lipoteichoic-acid and Periapical-Periodontitis

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]. CMP was effective in reducing the microbial load in both groups (P < 0.05). LPS levels were higher in GI than in GII (P < 0.05). There was a significant reduction in the LPS levels after CMP and ICM (P < 0.05) in GI and GII. LTA levels were significantly reduced in GI after ICM and in GII after CMP and ICM (both P < 0.05). Fusobacterium nucleatum and Enterococcus faecalis were frequently identified in both groups, alone or in combination with each other.. Different species were detected in all stages of the endodontic treatment. CMP was able to reduce bacterial content and the levels of LPS, but not of LTA in the symptomatic group. High levels of LPS were correlated with spontaneous pain and pain to percussion in the symptomatic group.. This clinical study showed that chemo-mechanical preparation was able to reduce bacterial load and levels of LPS, but not of LTA in the symptomatic group. Elevated levels of LPS were correlated with the presence of symptomatology.

Topics: Bacteria; Dental Pulp Cavity; Humans; Lipopolysaccharides; Periapical Periodontitis; Root Canal Irrigants; Teichoic Acids

To investigate the microbial profile, and levels of endotoxin (LPS) and lipoteichoic acid (LTA), in infected dentine (ID) and root canals (RC) at different phases of root canal treatment in teeth with symptomatic irreversible pulpitis.. Ten volunteers were included, and samples were collected from infected dentine (ID) and the root canal lumen (RC) using sterile excavators and paper points, respectively. RC samples were taken before (S1) and after (S2) chemo-mechanical canal preparation (CMP), and after intracanal medication (ICM; S3). Checkerboard DNA-DNA hybridization was used for microbial analysis. The levels of LPS and LTA were evaluated using the limulus amebocyte lysate assay and ELISA, respectively. Shapiro-Wilk's test was used to verify data normality. Friedman's test was used to evaluate statistical differences using checkerboard DNA-DNA hybridization in the ID and RC at the different phases of the RC treatment. Post hoc Dunn's multiple comparison test was used to verify significant differences recorded at the different time-points. The levels of LPS and LTA were analysed statistically by using repeated measures anova and Tukey's post hoc test to evaluate differences in both sites. The significance level was set at 5% (P < 0.05).. A total of 40 DNA probes were used for microbial investigation of ID and RC samples using checkerboard DNA-DNA hybridization. The levels and complexity of bacteria were similar in the ID and initial RC samples. The levels of LPS and LTA in ID were significantly higher than the initial RC samples (S1; P < 0.05). Canal preparation was effective in significantly decreasing the levels of bacteria, LPS and LTA (P < 0.05). ICM did not provide additional reduction in the levels of bacteria and LPS (P > 0.05). However, a significant reduction in the levels of LTA was observed after ICM (P < 0.05).. The microbial profile of infected dentine and root canals of teeth with irreversible pulpitis was complex, harbouring different species including Gram-positive and Gram-negative, cocci and bacilli, and facultative and strict anaerobes. Root canal preparation was effective in reducing the levels of bacteria, LPS and LTA from the root canals of teeth with pulpitis.

Topics: Dental Pulp Cavity; Endotoxins; Humans; Lipopolysaccharides; Periapical Periodontitis; Pulpitis; Root Canal Irrigants; Root Canal Preparation; Teichoic Acids

The purpose of this study was to evaluate the effect of diode laser irradiation on Enterococcus faecalis (E. faecalis) and its lipoteichoic acid (LTA). Ninety-six freshly extracted single-rooted teeth were divided into six groups, n = 8 per group. Groups 1, 2, 3, and 4 as laser group (810 nm PILOT™ Diode Laser, 400 μm fiber diameter, continuous mode, 30 s time) with powers at 1.0 W, 1.5 W, 2.0 W, and 2.5 W respectively. Group 5 or positive control group (3 ml of 1% sodium hypochlorite (NaOCl) irrigation) and group 6 or negative control group (3 ml of normal saline (0.9% NaCl) irrigation). Root canal samples were collected before and after receiving laser irradiation and irrigation solution. Cultivable bacteria were determined by counting the colony (CFU/ml). Evaluation of temperature on the external root surface of teeth was done with K type thermocouple using laser at different powers. Enzyme-linked immunosorbant assay (ELISA) was performed to measure the LTA levels and the correlations between E. faecalis count, LTA levels, and rise in temperature were observed using Pearson's correlation test. E. faecalis LTA was subjected to laser irradiation and its structural damage was examined by thin layer chromatography (TLC). Compared with the control groups, all laser groups showed a decreased colony counts and decreased LTA levels with statistically significant difference (p ˂ 0.05). The bactericidal effect and LTA reduction of laser was better at 2.5 W power. Laser at 2.5 W power had temperature rise of more than 7 °C which is beyond the safe thermal threshold level. No statistically significant correlation was found between E. faecalis count, levels of LTA, and rise in external root surface temperature (p ˃ 0.05). TLC results showed a structural damage in the glycolipid moiety of E. faecalis LTA. Diode laser can effectively reduce the E. faecalis count and its LTA levels.

