: Intragroup analysis showed that both protocols promoted a highly significant bacterial reduction (P < 0.001). Intergroup analysis (S2 samples) showed no significant differences between the two instrumentation systems (P > 0.05). As for the quantification methods, qPCR revealed significantly higher counts of E. faecalis in S1 than culture (P < 0.05), but no significant differences occurred for S2 (P > 0.05). CONCLUSION: The single-file technique was comparable with the conventional technique in oval-shaped canals provided the width of apical preparation, volume of irrigants and duration of irrigation are kept similar. No significant difference was observed for qPCR and culture in post-instrumentation samples, indicating that both methods can be reliably used for studies of antibacterial effectiveness.

Molecular survey of atheromatous plaques for the presence of DNA from periodontal bacterial pathogens, archaea and fungi.

BACKGROUND AND OBJECTIVE: Chronic infections, such as periodontitis, have been associated with the development and progression of atherosclerosis. The mechanisms through which this occurs have yet to be elucidated. This study was carried out to detect periodontopathic bacteria as well as archaea and fungi in atheromatous plaques and search for factors associated with their occurrence in atheromas. MATERIAL AND METHODS: A cross-sectional study was carried out including 30 patients diagnosed with atherosclerosis in the carotid, coronary or femoral arteries. Plaques were collected during surgery and analysed using PCR to detect Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and members of the Synergistetes group. Samples were also surveyed with universal primers for bacterial, archaeal and fungal DNA. Patients responded to a questionnaire to determine factors associated with PCR results. RESULTS: All dentate individuals (66.7%) had periodontal disease, 95% of which was severe and 65% extensive. None of the targeted periodontopathic bacteria was found in the atheromas. No sample yielded positive results for fungal and archaeal DNA. Four samples (13%) were positive for the presence of bacterial DNA. Of these, three participants were dentate (two with severely chronic generalized periodontitis and one with severely chronic localized periodontitis). CONCLUSION: This study did not confirm previous findings of periodontal pathogens in atheromas, making it impossible to establish factors associated with their presence in plaques. Presence of bacterial DNA in some samples indicates that periodontal or nonoral bacterial species other than the ones targeted in this study may be involved with some cases of atherosclerosis.

Microbiota of dentinal caries as assessed by reverse-capture checkerboard analysis.

BACKGROUND/AIMS: This study aimed to identify the microbiota of different layers of dentinal caries by using a culture-independent molecular biology approach. METHODS: DNA was extracted from samples taken from 3 distinct layers (superficial, middle and deep) of advanced occlusal caries and analyzed for the presence and relative levels of 28 oral bacterial species/phylotypes using a reverse-capture checkerboard hybridization assay. RESULTS: The mean number of target taxa per layer was 7.7 (± 3.96) in the superficial, 7 (± 3.4) in the middle, and 6.3 (± 3.04) in the deep layer. No statistical significance was observed for these differences (p = 0.36). Overall, the most prevalent taxa in the 3 layers were Atopobium genomospecies C1 (72.5%), Fusobacterium nucleatum (69%), Lactobacillus casei (68%), Veillonella species (55%) and Lactobacillus fermentum (52%). No differences were found in the prevalence rates of the most frequent target species in the 3 layers. The most prevalent taxa found at levels above 10(5) in the advanced front line of deep-dentin caries were Atopobium genomospecies C1, F. nucleatum, Streptococcus mutans, Streptococcus species and Veillonella species. CONCLUSION: The present results revealed that the prevalences of several established or candidate caries pathogens do not differ significantly in the different zones of dentinal caries lesions. The finding that some as-yet-uncharacterized species and novel species were found in high frequencies join other molecular studies to include them in the set of candidate caries pathogens.

Diversity of endodontic microbiota revisited.

