Current knowledge among Japanese experienced general dentists regarding prevention of infective endocarditis.

Infective endocarditis (IE), a life-threatening condition predominantly occurring in patients with underlying heart disease, is mainly caused by bacteremia induced by invasive dental treatment. However, the amount of related information shared between cardiologists and dentists appears to be inadequate. In the present study, a survey regarding prevention of IE, composed of 13 major questions, 2 of which also allowed free comments, was sent to approximately 3000 dentists belonging to a prefectural dental association in Japan. Of the 13.6% who returned the forms, more than 80% were general dentists with more than 20 years of experience. Approximately, 55% of the responders reported that they had opportunities to prescribe antibiotics prior to performing treatments with risk of IE, though noted difficulties with designation of which patients with heart disease were at risk. Most of the dentists considered that oral surgery procedures have a high risk for IE, whereas less invasive procedures were considered to be not associated with the disease. Approximately, 35% selected oral amoxicillin, with a dose of 2.0 g (20%) or 500 mg (27%) prescribed for adults, and 50 mg (10%) or 30 mg (12%) per kg of body weight for children. However, the timing of the antibiotics administration varied. The present results reveal current knowledge regarding prevention of IE among general dentists in Japan, and should be valuable for construction of a protocol to establish consensus between dentists and cardiologists.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates.

Contribution of the interaction of Streptococcus mutans serotype k strains with fibrinogen to the pathogenicity of infective endocarditis.

Streptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotype k-specific bacterial DNA is frequently detected in S. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE from S. mutans strains, focusing on the characterization of serotype k strains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm(+)/PA(-) group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm(+)/PA(-) strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm(+)/PA(-) strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotype k DNA in S. mutans-positive heart valve clinical specimens.

α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae α-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae α-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that α-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared with untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that α-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that α-enolase increased neutrophil extracellular trap-dependent killing of S. pneumoniae in human blood. Moreover, pulldown assay and mass spectrometry results identified myoblast antigen 24.1D5 as an α-enolase-binding protein on human neutrophils, whereas flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that α-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to α-enolase of S. pneumoniae.

Krüppel-like factor 4 regulates membranous and endochondral ossification.

Krüppel-like factor 4 (KLF4/GKLF/EZF) is a zinc finger type of transcription factor highly expressed in the skin, intestine, testis, lung and bone. The role played by Klf4 has been studied extensively in normal epithelial development and maintenance; however, its role in bone cells is unknown. Previous reports showed that Klf4 is expressed in the developing flat bones but its expression diminishes postnatally. We now show that in the developing long bones, Klf4 is expressed in the perichondrium, trabecular osteoblasts and prehypertrophic chondrocytes. In contrast, osteoblasts lining at the surface of the bone collar showed extremely low levels of Klf4 expression. To investigate the possible roles played by Klf4 during skeletal development, we generated transgenic mice expressing Klf4 under mouse type I collagen regulatory sequence. Transgenic mice exhibited severe skeletal deformities and died soon after birth. Transgenic mice showed delayed formation of the calvarial bones; and over-expressing Klf4 in primary mouse calvarial osteoblasts in culture resulted in strong repression of mineralization indicating that this regulation of Klf4 is through an osteoblast-autonomous effect. Surprisingly, long bones of the transgenic mice exhibited delayed marrow cavity formation. Even at E18.5, the presumptive marrow space was occupied by cartilage anlage and invasion of the vascular endothelial cells and osteoclasts were seldom observed. Instead of entering the cartilage anlage, osteoclasts accumulated at the periosteum in the transgenic mice. Significantly, osteocalcin, which is known to chemotact osteoclasts, was up-regulated at the perichindrium as early as E14.5 in the mutants. In vitro studies showed that this induction of osteocalcin by Klf4 was regulated at its transcriptional level. Our results demonstrate that Klf4 regulates normal skeletal development through coordinating the differentiation and migration of osteoblasts, chondrocytes, vascular endothelial cells and osteoclasts.

[Accuracy estimation of non-invasive X-ray output analyzer].