Topics: Chronic Disease; Enterococcus faecalis; Humans; Lasers, Semiconductor; Lipopolysaccharides; Periapical Periodontitis; Teichoic Acids

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.. 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.. 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.. 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.

Topics: Dental Pulp Cavity; Gemella; Humans; Lipopolysaccharides; Periapical Periodontitis; Teichoic Acids

To compare the microbial load and composition and to determine the lipopolysaccharides (LPS) and lipoteichoic acid (LTA) concentrations found in primary apical periodontitis (PAP) and post-treatment apical periodontitis (PTAP), correlating these findings with clinical/tomographic features.. Sixty patients with PAP (31) and PTAP (29) were submitted to clinical and tomographic assessment. Samples were collected from each root canal using paper points for microbiological assessment (culture technique and Checkerboard DNA-DNA hybridization) and determination of LPS and LTA levels (limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively). Data were correlated with clinical/tomographic findings and statistically analyzed using the Mann-Whitney and Pearson correlation tests (α = 5%).. A higher number of cultivable bacteria and LPS were found in PAP (p < 0.05). The median number of species per root canal found in PAP and PTAP was 9 and 22, respectively (p < 0.05). LPS was positively correlated with a larger periapical lesion volume (p < .05). LTA levels were similar in both infections and had no correlation with signs and symptoms. In PAP, gram-positive bacteria were correlated with spontaneous pain (p < .05) and exudate (p < .05). Tenderness to percussion and pain on palpation were correlated to the presence of both gram-positive and negative bacteria. In PTAP, a positive correlation was observed between both gram-positive and gram-negative bacteria with exudate and periapical lesion volume (p < .05).. PAP had higher contents of microbial load and LPS compared with PTAP. However, PTAP presented a more diverse microbiota compared with PAP. Higher content of LPS was positively correlated with larger periapical bone destruction, whereas signs and symptoms with specific microorganisms.. It was verified that PAP and PTAP are polymicrobial infections with predominance of gram-negative bacteria and a more diverse bacterial population found in PTAP. A wide interaction of specific microbial species resulted in different clinical features in both infections.

Topics: Anti-Bacterial Agents; Dental Pulp Cavity; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Lipopolysaccharides; Periapical Periodontitis; Teichoic Acids

Apical periodontitis is caused by biofilm-mediated root canal infection. Early phase oral bacterial biofilms are inhibited by Lactobacillus plantarum lipoteichoic acid (Lp.LTA). However, mature biofilms that develop over 3 weeks are more resistant to traditional endodontic medicaments. Therefore, this study examined the effectiveness of Lp.LTA on disrupting mature Enterococcus faecalis biofilms, and on enhancing the effects of endodontic medicaments. LTA was purified from L. plantarum through butanol extraction followed by hydrophobic and ion-exchange chromatography. E. faecalis biofilms were formed over 3 weeks on glass bottom dishes and in dentin blocks obtained from human single-rooted premolars. These mature biofilms were treated with or without Lp.LTA for 1 h, followed by additional treatment with either chlorhexidine digluconate (CHX), calcium hydroxide (CH), or triple antibiotics for 24 h. Biofilms on glass were live/dead stained and quantified by ZEN through confocal laser microscopy. Bio-films in dentin were fixed, sputter coated and analyzed by ImageJ with scanning electron microscopy. Preformed E. faecalis mature biofilms on the culture dishes were dose-dependently disrupted by Lp.LTA. Lp.LTA potentiated the effects of CHX or CH on the disruption of mature biofilm. Interestingly, CHX-induced disruption of preformed E. faecalis mature biofilms was synergistically enhanced only when pre-treated with Lp.LTA. Furthermore, in the dentin block model, Lp.LTA alone reduced E. faecalis mature biofilm and pre-treatment with Lp.LTA promoted the anti-biofilm activity of CHX. Lp.LTA could be an anti-biofilm or supplementary agent that can be effective for E. faecalis-biofilm-induced diseases.

