A novel 12-membered ring non-antibiotic macrolide EM982 attenuates cytokine production by inhibiting IKKß and IκBα phosphorylation.

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.

Degradation of EGFR on lung epithelial cells by neutrophil elastase contributes to the aggravation of pneumococcal pneumonia.

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.

Clinical Investigation of the Inhibitory Effects of Tooth-Coating Materials on Initial Active Root Caries: A Pilot Randomized Controlled Trial.

Background and Objectives: Caredyne ZIF-C is a novel, capsule-mixed zinc-containing prototype glass ionomer cement (GIC). Zinc ions are reported to inhibit root dentin demineralization, dentin collagen degradation, bacterial growth, acid production, and in vitro bacterial biofilm formation. However, the effectiveness of GICs against initial root caries lesions is unclear. Therefore, this study aimed to evaluate the efficacy of GICs, especially the new zinc-containing Caredyne ZIF-C GIC, as tooth-coating materials in patients with initial active root caries. Materials and Methods: A total of 58 lesions in 47 older adults (age > 65 years) were randomly allocated to one of the following three groups: Caredyne ZIF-C, Fuji VII (a conventional GIC), and sodium fluoride (NaF). All the lesions were treated with the assigned materials without removing the infected dentin, and the rates of dental plaque attachment and coating material fall-out were evaluated after 3, 6, and 12 months. The failure rate was defined as the number of teeth that needed restoration due to caries progression. Results: The plaque attachment rates tended to be lower in the material-coated root surfaces than in the healthy exposed root surfaces after 3, 6, and 12 months, although the differences among the three groups were not significant. Moreover, the coating material fall-out rate tended to be lower in the Caredyne ZIF-C group than in the Fuji VII group. There was no significant difference in the failure rate among the three groups at the 12 months mark. Conclusions: Though this pilot study offers a new direction for suppressing the progression of initial active root caries by controlling plaque attachment using GICs including Caredyne ZIF-C, clinical studies with a larger sample size are needed.

Analysis of Genetic Relatedness between Gastric and Oral Helicobacter pylori in Patients with Early Gastric Cancer Using Multilocus Sequence Typing.

The oral cavity is the second most colonized site of Helicobacter pylori after the stomach. This study aimed to compare the genetic relatedness between gastric and oral H. pylori in Japanese patients with early gastric cancer through multilocus sequence typing (MLST) analysis using eight housekeeping genes. Gastric biopsy specimens and oral samples were collected from 21 patients with a fecal antigen test positive for H. pylori. The number of H. pylori allelic profiles ranged from zero to eight since the yield of DNA was small even when the nested PCR was performed. MLST analysis revealed that only one patient had a matching oral and gastric H. pylori genotype, suggesting that different genotypes of H. pylori inhabit the oral cavity and gastric mucosa. The phylogenetic analysis showed that oral H. pylori in six patients was similar to gastric H. pylori, implying that the two strains are related but not of the same origin, and those strains may be infected on separate occasions. It is necessary to establish a culture method for oral H. pylori to elucidate whether the oral cavity acts as the source of gastric infection, as our analysis was based on a limited number of allele sequences.

Comparison of calcium and hydroxyl ion release ability and in vivo apatite-forming ability of three bioceramic-containing root canal sealers.