This study estimated the accuracy of an X-ray analyzer by comparing it with an ionization chamber and a tube voltage current meter, and investigated whether it was usable as a substitute for a reference meter for output measurements for quality control purposes. The X-ray output analyzer used was a Piranha (RTI Electronics), a non-invasive instrument. The two subjects of measurements were as follows: the tube voltage, exposure, and half-value layer used in ordinary X-ray radiographic system equipment, and the exposure and half-value layer in X-ray equipment for mammographic systems. The results for a conventional radiographic system showed the error rates for tube voltage, exposure, and half value layer to be within ±1.0%, ±1.8%, and ±4.3%, respectively. The Piranha is not influenced by the dependence of the beam quality in a range of the tube voltage in clinical use. In X-ray equipment for mammographic system results, error rates for exposure and half value layer were ±2.2% and within ±4.0%, respectively. We conclude that it is possible to use the Piranha as an alternative reference meter for quality control of X-ray equipment for typical radiographic and mammographic systems.

Complete genome sequence of the serotype k Streptococcus mutans strain LJ23.

Streptococcus mutans is the major pathogen of dental caries and occasionally causes infective endocarditis. Here we report the complete genome sequence of serotype k S. mutans strain LJ23, which was recently isolated from the oral cavity of a Japanese patient.

Cellular ATP synthesis mediated by type III sodium-dependent phosphate transporter Pit-1 is critical to chondrogenesis.

Disturbed endochondral ossification in X-linked hypophosphatemia indicates an involvement of P(i) in chondrogenesis. We studied the role of the sodium-dependent P(i) cotransporters (NPT), which are a widely recognized regulator of cellular P(i) homeostasis, and the downstream events in chondrogenesis using Hyp mice, the murine homolog of human X-linked hypophosphatemia. Hyp mice showed reduced apoptosis and mineralization in hypertrophic cartilage. Hyp chondrocytes in culture displayed decreased apoptosis and mineralization compared with WT chondrocytes, whereas glycosaminoglycan synthesis, an early event in chondrogenesis, was not altered. Expression of the type III NPT Pit-1 and P(i) uptake were diminished, and intracellular ATP levels were also reduced in parallel with decreased caspase-9 and caspase-3 activity in Hyp chondrocytes. The competitive NPT inhibitor phosphonoformic acid and ATP synthesis inhibitor 3-bromopyruvate disturbed endochondral ossification with reduced apoptosis in vivo and suppressed apoptosis and mineralization in conjunction with reduced P(i) uptake and ATP synthesis in WT chondrocytes. Overexpression of Pit-1 in Hyp chondrocytes reversed P(i) uptake and ATP synthesis and restored apoptosis and mineralization. Our results suggest that cellular ATP synthesis consequent to P(i) uptake via Pit-1 plays an important role in chondrocyte apoptosis and mineralization, and that chondrogenesis is ATP-dependent.

Trps1 is necessary for normal temporomandibular joint development.

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

Involvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that is closely associated with multiple factors such as obesity, hyperlipidemia and type 2 diabetes mellitus. However, other risk factors for the development of NAFLD are unclear. With the association between periodontal disease and the development of systemic diseases receiving increasing attention recently, we conducted this study to investigate the relationship between NAFLD and infection with Porphyromonas gingivalis (P. gingivalis), a major causative agent of periodontitis. METHODS: The detection frequencies of periodontal bacteria in oral samples collected from 150 biopsy-proven NAFLD patients (102 with non-alcoholic steatohepatitis (NASH) and 48 with non-alcoholic fatty liver (NAFL) patients) and 60 non-NAFLD control subjects were determined. Detection of P. gingivalis and other periodontopathic bacteria were detected by PCR assay. In addition, effect of P. gingivalis-infection on mouse NAFLD model was investigated. To clarify the exact contribution of P. gingivalis-induced periodontitis, non-surgical periodontal treatments were also undertaken for 3 months in 10 NAFLD patients with periodontitis. RESULTS: The detection frequency of P. gingivalis in NAFLD patients was significantly higher than that in the non-NAFLD control subjects (46.7% vs. 21.7%, odds ratio: 3.16). In addition, the detection frequency of P. gingivalis in NASH patients was markedly higher than that in the non-NAFLD subjects (52.0%, odds ratio: 3.91). Most of the P. gingivalis fimbria detected in the NAFLD patients was of invasive genotypes, especially type II (50.0%). Infection of type II P. gingivalis on NAFLD model of mice accelerated the NAFLD progression. The non-surgical periodontal treatments on NAFLD patients carried out for 3 months ameliorated the liver function parameters, such as the serum levels of AST and ALT. CONCLUSIONS: Infection with high-virulence P. gingivalis might be an additional risk factor for the development/progression of NAFLD/NASH.

Establishment of Streptococcus mutans in infants induces decrease in the proportion of salivary α-haemolytic bacteria.

