An extensive description of the microbiological effects of silver diamine fluoride on dental biofilms using an oral in situ model.

Silver diamine fluoride (SDF) has been long studied in laboratories, and its clinical effectiveness in the treatment and prevention of root caries has been reported. In the present study, we assessed the microbiological effects of SDF on dental biofilms grown on demineralized dentin in situ. Specifically, demineralized bovine root dentin slabs used as biofilm substrates were treated with 38% SDF, and the biofilms formed after this treatment were analyzed via real-time PCR, DEAD/LIVE cell staining, and SEM. Next, the viable cell count was determined, and microbial profiles were compared using 16S rRNA gene sequencing. Untreated slabs were used as controls. We observed significant decreases in viable cell counts (p < 0.05), number of biofilm-forming cells (p < 0.01), biofilm thickness (p < 0.01), and high proportion of dead cells with SDF treatment (p < 0.01). The microcolonies in the SDF-treated biofilms showed less complexity, and only a limited number of genera were differentially abundant between the groups. Microbial diversity index comparisons showed no significant differences between the groups with respect to treatments days (p = 0.362). Thus, SDF negatively influenced dental biofilm growth on demineralized root dentin in situ; however, its antimicrobial action did not target a specific oral taxon.

Impact of sleep on the microbiome of oral biofilms.

Dysbiosis of the oral microbiome is associated with diseases such as periodontitis and dental caries. Because the bacterial counts in saliva increase markedly during sleep, it is broadly accepted that the mouth should be cleaned before sleep to help prevent these diseases. However, this practice does not consider oral biofilms, including the dental biofilm. This study aimed to investigate sleep-related changes in the microbiome of oral biofilms by using 16S rRNA gene sequence analysis. Two experimental schedules-post-sleep and pre-sleep biofilm collection-were applied to 10 healthy subjects. Subjects had their teeth and oral mucosa professionally cleaned 7 days and 24 h before sample collection. Samples were collected from several locations in the oral cavity: the buccal mucosa, hard palate, tongue dorsum, gingival mucosa, tooth surface, and saliva. Prevotella and Corynebacterium had higher relative abundance on awakening than before sleep in all locations of the oral cavity, whereas fluctuations in Rothia levels differed depending on location. The microbiome in different locations in the oral cavity is affected by sleep, and changes in the microbiome composition depend on characteristics of the surfaces on which oral biofilms form.

Duration of dentin tubule occlusion by the calcium phosphate precipitation method: An in vivo study in beagle dogs.

The aim of the present study was to assess the duration of dentin tubule occlusion by the calcium phosphate precipitation (CPP) method in the vital teeth of beagle dogs. Vital teeth were treated using the CPP method, potassium oxalate, or a bonding agent (Liner bond II) after cavity preparation and acid etching. The dentin tubules of all groups, except for the bonding agent, opened more widely with time in the absence of plaque control. Dentin tubules treated with the CPP method were open and no precipitate remained in the absence of plaque control. Differences were observed in dentin tubule occlusion when plaque control was achieved by daily tooth brushing. The majority of dentin tubules were occluded with an apatitic precipitate seven days after the CPP method with plaque control. The present results demonstrated that the CPP method is useful with proper plaque control.

Impacts of sleep on the characteristics of dental biofilm.

Dental biofilm present on the tooth surface is associated with oral diseases, such as dental caries and periodontal disease. Because bacterial numbers rapidly increase in saliva during sleep, oral care before sleeping is recommended for the prevention of chronic oral diseases. However, temporal circadian changes in the quantity and quality of dental biofilms are poorly understood. This study aimed to investigate the impacts of sleeping on dental biofilm amounts and compositions by using an in situ model. The use of this in situ model enabled us to investigate dental biofilm formed in the oral cavity and to perform a quantitative analysis. Subjects began wearing oral splints in the morning or before sleeping, and biofilm samples were collected at 8, 16, and 24 h after the subjects began wearing oral splints; these samples were then used in various experiments. No significant changes in the numbers of biofilm-forming bacteria were caused by sleep. However, the relative abundances of genera related to periodontitis (i.e., Fusobacterium and Prevotella) increased after awakening. In conclusion, the numbers of biofilm-forming bacteria were not affected by sleep, and the abundances of obligate anaerobes increased after sleep. This research may aid in defining efficacious preventive oral care.

Next-Generation Sequencing for Determining the Effect of Arginine on Human Dental Biofilms Using an In Situ Model.