OBJECTIVE: Bioceramic-containing root canal sealers promote periapical healing via Ca2+ and OH- release and apatite formation on the surface. This study aimed to compare Ca2+ and OH- release and in vivo apatite formation of three bioceramic-containing root canal sealers: EndoSequence BC sealer (Endo-BC), MTA Fillapex (MTA-F), and Nishika Canal Sealer BG (N-BG). MATERIALS AND METHODS: Polytetrafluoroethylene tubes filled with sealers were immersed in distilled water for 6 and 12 h and for 1, 7, 14, and 28 days to measure Ca2+ and OH- release. Additionally, tubes filled with sealers were implanted in the backs of rats for 28 days, and in vivo apatite formation was analyzed using an electron probe microanalyzer. RESULTS: Endo-BC released significantly more Ca2+ than the other sealers at 6 and 12 h and 1 day. Ca2+ release was significantly lower from N-BG than from Endo-BC and MTA-F at 14 and 28 days. OH- release was significantly higher from Endo-BC than from the other sealers throughout the experiment, except at 1 day. OH- release was lower from N-BG than from MTA-F at 6 h and 7 days. Only Endo-BC implants exhibited apatite-like calcium-, phosphorus-, oxygen-, and carbon-rich spherulites and apatite layer-like calcium- and phosphorus-rich, but radiopaque element-free, surface regions. CONCLUSIONS: Ca2+ and OH- release is ranked as follows: Endo-BC > MTA-F > N-BG. Only Endo-BC demonstrated in vivo apatite formation. CLINICAL RELEVANCE: Endo-BC could promote faster periapical healing than MTA-F and N-BG.

Effects of rice fermented extracts, "Sake Lees", on the functional activity of odontoblast-like cells (KN-3 cells).

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

An experimental intraradicular biofilm model in the pig for evaluating irrigation techniques.

BACKGROUND: We established an in vivo intraradicular biofilm model of apical periodontitis in pigs in which we compared the efficacy of different irrigant activation techniques for biofilm removal. METHODS: Twenty roots from the deciduous mandibular second premolar of 5 male pigs were used. After pulpectomy, canals were left open for 2 weeks and then sealed for 4 weeks to enable the development of an intracanal biofilm. The intraradicular biofilms was evaluated using SEM and bacterial 16S rRNA gene-sequencing. To investigate the efficacy of biofilm removal, root canal irrigations were performed using conventional needle, passive ultrasonic, subsonic, or laser-activated irrigation. Real-time PCR was conducted to quantitate the remaining biofilm components. Statistical analysis was performed using ANOVA followed by a Tukey kramer post-hoc test with α = 0.05. RESULTS: The pulp exposure model was effective in inducing apical periodontitis and SEM analysis revealed a multi-layer biofilm formation inside the root canal. 16S rRNA sequence analysis identified Firmicutes, Bacteroidetes, and Fusobacteria as the predominant bacterial phyla components, which is similar to the microbiome profile seen in humans. None of the tested irrigation techniques completely eradicated the biofilm components from the root canal, but the subsonic and laser-activated irrigation methods produced the lowest bacterial counts (p < 0.05). CONCLUSIONS: An experimental intraradicular biofilm model has been successfully established in pigs. Within the limitations of the study, subsonic or laser-activated irrigation demonstrated the best biofilm removal results in the pig system.

Sulfated vizantin causes detachment of biofilms composed mainly of the genus Streptococcus without affecting bacterial growth and viability.

BACKGROUND: Sulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva. RESULTS: The biofilm, composed mainly of the genus Streptococcus and containing 50 µM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the Streptococcus species, revealed that a variety of Streptococcus species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus Streptococcus, the expression of luxS in three species, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mutans, was significantly downregulated in the presence of 50 µM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 µM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence. CONCLUSION: Sulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.

Effect of water aging on the anti-biofilm properties of glass ionomer cement containing fluoro-zinc-silicate fillers.

A previous study has reported that a novel fluoro-zinc-silicate glass ionomer cement (Caredyne Restore) showed superior anti-biofilm effects by interfering with bacterial adhesion. However, the active ions may degrade with time. This study aimed to assess the valid anti-biofilm effects of Caredyne Restore after being aged by water immersion for 3 weeks. Streptococcus mutans biofilm was allowed to grow on the surface before and after water aging for 24 h using a modified Robbins device flow-cell system. The results showed water aging promoted biofilm formation. Insufficient amount of fluoride and zinc ions were released from Caredyne Restore after water aging under neutral pH condition. An acidic pH is needed to exert effective anti-biofilm properties. As the release of active ions from Caredyne Restore will gradually decrease after the restoration,  the restoration may not prevent biofilm formation after 3 weeks while neutral pH is maintained by the buffering capacity of saliva.

