Metatranscriptome and Resistome of the Endodontic Microbiome.

INTRODUCTION: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections. METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification. RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms. CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.

Enhancing Antibiotic Efficacy in Regenerative Endodontics by Improving Biofilm Susceptibility.

INTRODUCTION: Various strategies have been researched to enhance the susceptibility of biofilms, given their tolerance to antibiotics. This study evaluated the effect of the anti-microbial peptide nisin in association with antibiotics used in regenerative endodontics, exploring different treatment times and biofilm growth conditions. METHODS: A mixture of 10 bacterial species was cultivated on dentin specimens anaerobically for 21 days. Biofilms were treated with 1 mL of high-purity nisin Z (nisin ZP, 200 µg/mL) and a triple antibiotic mixture (TAP: ciprofloxacin + metronidazole + minocycline, 5 mg/mL), alone or in combination. The effectiveness of antimicrobial agents was assessed after 1 and 7 days. During the 7-day period, biofilms were treated under 2 conditions: a single dose in a nutrient-depleted setting (ie, no replenishment of growth medium) and multiple doses in a nutrient-rich environment (ie, renewal of medium and antimicrobial agents every 48 h). After treatments, biofilm cells were dispersed, and total colony-forming units were counted. RESULTS: After 1 d-treatment, nisin ZP + TAP resulted in 2-log cell reduction compared to TAP alone (P < .05). After 7 d-treatment with a single dose, nisin ZP + TAP and TAP reduced bacteria to nonculturable levels (P < .05), whereas repeated antimicrobial doses did not eliminate bacteria in a nutrient-rich environment. No bacterial reduction was observed with nisin ZP alone in any treatment time. CONCLUSIONS: The additional use of nisin improved the TAP activity only after a short exposure time. Longer exposure to TAP or nisin + TAP in a nutrient-deprived environment effectively eliminated biofilms.

Antibiofilm Activity of LL-37 Peptide and D-Amino Acids Associated with Antibiotics Used in Regenerative Endodontics on an Ex Vivo Multispecies Biofilm Model.

The antimicrobial peptide LL-37 and D-amino acids (D-AAs) have been proposed as antibiofilm agents. Therefore, this study aimed to test the antimicrobial effect of antibiofilm agents associated with antibiotics used in regenerative endodontic procedures (the triple antibiotic paste­TAP: ciprofloxacin + metronidazole + minocycline). An endodontic-like biofilm model grown on bovine dentin discs was used in this study. After 21-day growth, the biofilms were treated with 1 mg/mL TAP, 10 µM LL-37, an association of LL-37 + TAP, 40 mM D-AAs solution, an association of D-AAs + TAP, and phosphate-buffered saline (negative control). Colony forming unit (CFU) data were analyzed by two-way ANOVA and Tukey's multiple comparison test (p < 0.05). LL-37 + TAP showed the best antibacterial activity (7-log10 CFU/mL ± 0.5), reaching a 1 log reduction of cells in relation to the negative control (8-log10 CFU/mL ± 0.7) (p < 0.05). In turn, no significant reduction in bacterial cells was observed with TAP, LL-37, D-AAs, and D-AAs + TAP compared to the negative control. In conclusion, the combination of antibiotics and LL-37 peptide showed mild antibacterial activity, while the combination of antibiotics and D-AAs showed no activity against complex biofilms.

Nature and Prevalence of Bacterial Taxa Persisting after Root Canal Chemomechanical Preparation in Permanent Teeth: A Systematic Review and Meta-analysis.

