Identification of Culturable and Nonculturable Microorganisms, Lipopolysaccharides, and Lipoteichoic Acids From Root Canals of Teeth With Endodontic Failure.

AIM: To elucidate the presence of apical periodontitis in the root canal of teeth with secondary/persistent infection, including composition of microbiota, levels of endotoxins and lipoteichoic acid (LTA), and clinical implications of these findings. METHOD: Samples were collected from root canals of 50 patients who needed endodontic retreatment and had radiographic evidence of apical periodontitis. Microorganisms were identified by using the culture technique and biochemical tests. Nested-polymerase chain reaction (nested-PCR) was used to identify 17 species of specific bacteria. Lipopolysaccharides (LPS) and LTAs were quantified by using, respectively, limulus amebocyte lysate and enzyme-linked immunosorbent assay tests. RESULTS: Bacteria were detected in all samples by culture and molecular methods. A total of 154 gram-positive strains, of 188 strains isolated, were found in the root canals by culture. Enterococcus faecalis and Gemella morbillorum were the most prevalent species identified by the biochemical tests, whereas molecular analyses (nested-PCR) showed a high frequency of P. gingivalis, E. faecalis, and Fusobacterium nucleatum. LPS and LTA were detected in all samples, with mean values being 3.52 EU/mL and 597.83 pg/mL, respectively. Significant statistical correlations were found between levels of LTA and clinical features. CONCLUSION: Despite the prevalence of gram-positives, the microbiota present in secondary/persistent infections showed a large variety of species. Within this diversity, associations were found between specific bacteria and clinical features. In addition, higher levels of LTA were statistically associated with larger periapical radiolucent areas, but no correlation between this feature and LPS was found.

Pathogenic potential of Enterococcus faecalis strains isolated from root canals after unsuccessful endodontic treatment.

OBJECTIVES: To evaluate strains of Enterococcus faecalis isolated from endodontic failures cases for (a) presence of virulence genes, namely, gelatinase production (gelE), surface protein (esp), collagen-binding adhesin (ace), cytolysin activator (cylA), E. faecalis antigen A (efaA) and aggregation substance (asa), all by using PCR; (b) biofilm formation capacity; and (c) activity of gelatinase and ß-lactamase. MATERIALS AND METHODS: Twenty-five strains of E. faecalis were tested. The DNA extracted from these strains was used for identification of virulence genes by PCR and 1% agarose gel. Biofilm formation was performed on polystyrene microplates by using the violet crystal staining method. For assessment of the gelatinase activity, inoculum of pure cultures was deposited in tubes containing gelatin and a nutrient broth, whereas nitrocefin disks were used to assess the ß-lactamase action. RESULTS: The virulence genes efaA and cylA were detected in 100% of the strains, whereas gelE was present in 84%, ace in 68%, esp in 56% and asa in 48%. Four strains had no biofilm formation, 17 had poor formation and four had moderate formation. Gelatinase production was observed in three strains and ß-lactamase resistance in five strains of E. faecalis. TOPIC: Diverse patterns of virulence gene detection were observed among the E. faecalis strains, with predominance of those capable of forming biofilm. A few strains have been found to hydrolyze gelatin proteins, whereas ß-lactamase resistance was detected in different isolates. CLINICAL RELEVANCE: To understand the influence of virulence factors in E. faecalis on the host heath status.

Detection of Streptococcus mutans in symptomatic and asymptomatic infected root canals.

OBJECTIVES: To investigate the presence of Streptococcus mutans in root canals of symptomatic necrotic teeth (SNT) and their associated acute apical abscesses (AAA) and in the root canals of asymptomatic necrotic teeth (ANT). It also aimed to investigate the presence of the cnm and cbm genes in specimens that harbored S. mutans. MATERIALS AND METHODS: DNA was extracted from samples collected from 10 patients presenting pulpal necrosis associated with radiographic evidence of apical periodontitis (ANT) and from 10 patients in need of endodontic therapy due to the presence of pulpal necrosis (SNT) and AAA. The control group consisted of 10 patients with teeth with normal vital pulp and requiring endodontic treatment for prosthetic reasons. The presence of S. mutans was detected by quantitative real-time-PCR (qPCR) using species-specific primers. Samples harboring S. mutans were further evaluated for the presence of CBP genes by qPCR as well. RESULTS: All studied sites showed a high prevalence of S. mutans, except the control group. Specifically, 60% of ANT and 70% of AAA/SNT paired samples were positive for S. mutans. The cnm gene was detected positive for S. mutans only in ANT samples (66.6%). The cbm gene was not detected in any of the investigated sites. CONCLUSIONS: S. mutans was found in high prevalence in both asymptomatic and symptomatic endodontic infections, including in abscesses, but it was not detected in the root canals of teeth with normal vital pulp. Interestingly, cnm+ S. mutans was only detected in asymptomatic/chronic primary endodontic infections associated with apical lesion. Therefore, it appears that cnm, and possibly other CBPs, may play an underestimated role in chronic endodontic infections. CLINICAL RELEVANCE: A high prevalence of Streptococcus mutans cnm+ gene was detected only in asymptomatic primary endodontic infections associated with apical lesion. Therefore, it appears that this collagen-binding protein gene plays an underestimated role in asymptomatic/chronic endodontic infections.