Topics: Anti-Bacterial Agents; Bicuspid; Biofilms; Calcium Hydroxide; Chlorhexidine; Dentin; Enterococcus faecalis; Gram-Positive Bacterial Infections; Humans; Lactobacillus plantarum; Lipopolysaccharides; Periapical Periodontitis; Teichoic Acids

The aim of this in vivo study was to investigate the microbial profile as well as the levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) at different phases of endodontic treatment in teeth with vital pulp and associated periodontal disease.. Ten patients were selected for this clinical study. Samples were taken from periodontal pockets (PPs) and root canals (RCs) using sterile paper points before and after chemomechanical preparation and after intracanal medication. For microbiological analysis, nested polymerase chain reaction and checkerboard DNA-DNA hybridization were used. Levels of LPS and LTA were assessed using limulus amebocyte lysate and enzyme-linked immunosorbent assays, respectively. Data were statistically analyzed at a significance level of 5%.. Bacterial DNA from 17 of the 17 species investigated was detected in samples of PPs, whereas 6 of the 17 species were not present in the initial samples of RCs using nested polymerase chain reaction. In the initial samples, 38 of 40 probes were detected in PPs, whereas 12 of 40 probes were detected in RCs using checkerboard DNA-DNA hybridization. Overall, endodontic procedures were efficient in modifying the microbiota of PPs and RCs. Levels of LPS and LTA were reduced after the endodontic procedures, although higher concentrations of both had been found in PPs compared with RCs.. The microbiota of PPs and RCs in teeth with vital pulp and associated periodontal disease is polymicrobial, with the presence of gram-negative, gram-positive, facultative, and strict anaerobes. Chemomechanical preparation and calcium hydroxide-based intracanal medication allowed the reduction of infectious content in both sites.

Topics: Dental Pulp Cavity; Dental Pulp Necrosis; Endotoxins; Humans; Lipopolysaccharides; Periapical Periodontitis; Root Canal Irrigants; Teichoic Acids

Apical periodontitis is an inflammatory disease in the periradicular region of teeth that results from infection by multispecies bacterial biofilm residing in the root canal system. In this study, we investigated whether Lactobacillus plantarum lipoteichoic acid (Lp.LTA) could inhibit multispecies oral pathogenic bacterial biofilm formation.. Highly pure and structurally intact Lp.LTA was purified from L. plantarum. Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were co-cultured to form oral multispecies biofilm in the presence or absence of Lp.LTA on culture plates or human dentin slices. Preformed biofilm was treated with or without Lp.LTA, followed by additional treatment with intracanal medicaments such as calcium hydroxide or chlorhexidine digluconate. Confocal microscopy and crystal violet assay were performed to determine biofilm formation. Biofilm on human dentin slices was visualized with a scanning electron microscope.. Biofilm formation of multispecies bacteria on the culture dishes was dose-dependently reduced by Lp.LTA compared with the nontreatment control group. Lp.LTA also inhibited multispecies biofilm formation on the dentin slices in a dose-dependent manner. Interestingly, Lp.LTA was shown to reduce preformed multispecies biofilm compared with the nontreatment group. Moreover, Lp.LTA potentiated the effectiveness of the intracanal medicaments in the removal of preformed multispecies biofilm.. These results suggest that Lp.LTA is a potential anti-biofilm agent for treatment or prevention of oral infectious disease, including apical periodontitis, which is mainly caused by multispecies bacterial biofilm.

Topics: Actinomyces; Biofilms; Calcium Hydroxide; Chlorhexidine; Dentin; Depression, Chemical; Dose-Response Relationship, Drug; Enterococcus faecalis; Humans; Lactobacillus plantarum; Ligilactobacillus salivarius; Lipopolysaccharides; Periapical Periodontitis; Root Canal Irrigants; Teichoic Acids