INTRODUCTION: Culture-independent molecular studies have shown a broad spectrum of bacterial taxa that persist after chemomechanical procedures (CMP). Therefore, this study systematically reviewed these reports to explore the prevalence of bacteria in post-instrumentation samples of root canals from permanent teeth, especially of as-yet-uncultivated/difficult-to-culture bacteria. METHODS: Electronic databases were searched from 2007 to January 2021. Clinical studies using culture-independent molecular methods to identify species-level taxa before and after CMP were included. Studies were critically appraised using the Joanna Briggs Institute Prevalence Critical Appraisal Checklist and the funnel plot analysis. The meta-analysis was performed on the prevalence of as-yet-uncultivated/difficult-to-culture bacterial taxa using RStudio. RESULTS: A total of 3781 titles were screened, but only 20 studies were included. The most frequent species in post-instrumentation samples were Streptococcus spp., Leptotrichia buccalis, Fusobacterium nucleatum, and Capnocytophaga ochracea. The detection frequency of some species increased after CMP, including mainly Firmicutes members such as streptococci, Enterococcus faecium, Selenomonas noxia, and Solobacterium moorei. The prevalence (confidence interval) of difficult-to-culture species was as follows: Dialister invisus, 17% (7%-29%); Solobacterium moorei, 14% (8%-23%); Bacteroidaceae [G-1] bacterium HMT 272, 13% (5%-23%); and Filifactor alocis, 11% (3%-23%). CONCLUSIONS: The prevalence of as-yet-uncultivated/difficult-to-culture bacterial taxa in post-instrumentation samples was low. The persistent species belonged mainly to the phylum Firmicutes, and streptococci were the major members. Future larger clinical studies on the composition of the whole bacterial community that persist after CMP are still necessary for a better understanding of bacterial interactions and their clinical significance in the treatment outcome.

Antibacterial Effect of Sodium Hypochlorite and EDTA in Combination with High-Purity Nisin on an Endodontic-like Biofilm Model.

Antimicrobial peptides have been proposed as antibiofilm agents. Therefore, we evaluated the effect of endodontic irrigants combined or not with the antimicrobial peptide nisin against an endodontic biofilm model composed of eleven bacterial species. Biofilms were grown on hydroxyapatite discs for 3, 15 and 21 days and treated with 1.5% sodium hypochlorite (NaOCl) or 17% EDTA followed by high-purity nisin (nisin ZP) or saline for 5 min each. Differences between groups were tested by two-way ANOVA and Tukey's multiple comparisons test (p < 0.05). Treatment with 1.5% NaOCl completely eliminated 3-d and 15-d biofilms but did not eradicate 21-d biofilms. Treatment with 1.5% NaOCl and 17% EDTA was equally effective against 21-d biofilms, showing 5-log and 4-log cell reduction, respectively, compared to the untreated control (9 log10, p < 0.05). No significant difference was found between 1.5% NaOCl + nisin ZP and 1.5% NaOCl in 21-d biofilms (p > 0.05). Likewise, no significant difference was found between 17% EDTA + nisin ZP and 17% EDTA treatments (p > 0.05). In conclusion, 1.5% NaOCl or 17% EDTA were effective strategies to combat mature biofilms. The additional use of nisin did not improve the activity of conventional irrigants against multispecies biofilms.

Identification of Fusobacterium nucleatum in primary and secondary endodontic infections and its association with clinical features by using two different methods.

OBJECTIVE: Fusobacterium nucleatum is an important oral pathogen involved in endodontic infections. This study aimed to assess the frequency of Fusobacterium nucleatum in primary and secondary endodontic infections and its associations with the clinical features in a Brazilian population by using both culture and nested PCR methods. METHODS: A total of 100 microbial samples from patients with primary (n=50) and secondary endodontic infections (n=50) were analyzed by using culture and nested PCR methods. Strict anaerobic techniques were used for culture and identification of F. nucleatum. The DNA extracted from the samples was analyzed for the presence of target species by using species-specific primers. RESULTS: Culture and nested PCR methods detected F. nucleatum, respectively, in 11/100 and 82/100 root canals. F. nucleatum was isolated by culture from 10/50 (20%) root canals with primary infections and from 1/50 (2%) root canal with secondary/persistent infections. Nested PCR detected F. nucleatum in 42/50 (84%) root canals with primary infections and in 40/50 (80%) root canals with secondary/persistent endodontic infections. F. nucleatum was associated with spontaneous pain, tenderness to percussion, pain on palpation, swelling, tooth mobility, wet root canals, hemorrhagic exudate, tooth decay, inadequate restoration, and poor endodontic filling. CONCLUSION: F. nucleatum was found in more cases of primary endodontic infections than in cases of secondary/persistent ones. A higher prevalence of F. nucleatum was detected by using the nested PCR method than by using culture. The presence of F. nucleatum in the root canals was associated with several clinical features. CLINICAL RELEVANCE: The high prevalence of F. nucleatum in the root canals detected by molecular methods, and its association with several clinical features reveals the importance of these species in the development of apical pathologies and reinforces the need of an endodontic treatment directed to bacterial elimination.

