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This study aimed to detect the presence of specific bacteria and to evaluate the levels of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in symptomatic necrotic root canals associated with acute apical abscess (symptomatic group — GI). It also aimed to compare the findings with those presented by asymptomatic necrotic root canals (asymptomatic group — GII) in the different stages of the endodontic treatment. Microbiological samples were collected from 20 root canals, including purulent collection from acute apical abscesses, before and after chemo-mechanical preparation (CMP) preparation (CMP) with chlorhexidine gel 2% and after 30 days of intracanal medication (ICM) with (Ca[OH]2) + chlorhexidine. The culture method was used to evaluate the efficacy of CMP and ICM. Nested PCR was used to detect the presence of 17 specific bacteria. Levels of LPS were measured by using limulus amebocyte lysate, whereas enzyme-linked immunosorbent assay was used for the quantification of LTA. CMP was effective in reducing the microbial load in both groups (P

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 β-lactamase. 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 β-lactamase action. 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 β-lactamase resistance in five strains of E. faecalis. 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 β-lactamase resistance was detected in different isolates. To understand the influence of virulence factors in E. faecalis on the host heath status.

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.

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.

Streptococcus mutans plays an important role in caries etiology and eventually in systemic infections. However, it is often found in infected root canals, but the pathophysiological characteristics of strains residing in this site are largely unknown. Here, we characterized strains of S. mutans isolated from root canals of primary (PI) and secondary/persistent (SI) endodontic infections in relation to serotype and genotype; presence of genes coding for collagen binding proteins (CBPs); collagen binding activity and biofilm formation capacity; ability to withstand environmental stresses; systemic virulence in Galleria mellonella; and invasion of human coronary artery endothelial cells and human dental pupal fibroblasts. Samples from 10 patients with PI and 10 patients with SI were collected, and a total of 14 S. mutans isolates, belonging to 3 genotypes, were obtained. Of these, 13 were serotype c, and 1 was serotype k. When compared with the reference strains, the clinical isolates were hypersensitive to hydrogen peroxide. Remarkably, all 14 strains harbored and expressed the CBP-encoding gene cbm, showing increased binding to collagen, enhanced systemic virulence in G. mellonella, and ability to invade human coronary artery endothelial cells and human dental pupal fibroblasts when compared with CBP-negative strains. Whole genome sequence analysis of PI and SI isolates revealed that these strains are phylogenetically related but genetically distinct from each other. Our findings highlight the importance of CBPs in facilitating colonization and persistence of S. mutans in collagenous substrates such as root canals and their potential role in the pathogenesis of endodontic infections.

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.

Introduction The aim of this study was to evaluate the diversity and similarity of Enterococcus faecalis genotype isolates from multiple oral sites using repetitive sequence-based polymerase chain reaction and arbitrarily primed polymerase chain reaction (AP-PCR). Methods Forty-two endodontically treated teeth with apical periodontitis were selected. A total of 126 microbial samples were collected from 3 different sites (saliva, pulp chamber, and root canals, all n = 42) during the nonsurgical retreatment procedures. After growth on m-Enterococcus agar, the colonies were isolated, characterized as gram-positive catalase negative cocci, and identified using an API 20 Strep kit (bioMerieux, Marcy-l'Etoile, France). Seventy-four colonies from 10 patients were confirmed as E. faecalis by polymerase chain reaction (16S ribosomal RNA). Repetitive sequence-based polymerase chain reactions using ERIC and AP-PCR using RW3A primers were performed in all 74 colonies. Fingerprints were analyzed and separated into genotypic groups based on the Dice coefficient percentage of similarity (82% or greater) as determined by ERIC reproducibility assays involving E. faecalis controls. Results Seven different E. faecalis genotypes (GTs) (GT1 = 27%, GT2 = 17.6%, GT3 = 1.3%, GT4 = 18.9%, GT5 = 9.5%, GT6 = 14.9%, and GT7 = 10.8%) were observed in different subjects and oral sites associated with endodontic failure. Remarkably, in 4 of 5 patients, the same GTs present in the infected root canals were also isolated from either the pulp chamber or the saliva samples. In particular, GT6 was detected in all 3 oral sites of patient 37. Conclusions E. faecalis GTs isolated from saliva, the pulp chamber, and the root canal were similar using the Rep-PCR and AP-PCR methods. These findings suggest that coronal microleakage is a conceivable cause of endodontic failure.

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.