The infectious content of root canals, including bacteria and lipoteichoic acid (LTA), cause injuries to the periapical tissues. The purpose of this clinical study was to quantify the levels of both LTA and cultivable bacteria at the different phases of endodontic retreatment (ER) of teeth with post-treatment apical periodontitis. It also aimed to investigate the presence of gram-positive microorganisms before and after chemomechanical preparation (CMP) and intracanal medication (ICM).. Twenty infected root canals of single-rooted teeth were randomly assigned into 2 groups according to the chemical substance used for CMP (n = 10 per group): chlorhexidine (CHX) group, 2% CHX gel, and the sodium hypochlorite (NaOCl) group, 6% NaOCl. Root canal samples were taken using paper points before (S1) and after CMP (S2) and after 30 days of ICM with calcium hydroxide + 2% CHX gel (S3). Microorganisms were identified by the culture technique using biochemical tests. Cultivable bacteria were determined by counting the colony-forming unit. LTA levels were measured using the enzyme-linked immunosorbent assay (pg/mL).. A total of 70 gram-positive species, out of 102 species isolated, were found in the root canals (54 in S1, 4 in S2, and 12 in S3). Enterococcus faecalis was the most frequent isolated taxon in all phases of the ER. LTA (574.0 ± 94.7) and cultivable bacteria (101.2 ± 79.2) were present in all S1 samples. CMP decreased the overall levels of cultivable bacteria by 99.4% and LTA by 24.8% (P < .05), whereas the total overall reduction level of ICM on viable bacteria was 99.5% and on LTA it was 38.6% (P < .05). CMP with 2% CHX gel (CHX group, 99.3%) was more effective (P < .05) than 6% NaOCl (NaOCl group, 92.1%) on bacterial reduction. Likewise, ICM showed a 100% reduction in the CHX group and 98.5% in the NaOCl group. Regarding the reduction of LTA, CMP with 2% CHX gel (CHX group, 26.9%) was more effective (P < .05) than 6% NaOCl (NaOCl group, 22.6%). In addition, ICM showed a 43.2% reduction in the CHX group and 36.2% in the NaOCl group (P > .05).. The reduction rates of bacteria were higher than the LTA. Moreover, gram-positive microorganisms were present in all phases of the endodontic retreatment.

Topics: Adult; Bacteria; Chlorhexidine; Dental Pulp Cavity; Enterococcus faecalis; Gram-Positive Bacteria; Humans; Lipopolysaccharides; Middle Aged; Periapical Periodontitis; Random Allocation; Retreatment; Root Canal Irrigants; Root Canal Preparation; Root Canal Therapy; Sodium Hypochlorite; Teichoic Acids

We wished to examine the effects of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome on periapical periodontitis induced by Enterococcus faecalis and to investigate the molecular mechanisms of lipoteichoic acid (LTA) derived from E. faecalis on the expression of the NLRP3 inflammasome.. A model of periapical periodontitis by sealing E. faecalis into the pulp chambers of rats was established. We then examined the relationship between the expression, location, distribution, and concentration of NLRP3, caspase-1, and interleukin 1β with the inflammatory progression by immunohistochemistry and undertook correlation analyses. RAW264.7 cells were cultured in the absence or presence of LTA together with or without nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082; NLRP3 inflammasome expression was measured by Western blotting, the enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction. An immunofluorescence study was conducted to further detect whether NF-κB can be completely inhibited by BAY 11-7082 or activated by LTA.. An animal model of periapical periodontitis was established successfully. Expression of NLRP3, caspase-1, and interleukin 1β protein was observed in the inflamed area. The expression of these 3 proteins had a significant positive correlation (P < .05). Overall, our results showed that, compared with the negative control group, LTA could directly activate expression of messenger RNA and protein of the NLRP3 inflammasome (P < .05), whereas BAY 11-7082 inhibited it (P < .05).. Our results suggested that LTA can act as a directly stimulating factor associated with expression of the NLRP3 inflammasome during periapical periodontitis, which is mainly linked with the NF-κB signaling activation pathway.

Topics: Animals; Disease Models, Animal; Disease Progression; Enterococcus faecalis; Immunohistochemistry; Inflammasomes; Lipopolysaccharides; Male; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Periapical Periodontitis; Phagocytes; Rats; Rats, Sprague-Dawley; Teichoic Acids