Antibacterial Effect of High-Purity Nisin Alone and in Combination with D-Amino Acids or Chlorhexidine in an Endodontic-Like Biofilm Model.

New strategies to eradicate endodontic biofilms are needed. Therefore, we evaluated the effect of high-purity nisin alone and in combination with D-amino acids (D-AAs) or chlorhexidine (CHX) against an "endodontic-like" biofilm model. Biofilms were grown on hydroxyapatite discs for 64 h and treated with nisin, eight D-AAs mixture, nisin + eight D-AAs, 2% CHX, and nisin + 2% CHX. After the 5 min and 24 h treatments, biofilm cells were harvested and total colony-forming units were counted. Differences between groups were tested by two-way ANOVA followed by Tukey's multiple comparisons test (p < 0.05). Nisin and D-AAs, alone or in combination, were not effective in reducing bacteria after short or long exposure times. After 5 min, treatment with 2% CHX and nisin + 2% CHX resulted in 2 and 2.4-log cell reduction, respectively, compared with the no treatment control (p < 0.001). After 24 h, 2% CHX and nisin + 2% CHX drastically reduced bacterial counts. In conclusion, high-purity nisin alone or in combination with D-AAs did not show antibacterial activity against multispecies biofilms. Moreover, combined treatment using nisin and CHX showed similar antibiofilm activity compared with the use of CHX alone.

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

INTRODUCTION: Because 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. METHODS: Samples 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. RESULTS: After 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%). CONCLUSIONS: CMP 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.

Clinical and Molecular Microbiological Evaluation of Regenerative Endodontic Procedures in Immature Permanent Teeth.

INTRODUCTION: This study evaluated the bacterial levels after regenerative endodontic procedures and their correlation with the treatment outcome using molecular microbiology methods. METHODS: Root canal samples of 15 necrotic immature teeth were analyzed by quantitative polymerase chain reaction. Bacteria were counted before treatment (S1), after irrigation with 6% sodium hypochlorite (S2), and after intracanal dressing (S3) using either triple antibiotic paste (n = 7) or calcium hydroxide with chlorhexidine (n = 8). The Wilcoxon test for related samples and the Mann-Whitney test were used for statistical analysis (P < .05). After a follow-up period of 12-48 months, clinical and radiographic findings were correlated with microbiological data using a linear regression model (P < .05). RESULTS: All S1 and S2 samples were positive for bacteria, but the number of positive S3 samples decreased to 53.3% (P = .001). Overall, there was a significant reduction of bacterial levels after each treatment step (S1-S2, P = .001; S2-S3, P = .02). In the triple antibiotic paste and chlorhexidine groups, 57.1% and 50% of S3 samples were positive with median numbers of 6.97 × 103 and 3.59 × 104 bacterial cells, respectively. No significant differences were found between the groups. Periapical healing occurred in all cases despite the presence of low levels of residual bacteria. However, the latter had a negative impact on the thickness of dentinal walls (R2 = 0.0043). CONCLUSIONS: Although the bacterial levels were drastically reduced after the regenerative endodontic procedures, the residual bacteria influenced the thickness of the dentinal walls.

Investigation of Filifactor alocis in primary and in secondary endodontic infections: A molecular study.