Oral biofilms are associated with caries, periodontal diseases, and systemic diseases. Generally, antimicrobial therapy is used as the first line of treatment for infectious diseases; however, bacteria in biofilms eventually develop antibiotic resistance. This study aimed to apply our in situ biofilm model to verify whether an arginine preparation is useful for plaque control. Ten healthy subjects who did not show signs of caries, gingivitis, or periodontitis were recruited. The dental biofilms from the subjects were obtained using our oral device before and after gargling with arginine solution for 4 weeks. We found that 8% arginine solution significantly increased the concentration of ammonium ions (NH4 +) in vitro and in vivo in saliva (p < 0.05) and decreased the proportions of the genera Atopobium and Catonella in vivo. However, the viable count was unaffected by the mouthwash. Further, oral populations of the genera Streptococcus and Neisseria tended to increase with the use of arginine. Therefore, we concluded that using an 8% arginine solution decreased the NH4 + concentration in the oral cavity without affecting the number of viable bacteria, and that the diversity of oral bacterial flora changed. We suggest that arginine might help prevent mature biofilm formation.

Assessment of the functional efficacy of root canal treatment with high-frequency waves in rats.

The purpose of this study was to develop a high-frequency wave therapy model in rats and to investigate the influence of high-frequency waves on root canal treatment, which may provide a novel strategy for treating apical periodontitis. Root canal treatments with and without high-frequency wave irradiation were performed on the mandibular first molars of 10-week-old male Wistar rats. The mesial roots were evaluated radiologically, bacteriologically, and immunohistochemically. At 3 weeks after root canal treatment, lesion volume had decreased significantly more in the irradiated group than in the non-irradiated group, indicating successful development of the high-frequency therapy model. The use of high-frequency waves provided no additional bactericidal effect after root canal treatment. However, high-frequency wave irradiation was found to promote healing of periapical lesions on the host side through increased expression of fibroblast growth factor 2 and transforming growth factor-ß1 and could therefore be useful as an adjuvant nonsurgical treatment for apical periodontitis.

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.

Development of a root canal treatment model in the rat.

Root canal treatment is performed to treat apical periodontitis, and various procedures and techniques are currently used. Although animal models have been used in the developmental research of root canal treatment, little of this research has used small animals such as rats, because of their small size. In this study, root canal treatment was performed on the rat mandibular first molar, which had four root canals, using a microscope, and the therapeutic effect was evaluated bacteriologically, radiologically and histopathologically. By performing root canal treatment, the level of bacteria in the mesial root of the treated teeth was reduced by 75% compared with the control. Additionally, the volume of the periapical lesions of the treated teeth as measured by micro-computed tomography decreased significantly 2 weeks after the root canal treatment when compared with the control. Histological evidence of healing was observed in the treatment group 8 weeks after root canal treatment. These results suggest that a root canal treatment model using rats can be used in developmental research for novel methods of root canal treatment.

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.

Statistical Analysis for Subjective and Objective Evaluations of Dental Drill Sounds.

The sound produced by a dental air turbine handpiece (dental drill) can markedly influence the sound environment in a dental clinic. Indeed, many patients report that the sound of a dental drill elicits an unpleasant feeling. Although several manufacturers have attempted to reduce the sound pressure levels produced by dental drills during idling based on ISO 14457, the sound emitted by such drills under active drilling conditions may negatively influence the dental clinic sound environment. The physical metrics related to the unpleasant impressions associated with dental drill sounds have not been determined. In the present study, psychological measurements of dental drill sounds were conducted with the aim of facilitating improvement of the sound environment at dental clinics. Specifically, we examined the impressions elicited by the sounds of 12 types of dental drills in idling and drilling conditions using a semantic differential. The analysis revealed that the impressions of dental drill sounds varied considerably between idling and drilling conditions and among the examined drills. This finding suggests that measuring the sound of a dental drill in idling conditions alone may be insufficient for evaluating the effects of the sound. We related the results of the psychological evaluations to those of measurements of the physical metrics of equivalent continuous A-weighted sound pressure levels (LAeq) and sharpness. Factor analysis indicated that impressions of the dental drill sounds consisted of two factors: "metallic and unpleasant" and "powerful". LAeq had a strong relationship with "powerful impression", calculated sharpness was positively related to "metallic impression", and "unpleasant impression" was predicted by the combination of both LAeq and calculated sharpness. The present analyses indicate that, in addition to a reduction in sound pressure level, refining the frequency components of dental drill sounds is important for creating a comfortable sound environment in dental clinics.