Enterococcus faecalis is a pathogenic gram-positive bacterium closely associated with apical periodontitis. Although sodium hypochlorite (NaOCl) has been used as a common endodontic irrigant to eradicate bacteria in the root canal, it has not been elucidated whether NaOCl attenuates the inflammatory response induced by the E. faecalis virulence factor lipoteichoic acid (EfLTA).. Structurally intact EfLTA purified from E. faecalis was treated with NaOCl at various concentrations and time periods. Murine macrophage cell line RAW 264.7 was treated with interferon gamma followed by treatment with intact or NaOCl-treated EfLTA to determine the inducibility of inflammatory mediators such as nitric oxide, interferon gamma-inducible protein 10, and macrophage inflammatory protein-1α. Reporter gene assays assessed by flow cytometry were used to examine the ability of intact or NaOCl-treated EfLTA to activate Toll-like receptor 2 (TLR2), which is known to recognize EfLTA on host cells. Structural damage of EfLTA by NaOCl was examined using silver staining and thin-layer chromatography.. NaOCl-treated EfLTA showed markedly less induction of nitric oxide, interferon gamma-inducible protein 10, and macrophage inflammatory protein-1α in RAW 264.7 cells compared with intact EfLTA. In contrast to intact EfLTA that potently stimulated TLR2 activation, NaOCl-treated EfLTA did not activate TLR2. Structural analysis showed that NaOCl damaged EfLTA structure by deacylation.. NaOCl deacylates the glycolipid moiety of EfLTA, which fails to activate TLR2, leading to the reduced production of inflammatory mediators.

Topics: Animals; Chemokine CCL3; Chemokine CXCL10; CHO Cells; Cricetulus; Enterococcus faecalis; Lipopolysaccharides; Macrophages; Mice; Nitric Oxide; Periapical Periodontitis; RAW 264.7 Cells; Root Canal Irrigants; Sodium Hypochlorite; Teichoic Acids; Toll-Like Receptor 2

Enterococcus faecalis is a frequently isolated microorganism in persistent periapical lesion or secondary infection. However, no evidence has demonstrated that E. faecalis induced inflammation directly in the apical area. This study aimed to explore the mechanism of the inflammatory responses of human periodontal ligament fibroblasts (PDLs) to E. faecalis.. PDLs were stimulated with heat-killed E. faecalis (HKEF) or lipoteichoic acid from E. faecalis (LTA) with or without silencing of tumor necrosis factor receptor-associated factor 6 (TRAF6). The expressions of toll-like receptor 2/4, nucleotide-binding oligomerization domain 1/2, and TRAF6 were detected by using quantitative real-time polymerase chain reaction and Western blot. The secretions of proinflammatory cytokines, including interleukin-1β, interleukin-6, interleukin-8, and tumor necrosis factor-α, were determined in the cell supernatants with enzyme-linked immunosorbent assay.. Both HKEF and LTA stimulated the expression of toll-like receptor 2 and TRAF6 in a time-dependent manner. The secretions of proinflammatory cytokines were also increased. After silencing TRAF6, the upregulations of proinflammatory cytokines induced by HKEF or LTA were attenuated.. TRAF6 plays a pivotal role in inflammation induced by E. faecalis or its LTA in PDLs.

Topics: Cells, Cultured; Enterococcus faecalis; Fibroblasts; Humans; Immunity, Innate; Lipopolysaccharides; Periapical Periodontitis; Periodontal Ligament; Teichoic Acids; TNF Receptor-Associated Factor 6

Enterococcus faecalis, a pathogenic gram-positive bacterium, is closely related to refractory apical periodontitis. Because lipoteichoic acid (LTA) is considered a major virulence factor of gram-positive bacteria, in the present study, highly pure LTA from E. faecalis was prepared, and its ability to stimulate murine macrophages was investigated in comparison with those of the killed whole cells. Upon exposure to E. faecalis LTA, RAW 264.7 (a murine macrophage cell line) produced a significantly (p < 0.05) high level of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in a concentration-dependent manner. It is to note that the LTA was able to stimulate Toll-like receptor 2 (TLR2) but not TLR4. Concomitantly, LTA enhanced the DNA-binding activity of a transcription factor, nuclear factor-kappa B (NF-kappaB), which plays an important role in the transcriptional activation of genes encoding inflammatory mediators. In contrast, heat-killed E. faecalis stimulated both TLR2 and TLR4, whereas the killed E. faecalis whole cells induced significant (p < 0.05) levels of TNF-alpha and NO in RAW 264.7 cells as their LTA did. These results suggest that LTA partially contributes to E. faecalis-induced inflammatory responses.

Topics: Animals; Cell Line; Enterococcus faecalis; Immunity, Innate; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; Nitric Oxide; Periapical Periodontitis; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Virulence Factors