OBJECTIVE: Identification of specific bacteria in root canals (RCs) in distinct clinical conditions can support the comprehension of pathological processes. Thus, the objective of this clinical study was to investigate the presence of F. alocis in RCs of teeth with primary endodontic infection (PEI) and with persistent/secondary endodontic infection (SEI) by using molecular techniques. It was also aimed to associate its presence with the clinical features. In addition, the levels of F. alocis as well as the total bacterial cells in the samples were also quantitated. DESIGN: One hundred teeth (50 PEI and 50 SEI) were included. Microbial samples were performed using sterile paper points and assessed by using nested PCR and quantitative Real Time PCR (qPCR). The prevalence of F.alocis in RCs from PEI and SEI were compared by chi-square analysis. Fisher´s exact test or Pearson Chi-square, when appropriate, was used to test associations between clinical and radiographic features and the presence of F. alocis. Significance level was set at 5%. RESULTS: F. alocis was detected in 23 and 28 (PEI) and 12 and 11 (SEI) RCs using Nested PCR and qPCR, respectively. Statistically significant associations were found between the presence of F. alocis and PEI, pain, wet canals, swelling, abscess and purulent exudate (P < 0.05). Total bacterial count was similar in both conditions (P > 0.05). CONCLUSIONS: PEI harbour a significantly higher number of F. alocis than those with SEI. Filifactor alocis was significantly associated with clinical features in primary endodontic infections. Total bacterial count was similar in both clinical conditions.

Next-Generation Sequencing to Assess Potentially Active Bacteria in Endodontic Infections.

INTRODUCTION: Because active bacteria present a higher abundance of ribosomal RNA (rRNA) than DNA (rRNA gene), the rRNA/DNA ratio of next-generation sequencing (NGS) data was measured to search for active bacteria in endodontic infections. METHODS: Paired complementary DNA and DNA samples from 5 root canals of teeth with apical periodontitis were subjected to polymerase chain reaction with bar-coded primers amplifying the 16S rRNA gene hypervariable regions V4-V5. High-throughput sequencing was performed using MiSeq (Illumina, San Deigo, CA), and data were analyzed using Quantitative Insights Into Microbial Ecology and Human Oral Microbiome Database. Statistical analysis was performed for relative abundance of bacteria in the DNA- and rRNA-based NGS data using the Mann-Whitney test, whereas differences in the diversity and richness indexes were assessed using a nonparametric 2-sample t test (P < .05). For bacterial taxa detected in both approaches, the rRNA/DNA ratios were calculated by dividing the average abundance of individual species in the respective analysis. RESULTS: Although no significant difference was found in the indexes of bacterial richness and diversity, the relative abundance of bacterial members varied in both analyses. Comparing rRNA with DNA data, there was a significant decrease in the relative abundance of Firmicutes (P < .05). The bacterial taxa Bacteroidales [G-2] bacterium HMT 274, Porphyromonas endodontalis, Tannerella forsythia, Alloprevotella tannerae, Prevotella intermedia, Pseudoramibacter alactolyticus, Olsenella sp. HMT 809, Olsenella sp. HMT 939, Olsenella uli, and Fusobacterium nucleatum subsp. animalis were both dominant (DNA ≥ 1%) and active (rRNA/DNA ≥ 1). CONCLUSIONS: The integrated DNA- and rRNA-based NGS strategy was particularly important to disclose the activity of as-yet-uncultivated or difficult-to-culture bacteria in endodontic infections.

Effects of Contemporary Irrigant Activation Schemes and Subsequent Placement of an Interim Dressing on Bacterial Presence and Activity in Root Canals Associated with Asymptomatic Apical Periodontitis.

New tools for activating endodontic irrigants have evolved, yet their impact on root canal disinfection, in comparison to the passive placing of an inter-visit medication, have not yet been fully elucidated. The use of DNA- and rRNA-based methods may cast some new light on this issue, as they allow a comparison to be made between microbial presence and activity. Therefore, the aim of this single-arm intervention trial is to evaluate the antibacterial effect of endodontic procedures using both molecular methods. Root canal samples were obtained from 20 patients with asymptomatic apical periodontitis after each treatment step: access cavity, chemo-mechanical preparation, adjunctive procedures (XP-endo Finisher file and passive ultrasonic irrigation), calcium hydroxide medication, and 2nd-visit root canal preparation. DNA and cDNA from the samples were subjected to quantitative polymerase chain reaction with universal primers for the bacterial 16S rRNA gene. Chemo-mechanical preparation promoted a drastic reduction in bacterial levels and activity, whereas the adjunctive procedures did not make a significant contribution to further disinfection. At the 2nd visit, bacteria were active after the use of calcium hydroxide medication; however, they were significantly reduced after a 2nd-visit preparation. Consequently, the lowest bacterial levels were found at the end of the treatment. This clinical trial, which used an rRNA and rDNA combined approach, confirmed previous studies showing that root canal preparation represents the main strategy for root canal disinfection.