Temporal dynamics of bacterial microbiota in the human oral cavity determined using an in situ model of dental biofilms.

Numerous studies on oral biofilms have been performed in vitro, although it is difficult to mimic the oral environment. Here we used an in situ model to conduct a quantitative analysis and comprehensive identification of bacterial communities over time by performing deep sequencing of 16S rRNA genes. We show here that the number of viable bacteria in supragingival biofilms increased in two steps. Using scanning and transmission electron microscopy, as well as confocal laser scanning microscopy, we detected gram-positive cocci during the first 8 h. The biofilm was subsequently covered with a thick matrix-like structure composed of different bacterial morphotypes that diversified as the number of bacteria increased. Streptococcus accounted for >20% of the population until 16 h, and obligate anaerobes such as Fusobacterium, Prevotella and Porphyromonas predominated after 48 h, and this increase was statistically significant after 96 h (P<0.05). Together, our data demonstrate that an initial population of facultative anaerobic bacteria was replaced with a population of gram-negative anaerobic bacteria during oral biofilm formation. This study, therefore, contributes to a comprehensive understanding of the composition of the bacterial microbiota involved in the health of the human oral cavity.

RabGDIα is a negative regulator of interferon-γ-inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii.

IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIß, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.

Simple observation of Streptococcus mutans biofilm by scanning electron microscopy using ionic liquids.

Scanning electron microscopy (SEM) has been successfully used to image biofilms because of its high resolution and magnification. However, conventional SEM requires dehydration and metal coating of biological samples before observation, and because biofilms consist mainly of water, sample dehydration may influence the biofilm structure. When coated with an ionic liquid, which is a kind of salt that exists in the liquid state at room temperature, biological samples for SEM observation do not require dehydration or metal coating because ionic liquids do not evaporate under vacuum conditions and are electrically conductive. This study investigates the ability of ionic liquids to allow SEM observation of Streptococcus mutans biofilms compared with conventional coating methods. Two hydrophilic and two hydrophobic ionic liquids, all of which are electronic conductors, are used. Compared with samples prepared by the conventional method, the ionic-liquid-treated samples do not exhibit a fibrous extracellular matrix structure and cracking on the biofilm surface. The hydrophilic ionic liquids give clearer images of the biofilm structure than those of the hydrophobic ionic liquids. This study finds that ionic liquids are useful for allowing the observation of biofilms by SEM without preparation by dehydration and metal coating.

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.

Role of mouse and human autophagy proteins in IFN-γ-induced cell-autonomous responses against Toxoplasma gondii.

IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti-T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-γ-inducible GTPases. IFN-γ-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ-induced recruitment of the immunity-related GTPases to the intracellular pathogen.

The periodontopathogenic bacterium Eikenella corrodens produces an autoinducer-2-inactivating enzyme.

Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)-furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.

LuxS affects biofilm maturation and detachment of the periodontopathogenic bacterium Eikenella corrodens.

Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens.

Porphyromonas gingivalis biofilms persist after chlorhexidine treatment.

Chlorhexidine (CHX) gluconate effectively reduces the viability of biofilm-forming bacteria, such as Porphyromonas gingivalis. However, it is impossible to completely remove biofilms. The goal of the present study was to assess the potential pathogenicity of residual P. gingivalis biofilms in vitro after treatment with CHX gluconate. Scanning and transmission electron microscopy and confocal laser imaging revealed that treatment with CHX gluconate disrupted individual biofilm-forming P. gingivalis cells but did not destroy the biofilms. The volumes of the protein and carbohydrate constituents in the residual biofilms were not significantly different from those of the controls. The physical resistance of the residual biofilms to ultrasonication was significantly higher than that of controls. The volume of P. gingivalis adherent to the residual biofilms was higher than that to saliva-coated wells. These findings suggest that although CHX gluconate caused disruption of biofilm-forming cells, the constituents derived from disrupted cells were maintained in the biofilms, which sustained their external structures. Moreover, the residual biofilms could serve as a scaffold for the formation of new biofilms.

The sinR ortholog PGN_0088 encodes a transcriptional regulator that inhibits polysaccharide synthesis in Porphyromonas gingivalis ATCC 33277 biofilms.

Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.