Effects of a sub-minimum inhibitory concentration of chlorhexidine gluconate on the development of in vitro multi-species biofilms.

Following antimicrobial administrations in oral environments, bacteria become exposed to a sub-minimum inhibitory concentration (sub-MIC), which can induce in vitro single-species biofilms. This study explored the effects of chlorhexidine gluconate (CHG) at a sub-MIC on in vitro multi-species biofilms comprising Streptococcus mutans, Streptococcus oralis and Actinomyces naeslundii. CHG at a sub-MIC was found to induce in vitro biofilm growth, although the bacterial growth was not significantly different from that in the control. The gene transcription related to S. mutans multi-species biofilm formation with CHG at a sub-MIC was significantly higher than that of the control, but this was not found in S. mutans single-species biofilms. The bio-volume of extracellular polysaccharides with CHG at a sub-MIC was significantly higher than that of the control. This suggests that CHG at a sub-MIC may promote the development of multi-species biofilms by affecting the gene transcription related to S. mutans biofilm formation.

Immunohistochemistry and gene expression of GLUT1, RUNX2 and MTOR in reparative dentinogenesis.

OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

Effect of a novel glass ionomer cement containing fluoro-zinc-silicate fillers on biofilm formation and dentin ion incorporation.

OBJECTIVES: This study is aimed at evaluating the effect of a new glass ionomer cement (GIC) containing fluoro-zinc-silicate fillers on biofilm formation and ion incorporation. MATERIALS AND METHODS: Streptococcus mutans biofilms were developed on two GIC materials: Caredyne Restore (CD) and Fuji VII (FJ); and hydroxyapatite (HA) for 24 h at 37 °C using a flow cell system. The morphological structure and bacterial viability were analyzed using a confocal laser scanning microscopy. Bacterial adhesion during the initial 2 h was also assessed by viable cell counting. To study the ion incorporation, restored cavities prepared on the root surfaces of human incisors were subjected to the elemental mapping of the zinc and fluoride ions in the GIC-dentin interface using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. RESULTS: Morphological observations revealed that biofilm formation in the CD group was remarkably inhibited compared with the HA and FJ groups, exhibiting sparse, thinner biofilm clusters. The microorganisms adhering to the CD group were significantly inhibited, revealing 2.9 ± 0.4 for CD, 4.9 ± 0.2 for FJ, and 5.4 ± 0.4 log colony-forming units (CFU) for HA. The CD zinc ion incorporation depth was 72.2 ± 8.0 µm. The fluoride penetration of CD was three times deeper than that of FJ; this difference was statistically significant (p < 0.05). CONCLUSIONS: Enhanced by the incorporation of zinc and fluoride ions, the new GIC inhibited biofilm formation by interfering with bacterial adhesion. CLINICAL RELEVANCE: A novel GIC comprised of fluoro-zinc-silicate fillers may improve clinical outcomes, such as root caries and minimally invasive dentistry.

Adjunct use of mouth rinses with a sonic toothbrush accelerates the detachment of a Streptococcus mutans biofilm: an in vitro study.

BACKGROUND: The aim of this in vitro study was to examine the possible enhancement of the biofilm peeling effect of a sonic toothbrush following the use of an antimicrobial mouth rinse. METHODS: The biofilm at a noncontact site in the interdental area was treated by sound wave convection with the test solution or by immersion in the solution. The biofilm peeling effect was evaluated by determining the bacterial counts and performing morphological observations. A Streptococcus mutans biofilm was allowed to develop on composite resin discs by cultivation with stirring at 50 rpm for 72 h. The specimens were then placed in recesses located between plastic teeth and divided into an immersion group and a combination group. The immersion group was treated with phosphate buffer, chlorhexidine digluconate Peridex™ (CHX) mouth rinse or Listerine® Fresh Mint (EO) mouth rinse. The combination group was treated with CHX or EO and a sonic toothbrush. RESULTS: The biofilm thickness was reduced by approximately one-half compared with the control group. The combination treatment produced a 1 log reduction in the number of bacteria compared to the EO immersion treatment. No significant difference was observed in the biofilm peeling effect of the immersion group compared to the control group. CONCLUSIONS: The combined use of a sonic toothbrush and a mouth rinse enhanced the peeling of the biofilm that proliferates in places that are difficult to reach using mechanical stress.