Evaluation of the Propidium Monoazide-quantitative Polymerase Chain Reaction Method for the Detection of Viable Enterococcus faecalis.

INTRODUCTION: One limitation of DNA-based molecular assays is their inability to distinguish between live and dead cells. A sample treatment with propidium monoazide (PMA) before DNA amplification has been proposed to overcome this problem. The aim of this in vitro study was to test different concentrations of PMA coupled with quantitative polymerase chain reaction (qPCR) for the detection of viable Enterococcus faecalis. METHODS: Viable or heat-killed suspensions of E. faecalis (106 colony-forming units/mL) were treated with PMA at 10, 50, and 100 µg/mL before DNA extraction. qPCR was performed using primers complementary for E. faecalis 16S ribosomal RNA sequence. PMA was also tested on bacteria suspensions containing different proportions of viable and dead cells. Bacterial suspensions without PMA treatment were used as positive controls. RESULTS: The treatment of heat-killed suspensions with PMA at different concentrations significantly reduced the DNA amplification when compared with the group without treatment (P < .0001), indicating that DNA from dead cells was not used as templates. The greatest reduction in qPCR amplification of dead cell DNA was found when 100 µg/mL PMA was used (P < .005). In mixtures containing live/dead cells, PMA allowed selective detection of viable cells. CONCLUSIONS: PMA was effective in inhibiting qPCR amplification from the DNA of dead cells, enabling in vitro detection and quantification of viable cells of E. faecalis.

RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures.

INTRODUCTION: Because ribosomal RNA (rRNA) indicates metabolic cell activity, this study aimed to evaluate the sensitivity of rRNA-based quantitative polymerase chain reaction (RT-qPCR) for the identification of active Enterococcus faecalis in root canals samples compared with a method based on ribosomal DNA (rDNA) (rRNA genes). METHODS: Samples were taken from 18 teeth with persistent/secondary intraradicular infection before (S1) and after (S2) chemomechanical preparation. RNA and DNA were extracted, and complementary DNA was synthesized from RNA using RT-PCR. Complementary DNA and genomic DNA were subjected to quantitative polymerase chain reaction with primers complementary for E. faecalis 16S rRNA sequence. RESULTS: E. faecalis was detected in 77.8% and 72.2% of S1 samples using rRNA- and rDNA-based assays, respectively. In contrast, E. faecalis was detected in only 33.3% of S2 samples using rDNA as the template compared with 61.1% using the rRNA-based method. The median concentration of rRNA copies of E. faecalis was significantly higher than rDNA copies, indicating a higher sensitivity for the method targeting rRNA in both S1 (P < .01) and S2 samples (P < .05). After chemomechanical preparation, the number of rRNA and rDNA copies was significantly reduced (P < .05). The high ratio of rRNA to rDNA copies in S2 samples suggested that active E. faecalis persisted in root canals after chemomechanical preparation. CONCLUSIONS: The RT-qPCR assay provides a sensitive method for the identification of active E. faecalis from endodontic samples. Furthermore, the rRNA-based assay indicated that E. faecalis viable cells persisted in treated root canals, suggesting that it may be a useful tool for monitoring microbial load during endodontic treatment.

Analysis of genetic lineages and their correlation with virulence genes in Enterococcus faecalis clinical isolates from root canal and systemic infections.