Salivary proteomic profile of dogs with and without dental calculus.

BACKGROUND: Dogs' saliva is a complex mixture of inorganic and organic constituents, rich in proteins. Therefore, knowing the saliva composition of these animals is extremely important to identify the presence of proteins that may be involved in physiological and pathological mechanisms of their oral cavity. The present study aimed to characterize the proteomic profile of saliva from dogs with and without dental calculus. RESULTS: Saliva samples were collected from 20 dogs. Before the collection, a visual clinical examination was performed and 8 subjects (40%) did not present any signs of dental calculus, while 12 (60%) presented dental calculus. After saliva collection, the samples were submitted to protein quantification (mBCA), and then they were prepared for analysis by nLC-ESI-MS/MS. A total of 658 unique proteins were identified, of which 225 were specific to dogs without dental calculus, 300 were specific to dogs with dental calculus, and 133 were common to all subjects. These proteins presented functions including transportation, immune response, structural, enzymatic regulation, signal transduction, transcription, metabolism, and some proteins perform functions as yet unknown. Several salivary proteins in dogs with dental calculus differed from those found in the group without dental calculus. Among the abundant proteins detected in periodontal affected cases, can be highlighting calcium-sensing receptor and transforming growth factor beta. Enrichment analysis reveled the presence of Rho GTPases signaling pathway. CONCLUSIONS: This research identified salivary proteins, that should be further investigated as potencial biomarkers of chronic periodontits with dental calculus formation in dogs.

Effectiveness of XP-Endo Finisher in the reduction of bacterial load in oval-shaped root canals.

This study investigated the effectiveness of XP-Endo Finisher (XPF) associated with XP-Endo Shaper (XPS) or Reciproc Blue (RB) files in reducing bacterial load in oval-shaped root canals (RC) during chemomechanical preparation (CMP) using 0.9% saline solution (NaCl) or 2.5% sodium hypochlorite (NaOCl). Eighty mandibular incisors with single oval-shaped RC were contaminated with Enterococcus faecalis. The teeth were randomly assigned to eight experimental groups (n = 10) according to the CMP, as follows: G1: XPS, G2: XPS + XPF, G3: RB, and G4: RB + XPF. CMP was performed with NaCl or NaOCl. The reduction of bacterial load was assessed by colony-forming unit count before (S1) and after (S2) CMP. Data normality was verified by using Shapiro-Wilk test. ANOVA, Tukey's test, and Bonferroni post-hoc test were used at a 5% significance level. Culturable bacteria were present in all S1 samples (p>0.05). All instrumentation techniques were effective in reducing bacterial load, irrespective of the irrigating solution (p < 0.05). With the use of NaCl, RB was more effective than XPS (p = 0.035). With the use of NaOCl, XPS and RB presented similar effectiveness (p = 0.779). XPF enhanced the bacterial reduction of both systems tested (p < 0.05). The use of NaOCl improved the CMP, irrespective of the instrumentation technique used (p < 0.05). In conclusion, XPS and RB files are effective in reducing bacterial levels in oval-shaped RC. The use of XPF as a method of agitation of the irrigating solution improved the cleaning efficiency of both file systems tested. Mechanical preparation performed with saline solution decreased culturable bacteria from the root canal, but antimicrobial substances such as NaOCl should be used to achieve a significantly better disinfection.

Effectiveness of XP-Endo Finisher in the reduction of bacterial load in oval-shaped root canals

Abstract: This study investigated the effectiveness of XP-Endo Finisher (XPF) associated with XP-Endo Shaper (XPS) or Reciproc Blue (RB) files in reducing bacterial load in oval-shaped root canals (RC) during chemomechanical preparation (CMP) using 0.9% saline solution (NaCl) or 2.5% sodium hypochlorite (NaOCl). Eighty mandibular incisors with single oval-shaped RC were contaminated with Enterococcus faecalis. The teeth were randomly assigned to eight experimental groups (n = 10) according to the CMP, as follows: G1: XPS, G2: XPS + XPF, G3: RB, and G4: RB + XPF. CMP was performed with NaCl or NaOCl. The reduction of bacterial load was assessed by colony-forming unit count before (S1) and after (S2) CMP. Data normality was verified by using Shapiro-Wilk test. ANOVA, Tukey's test, and Bonferroni post-hoc test were used at a 5% significance level. Culturable bacteria were present in all S1 samples (p>0.05). All instrumentation techniques were effective in reducing bacterial load, irrespective of the irrigating solution (p < 0.05). With the use of NaCl, RB was more effective than XPS (p = 0.035). With the use of NaOCl, XPS and RB presented similar effectiveness (p = 0.779). XPF enhanced the bacterial reduction of both systems tested (p < 0.05). The use of NaOCl improved the CMP, irrespective of the instrumentation technique used (p < 0.05). In conclusion, XPS and RB files are effective in reducing bacterial levels in oval-shaped RC. The use of XPF as a method of agitation of the irrigating solution improved the cleaning efficiency of both file systems tested. Mechanical preparation performed with saline solution decreased culturable bacteria from the root canal, but antimicrobial substances such as NaOCl should be used to achieve a significantly better disinfection.