The effect of chlorhexidine on dental calculus formation: an in vitro study.

BACKGROUND: Chlorhexidine gluconate (CHG) has been proven to be effective in preventing and controlling biofilm formation. At the same time, an increase in calculus formation is known as one of considerable side effects. The purpose of this study was to investigate whether mineral deposition preceding a calculus formation would occur at an early stage after the use of CHG using an in vitro saliva-related biofilm model. METHODS: Biofilms were developed on the MBEC™ device in brain heart infusion (BHI) broth containing 0.5% sucrose at 37 °C for 3 days under anaerobic conditions. Biofilms were periodically exposed to 1 min applications of 0.12% CHG every 12 h and incubated for up to 2 days in BHI containing a calcifying solution. Calcium and phosphate in the biofilm were measured using atomic absorption spectrophotometry and a phosphate assay kit, respectively. Morphological structure was observed using a scanning electron microscope (SEM), and chemical composition was analyzed with an electron probe microanalyzer (EPMA). RESULTS: The concentrations of Ca and Pi following a single exposure to CHG increased significantly compared with the control. Repeatedly exposing biofilms to CHG dose-dependently increased Ca deposition, and the amount of Ca was five times as much as that of the control. Pi levels in CHG-treated biofilms were significantly higher than those from the control group (p < 0.05); however, the influence of the number of exposures was limited. Analyses using an SEM and EPMA showed many clusters containing calcium and phosphate complexes in CHG-treated biofilms. Upon composition analysis of the clusters, calcium was detected at a greater concentration than phosphate. CONCLUSIONS: Findings suggested that CHG may promote mineral uptake into the biofilm soon after its use. It is necessary to disrupt the biofilm prior to the start of a CHG mouthwash in order to reduce the side effects associated with this procedure. The management of patients is also important.

Factors that cause endodontic failures in general practices in Japan.

BACKGROUND: Bacterial biofilms that develop on root surfaces outside apical foramens have been found to be associated with refractory periapical periodontitis. However, several other factors cause endodontic failures apart from extraradicular biofilms. The aim of this study was to identify the factors causing endodontic failures in general practices in Japan. METHODS: Patients diagnosed as having refractory periapical periodontitis by general practitioners and who requested endodontic treatment at Osaka University Dental Hospital were selected by checking medical records from April 2009 to March 2013. Factors causing endodontic failures were identified. RESULTS: A total of 103 teeth were selected, and 76 teeth completed root-canal treatment. Tooth extractions were required for 18 teeth after or without endodontic treatment. Six teeth required apicoectomy after endodontic treatment. One tooth needed hemisection. One tooth needed intentional replantation. One tooth needed adhesion and replantation. The main causes of treatment failure were open apices (24 teeth), perforation (18 teeth), and root fracture (13 teeth). In six teeth with open apices that required apicoectomy or extraction, extraradicular biofilms may have been related to endodontic failure. CONCLUSIONS: Most endodontic cases diagnosed with refractory periapical periodontitis by general practitioners were compromised by any other factors rather than extraradicular biofilms.

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073.

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl ß-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.

Vizantin inhibits bacterial adhesion without affecting bacterial growth and causes Streptococcus mutans biofilm to detach by altering its internal architecture.