INTRODUCTION: Enterococcus faecalis is a member of the mammalian gastrointestinal microbiota but has been considered a leading cause of hospital-acquired infections. In the oral cavity, it is commonly detected from root canals of teeth with failed endodontic treatment. However, little is known about the virulence and genetic relatedness among E. faecalis isolates from different clinical sources. This study compared the presence of enterococcal virulence factors among root canal strains and clinical isolates from hospitalized patients to identify virulent clusters of E. faecalis. METHODS: Multilocus sequence typing analysis was used to determine genetic lineages of 40 E. faecalis clinical isolates from different sources. Virulence clusters were determined by evaluating capsule (cps) locus polymorphisms, pathogenicity island gene content, and antibiotic resistance genes by polymerase chain reaction. RESULTS: The clinical isolates from hospitalized patients formed a phylogenetically separate group and were mostly grouped in the clonal complex 2, which is a known virulent cluster of E. faecalis that has caused infection outbreaks globally. The clonal complex 2 group comprised capsule-producing strains harboring multiple antibiotic resistance and pathogenicity island genes. On the other hand, the endodontic isolates were more diverse and harbored few virulence and antibiotic resistance genes. In particular, although more closely related to isolates from hospitalized patients, capsule-producing E. faecalis strains from root canals did not carry more virulence/antibiotic genes than other endodontic isolates. CONCLUSIONS: E. faecalis isolates from endodontic infections have a genetic and virulence profile different from pathogenic clusters of hospitalized patients' isolates, which is most likely due to niche specialization conferred mainly by variable regions in the genome.

Capsule locus polymorphism among distinct lineages of Enterococcus faecalis isolated from canals of root-filled teeth with periapical lesions.

INTRODUCTION: Although Enterococcus faecalis is a member of the normal microbiota, it is also a major cause of nosocomial infections. Some strains of E. faecalis produce capsule, which contributes to pathogenesis through evasion of host defenses, and its production is dependent on the capsule (cps) operon polymorphism. This study investigated cps locus polymorphism in distinct lineages of E. faecalis isolated from canals of root-filled teeth with periapical lesions. METHODS: Twenty-two E. faecalis isolates were evaluated regarding the cps operon polymorphism and genetic diversity. The 3 known CPS types were determined by polymerase chain reaction. This information was correlated with multilocus sequence typing data, which were used to define genetic lineages. RESULTS: cpsA and cpsB were the only detected genes within the cps operon in 62.5% of E. faecalis strains (14/22), indicative of genotype CPS 1, which lacks capsule expression. The essential genes in the cps operon for capsule production were detected in the remaining strains, whereas 3 belonged to genotype CPS 5 and 5 strains to genotype CPS 2. A total of 14 sequence types (STs) were resolved in 22 E. faecalis isolates. Comparison with the E. faecalis international multilocus sequence typing database revealed that 9 STs were previously found, and that the 5 STs were novel. CONCLUSIONS: Certain E. faecalis genotypes from canals of root-filled teeth with periapical lesions belong to lineages associated with capsule expression and production of multiple virulence factors, which might account for their increased pathogenic potential.

Microbial analysis of canals of root-filled teeth with periapical lesions using polymerase chain reaction.

The objective of the present study was to investigate the presence of nine bacterial species in root-filled teeth associated with periapical lesions using a polymerase chain reaction analysis and to correlate these species with clinical features of the cases. DNA was extracted from 45 canal samples of root-filled teeth with periapical lesions. A PCR assay using species-specific primers of 16S rDNA and the downstream intergenic spacer region was used for microbial detection. Enterococcus faecalis was the most prevalent species, detected in 77.8% of the study teeth, followed by Peptostreptococcus micros, detected in 51.1%. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens were detected in 35.6%, 22.2%, 11.1%, and 11.1% of the sampled teeth, respectively. Moreover, PCR detected Filifactor alocis in 26.7%, Treponema denticola in 24.4%, and Tannerella forsythia in 4.4% of the samples. T. denticola and P. micros were statistically associated with tenderness to percussion (p < 0.05). P. nigrescens was associated with the presence of spontaneous pain and abscess (p < 0.05). P. endodontalis and P. nigrescens were associated with purulent exudates (p < 0.05). Synergistic relationship was also observed between some species. The results of this study indicated that E. faecalis was the most frequently identified test species by PCR in teeth with failing endodontic treatment.

The outcome of endodontic treatment: a retrospective study of 2000 cases performed by a specialist.