Although fungi, archaea, and viruses contribute to the microbial diversity in endodontic infections, bacteria are the most common micro-organisms occurring in these infections. Datasets from culture and molecular studies, integrated here for the first time, showed that over 460 unique bacterial taxa belonging to 100 genera and 9 phyla have been identified in different types of endodontic infections. The phyla with the highest species richness were Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. Diversity varies significantly according to the type of infection. Overall, more taxa have been disclosed by molecular studies than by culture. Many cultivable and as-yet-uncultivated phylotypes have emerged as candidate pathogens based on detection in several studies and/or high prevalence. Now that a comprehensive inventory of the endodontic microbial taxa has been established, future research should focus on the association with different disease conditions, functional roles in the community, and susceptibility to antimicrobial treatment procedures.

The microbiota of acute apical abscesses.

As the breadth of bacterial diversity in the oral cavity has been deciphered by molecular studies, several newly identified species/phylotypes have emerged as potential pathogens. We hypothesized that many of these species/phylotypes could also be involved with the etiology of endodontic abscesses. Abscess aspirates from 42 persons were analyzed for the presence of 81 species/phylotypes by means of a reverse-capture checkerboard hybridization assay. Associations between the most frequently detected taxa were calculated. The most prevalent taxa were Fusobacterium nucleatum, Parvimonas micra, and Porphyromonas endodontalis. Other frequently found taxa included Olsenella uli, streptococci, Eikenella corrodens, some as-yet-uncultivated phylotypes (Bacteroidetes clone X083 and Synergistes clone BA121), and newly named species (Prevotella baroniae and Dialister invisus). Several positive bacterial associations were disclosed. Findings not only strengthen the association of many cultivable species with abscesses, but also include some newly named species and uncultivated phylotypes in the set of candidate pathogens associated with this disease.

Root canal microbiota of teeth with chronic apical periodontitis.

Samples from infected root canals of 43 teeth with chronic apical periodontitis were analyzed for the presence and relative levels of 83 oral bacterial species and/or phylotypes using a reverse-capture checkerboard hybridization assay. Associations between the most frequently detected taxa were also recorded. The most prevalent taxa were Olsenella uli (74%), Eikenella corrodens (63%), Porphyromonas endodontalis (56%), Peptostreptococcus anaerobius (54%), and Bacteroidetes oral clone X083 (51%). When prevalence was considered only for bacteria present at levels >10(5), Bacteroidetes clone X083 was the most frequently isolated bacterium (37%), followed by Parvimonas micra (28%), E. corrodens (23%), and Tannerella forsythia (19%). The number of target taxa per canal was directly proportional to the size of the apical periodontitis lesion, with lesions >10 mm in diameter harboring a mean number of approximately 20 taxa. Several positive associations for the most prevalent taxa were disclosed for the first time and may have important ecological and pathogenic implications. In addition to strengthening the association of several cultivable named species with chronic apical periodontitis, the present findings using a large-scale analysis allowed the inclusion of some newly named species and as-yet-uncultivated phylotypes in the set of candidate pathogens associated with this disease.

Molecular analysis of the root canal microbiota associated with endodontic treatment failures.

INTRODUCTION: The failure of endodontic treatment is usually caused by persistent/secondary intraradicular infections and Enterococcus faecalis has been considered to be the main pathogen involved. Nevertheless, the breadth of bacterial diversity involved with endodontic treatment failures remains to be consistently explored by culture-independent approaches. METHODS: This study determined the intraradicular microbiota of root-canal-treated teeth with post-treatment apical periodontitis using 16S ribosomal RNA gene clone library analysis. RESULTS: Bacteria were present in all cases, confirming the infectious etiology of post-treatment disease. Seventy-four bacterial taxa belonging to six phyla were found in the nine cases investigated. Of these, 55% were identified as as-yet-uncultivated phylotypes, which also made up a significant proportion of the microbiota in many cases. Twenty-five new phylotypes were identified. Most teeth harbored a mixed consortium, with a mean number of 10 taxa per case. Only 11 taxa were found in more than one case, revealing a high interindividual variability in the composition of the microbiota. CONCLUSION: The current findings revealed new candidate endodontic pathogens, including as-yet-uncultivated bacteria and taxa other than E. faecalis, which may participate in the mixed infections associated with post-treatment apical periodontitis.