An ideal antibiofilm strategy is to control both in the quality and quantity of biofilm while maintaining the benefits derived from resident microflora. Vizantin, a recently developed immunostimulating compound, has also been found to have antibiofilm property. This study evaluated the influence on biofilm formation of Streptococcus mutans in the presence of sulfated vizantin and biofilm development following bacterial adhesion on a hydroxyapatite disc coated with sulfated vizantin. Supplementation with sulfated vizantin up to 50 µM did not affect either bacterial growth or biofilm formation, whereas 50 µM sulfated vizantin caused the biofilm to readily detach from the surface. Sulfated vizantin at the concentration of 50 µM upregulated the expression of the gtfB and gtfC genes, but downregulated the expression of the gtfD gene, suggesting altered architecture in the biofilm. Biofilm development on the surface coated with sulfated vizantin was inhibited depending on the concentration, suggesting prevention from bacterial adhesion. Among eight genes related to bacterial adherence in S. mutans, expression of gtfB and gtfC was significantly upregulated, whereas the expression of gtfD, GbpA and GbpC was downregulated according to the concentration of vizantin, especially with 50 µM vizantin by 0.8-, 0.4-, and 0.4-fold, respectively. These findings suggest that sulfated vizantin may cause structural degradation as a result of changing gene regulation related to bacterial adhesion and glucan production of S. mutans.

Inhibition of polysaccharide synthesis by the sinR orthologue PGN_0088 is indirectly associated with the penetration of Porphyromonas gingivalis biofilms by macrolide antibiotics.

Microbes commonly adhere to surfaces, aggregate in self-produced extracellular polymeric substances (EPS) and live in biofilms. Periodontitis is a serious oral infection that is initiated by the formation of biofilms by Porphyromonas gingivalis. EPS act as a barrier that protects biofilm-forming cells against sources of stress, including those induced by host immune cells and antimicrobial agents. Therefore, drugs intended to kill such micro-organisms cannot be used for the treatment of biofilm infections. Our previous studies revealed that subminimal inhibitory concentrations (subMIC) of two macrolide antibiotics (azithromycin, AZM and erythromycin, ERY) reduced P. gingivalis biofilms. Furthermore, we demonstrated that the Bacillus subtilis sinR orthologue (PGN_0088) inhibits the synthesis of carbohydrates that are components of EPS in P. gingivalis biofilms. Here, we constructed a novel sinR mutant from P. gingivalis ATCC 33277 and reveal that the increased abundance of carbohydrate in EPS of the mutant led to a reduced infiltration rate of AZM and ERY through EPS, and consequently elevated biofilm resistance to these macrolides. Detailed elucidation of the interaction between the product of the sinR gene and EPS will assist in the development of novel approaches that target EPS to prevent and inhibit the formation of biofilms.

Promotion of endodontic lesions in rats by a novel extraradicular biofilm model using obturation materials.

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (P < 0.05 by Welch's t test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.

Antibiofilm Properties and Demineralization Suppression in Early Enamel Lesions Using Dental Coating Materials.

This study aimed to investigate the effects of dental coating materials on Streptococcus mutans biofilm formation. The test materials were PRG Barrier Coat (PRG), BioCoat Ca (BioC), and FluorDental Jelly (FluorJ). Bovine enamel specimens were demineralized to mimic early enamel lesions. The biofilm was developed on a specimen treated with one of the materials by using a modified Robbins device flow-cell system. Scanning electron and fluorescence confocal laser scanning microscopy, viable and total cell counts, and gene expression assessments of the antibiofilm were performed. Ion incorporation was analyzed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyzer. All materials allowed biofilm formation but reduced its volume. FluorJ was the only material that inhibited biofilm accumulation and had a bactericidal effect, revealing 0.66 log CFU in viable cells and 1.23 log copy reduction in total cells compared with the untreated group after 24 h of incubation. The ions released from PRG varied depending on the element. BioC contributed to enamel remineralization by supplying calcium ions while blocking the acid produced from the biofilm. In summary, the dental coating materials physically prevented acid attacks from the biofilm while providing ions to the enamel to improve its mechanical properties.