The purpose of this study was to evaluate the treatment outcome of initial endodontic treatment and nonsurgical retreatment performed by an endodontic specialist in his private office. A total of 2,000 teeth were examined clinically and radiographically and the results were analyzed statistically by Pearson or Fisher's Exact test and multivariate logistic regression. The multivariate analysis evaluated joint associations among various factors, using logistic regression models. The dependent variable for this analysis was the dichotomous outcome: healed versus disease. The overall endodontic success rate was 91.45%, and the healed rate was significantly higher for initial endodontic treatments than for nonsurgical retreatments; teeth without lesion than for those with lesions; teeth treated without complications than for those with complications; recall period of 18-24 months than for other periods, and teeth with final coronal restoration than for those without. Of the 1376 teeth treated in the initial endodontic treatment sample, the success rate was 94.0%. Multivariate analysis identified the presence of procedural complications (file breakage, perforation and flare-up), as well as the absence of the restorations at follow-ups as the significant predictors of outcome, showing lower rates of success. Of the 624 teeth in the nonsurgical retreatment sample, 85.9% were successful. Stepwise logistic regression analysis revealed that preoperative radiolucency was a strong statistically significant factor to determine lower rates of success than in its absence. Two additional variables (age and tooth type) were found to have a significant influence on the outcome of the retreatment sample. A higher healed rate was observed for the 50-59 years age groups than others, while multirooted (molars) teeth revealed a significantly lower percentage of success than pre-molars and anterior teeth.

Molecular analysis of Filifactor alocis, Tannerella forsythia, and treponema denticola associated with primary endodontic infections and failed endodontic treatment.

The aim of this study was to investigate the presence of strict anaerobes such as Filifactor alocis, Tannerella forsythia, and Treponema denticola in primary and secondary root-infected canals with periapical lesions by molecular analysis and the association of these species with specific endodontic signs and symptoms. Microbial samples were taken from 100 root canals, 50 with necrotic pulp tissues (NPT, primary infection), and 50 with failed endodontic treatment (FET, secondary infection). DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens using species-specific primers and PCR. F. alocis were isolated from 23 canals with NPT and 12 canals with FET; T. forsythia from 12 canals with NPT and three canals with FET; T. denticola from 19 canals with NPT and 12 canals with TEP. Suggested associations were found between primary infection and the presence of F. alocis and T. forsythia (both p < 0.05). In particular, associations were found between: pain and F. alocis; swelling and F. alocis; tenderness to percussion and T. forsythia; mobility and T. forsythia and T. denticola; wet canals and F. alocis, T. forsythia, and T. denticola; purulent exsudate and F. alocis, T. forsythia and T. denticola; abscess and F. alocis, T. forsythia, and T. denticola (all p < 0.05). The findings of this study indicated that F. alocis, T. forsythia, and T. denticola seem to be associated with endodontic signs and symptoms. Additionally, F. alocis and T. forsythia were detected more frequently in teeth with necrotic pulp than in teeth with failing endodontic treatment.

Microleakage evaluation of intraorifice sealing materials in endodontically treated teeth.

OBJECTIVE: Evaluate intraorifice sealing materials Cavit, Vitremer, and Flow-It for the prevention of coronal microleakage in root-canal treatment. STUDY DESIGN: Root-canal treatment was performed on 80 extracted human molars. Three millimeters of coronal gutta-percha was removed from the coronal aspect of the root canal and replaced with one of the 3 filling materials. After thermocycling (5 degrees C to 55 degrees C) and 5 days of immersion in dye, the teeth were cleared for stereomicroscope evaluation for evidence of dye penetration into the sealing material and along canal walls. RESULTS: All groups showed dye penetration into the root canal. Cavit sealed significantly better than the other groups (P < .01), preventing the coronal leakage in 90% of the specimens. Flow-It exhibited the highest leakage (65% of specimens) and did not differ significantly from the Vitremer group, which showed dye penetration in 55% of specimens. CONCLUSION: Cavit sealed significantly better than Vitremer and Flow-It when used as intraorifice filling materials.