OBJECTIVE: For paediatric dentists, an indicator to assess caries risk of infants is very important. Conventionally, the number and/or proportions of Streptococcus mutans have been employed as risk indicator; however, because such figures reflect the existing situation, they are not suitable for assessing caries risk of infants that have not yet been infected with S. mutans. Thus, we searched for an indicator for the establishment of S. mutans. METHODS: To evaluate the changes caused by the establishment of S. mutans in the microbiota of the infant oral cavity, we monitored changes in the oral microbiota of two pre-dentate infants over a 3-year period and in a cross-sectional study of 40 nursery school-aged children by cultivation of saliva on nonselective blood agar, Mitis-Salivarius agar, and Mitis-Salivarius agar supplemented with bacitracin combined with identification of selected isolates. RESULTS: Two longitudinal observations suggested that the establishment of S. mutans would induce a decrease in α-haemolytic bacteria in the microbial population of the oral cavity. This suggestion was compensated with the results of cross-sectional study, and it was revealed that the establishment of 10(3) CFU/mL of mutans streptococci in saliva might be predicted by a microbiota comprising less than approximately 55% of α-haemolytic. CONCLUSION: Decrease in the proportion of α-haemolytic bacteria in saliva of infant was found to be applicable as an indicator to predict the establishment of S. mutans and to assess dental caries risk as a background for planning of dental care and treatment in the infants before infection with S. mutans.

Specific behavior of intracellular Streptococcus pyogenes that has undergone autophagic degradation is associated with bacterial streptolysin O and host small G proteins Rab5 and Rab7.

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Pili of oral Streptococcus sanguinis bind to salivary amylase and promote the biofilm formation.

Streptococcus sanguinis is a member of oral streptococci and one of the most abundant species found in oral biofilm called dental plaque. Colonization of the oral streptococci on the tooth surface depends on the adhesion of bacteria to salivary components adsorbed to the tooth surface. Recently, we identified unique cell surface long filamentous structures named pili in this species. Herein, we investigated the role of S. sanguinis pili in biofilm formation. We found that pili-deficient mutant, in which the genes encoding the three pilus proteins PilA, PilB and PilC have been deleted, showed an impaired bacterial accumulation on saliva-coated surfaces. Confocal microscopic observations suggested that the mutant was incapable of producing typical three-dimensional layer of biofilm. Ligand blot analysis showed that the ancillary pilus proteins PilB and PilC bound to human whole saliva. Additional analysis demonstrated that PilC bound to multiple salivary components, and one of which was found to be salivary α-amylase. These results indicate that pilus proteins are members of saliva-binding proteins of oral S. sanguinis, and suggest the involvement of pili in its colonization on saliva-coated tooth surfaces and in the human oral cavity.

Isolation and characterization of Streptococcus mitis from blood of child with osteomyelitis.

OBJECTIVES: Osteomyelitis is an inflammatory process accompanied by bone destruction that is caused by bacterial infection, with most child cases showing a haematogenous origin and metaphysis of the long bones. The aim of the present study was to characterize streptococcal strains isolated from the blood of a child diagnosed with osteomyelitis in a long bone and investigate the biological properties related to virulence of strains associated with osteomyelitis. METHODS: Blood isolate species were determined based on the 16S rRNA sequence. Next, the blood isolates were analysed for phagocytosis susceptibility by polymorphonuclear leukocytes, platelet aggregation, inhibitory effects on osteoblastic cells, and their properties of adhesion with cells, and compared to the reference strain Streptococcus mitis ATCC49456. RESULTS: The blood isolates were found to be a single clone (named SA1101), which was determined to be S. mitis. The phagocytosis susceptibility of SA1101 was significantly lower than that of ATCC49456, while its platelet aggregation rate was higher. Furthermore, SA1101 showed an inhibitory effect toward the growth of osteoblastic cells and had greater properties of adhesion to those cells as compared to ATCC49456. CONCLUSIONS: These results suggest that S. mitis SA1101 is a possible etiological agent and caused osteomyelitis in the present case.

Successful application of atelocollagen for treatment of perforated teeth.

OBJECTIVE: Cervical or furcal root perforation is a serious clinical problem and one of its treatment modalities is perforation repair with composite resin. However, many cases still progress in inevitable extraction. When primary teeth are affected, early tooth loss can cause problems related to the eruption space for the permanent successors. The aim of the present study was to evaluate a novel clinical treatment method for perforated teeth. STUDY DESIGN: Atelocollagen was applied to perforated furcal and cervical areas of 13 primary teeth in 13 children aged 4-9 years and 8 permanent teeth in 8 adults aged 35-69 years after debridement with an electric knife. Thereafter the final restorations were performed after confirming good tooth conditions. Clinical evaluations were performed at follow-up examinations at approximately 3-month intervals. RESULTS: None of the treated primary teeth showed any clinical problems throughout the observation period, with eruption of the permanent successors noted in 7 cases. In the permanent teeth, no clinical problems were identified in any of the cases during follow-up periods of 10-60 months. CONCLUSION: This novel method may enable preservation of perforated primary teeth for a longer duration.

Molecular detection of human periodontal pathogens in oral swab specimens from dogs in Japan.

Periodontal diseases are known to be major diseases in humans, and are also common in dogs. The purpose of the present study was to analyze the distribution of periodontitis-related bacterial species using oral swab specimens collected from 26 pet dogs. The distribution of an animal gingival organism Porphyromonas gulae, in addition to 10 human periodontitis-related bacterial species, including Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens, were evaluated by polymerase chain reaction with species-specific sets of primers. Porphyromonas gulae, Tannerella forsythia and Campylobacter rectus were detected in almost all dogs analyzed, all of which should be regarded as common members of oral flora in dogs. Then, isolation and identification of the Porphyromonas species in swab specimens were performed. There were 35 strains isolated from 22 dogs, and broad-range polymerase chain reaction and sequencing methods revealed that approximately 70% of them were Porphyromonas gulae. In contrast, the frequency of Porphyromonas gingivalis was extremely low. These findings indicate the presence of specific periodontitis-related pathogens in pet dogs, especially Porphyromonas gulae.

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion.

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Cell-surface structures of novel serotype k Streptococcus mutans strains and their correlation to virulence.

Streptococcus mutans is generally known as a pathogen of dental caries, and it is also considered to cause bacteremia and infective endocarditis (IE). S. mutans was previously classified into 3 serotypes, c, e, and f, due to the different chemical compositions of the serotype-specific polysaccharides, which are composed of a rhamnose backbone and glucose side chains. We recently designated non-c/e/f serotype S. mutans strains as novel serotype k, which is characterized by a drastic reduction in the amount of the glucose side chain. A common biological feature of novel serotype-k strains is a lower level of cariogenicity due to alterations of several major cell surface protein antigens. As for virulence in blood, these strains survive in blood for a longer duration due to lower antigenicity, while the detection rate of all strains carrying the gene encoding collagen-binding adhesin has been shown to be high. Furthermore, molecular biological analyses of infected heart valve specimens obtained from IE patients revealed a high detection rate of serotype-k S. mutans. Together, these findings suggest that serotype-k S. mutans strains show low cariogenicity but high virulence in blood as compared to the other serotypes, due to alterations of several cell surface structures.

Detection of oral streptococci with collagen-binding properties in saliva specimens from mothers and their children.

BACKGROUND: Approximately 10-20% of Streptococcus mutans strains have been reported to possess collagen-binding properties, whereas other species in the oral cavity with those properties remain to be elucidated. Aim. To identify strains with collagen-binding properties and analyse their characteristics in comparison with S. mutans. DESIGN: A total of 110 expectorated saliva specimens were collected from 55 pairs of mothers and their children. Bacterial strains with collagen-binding properties were isolated and the species specified. In addition, strains with collagen-binding properties isolated from mother-child pairs were analysed using molecular biological approaches. RESULTS: The detection frequency of strains with collagen-binding properties was shown to be 40.9%, among which S. salivarius was the most frequently detected, followed by S. mutans. The collagen-binding activity of the S. mutans group was the highest, followed by S. salivarius. In addition, S. mutans and S. salivarius strains from 3 and 1 mother-child pairs, respectively, were shown to be the same clones. CONCLUSIONS: Our results indicate that S. mutans and S. salivarius are major species with collagen-binding properties in the oral cavity, and that strains with such properties may be related to mother-child transmission.

[Genetic basis for skeletal disease. Dental management of patients with bone diseases].

Malformation of teeth can be found in patients with bone diseases, which was induced when the disease occurred during the tooth formation. The tooth malformation shows typical manifestations of the disease, which may demonstrate the occurrence of the bone disease. In this article, dental management of the patients with bone diseases such as X-linked hypophosphatemic rickets, osteogenesis imperfecta, and hypophosphatasia was presented.