Gene therapy improves dental manifestations in hypophosphatasia model mice.

BACKGROUND AND OBJECTIVE: Hypophosphatasia is a rare inherited skeletal disorder characterized by defective bone mineralization and deficiency of tissue non-specific alkaline phosphatase (TNSALP) activity. The disease is caused by mutations in the liver/bone/kidney alkaline phosphatase gene (ALPL) encoding TNSALP. Early exfoliation of primary teeth owing to disturbed cementum formation, periodontal ligament weakness and alveolar bone resorption are major complications encountered in oral findings, and discovery of early loss of primary teeth in a dental examination often leads to early diagnosis of hypophosphatasia. Although there are no known fundamental treatments or effective dental approaches to prevent early exfoliation of primary teeth in affected patients, several possible treatments have recently been described, including gene therapy. Gene therapy has also been applied to TNSALP knockout mice (Alpl-/- ), which phenocopy the infantile form of hypophosphatasia, and improved their systemic condition. In the present study, we investigated whether gene therapy improved the dental condition of Alpl-/- mice. MATERIAL AND METHODS: Following sublethal irradiation (4 Gy) at the age of 2 d, Alpl-/- mice underwent gene therapy using bone marrow cells transduced with a lentiviral vector expressing a bone-targeted form of TNSALP injected into the jugular vein (n = 3). Wild-type (Alpl+/+ ), heterozygous mice (Alpl+/- ) and Alpl-/- mice were analyzed at 9 d of age (n = 3 of each), while Alpl+/+ mice and treated or untreated Alpl-/- mice were analyzed at 1 mo of age (n = 3 of each), and Alpl+/- mice and Alpl-/- mice with gene therapy were analyzed at 3 mo of age (n = 3 of each). A single mandibular hemi-section obtained at 1 mo of age was analyzed using a small animal computed tomography machine to assess alveolar bone formation. Other mandibular hemi-sections obtained at 9 d, 1 mo and 3 mo of age were subjected to hematoxylin and eosin staining and immunohistochemical analysis of osteopontin, a marker of cementum. RESULTS: Immunohistochemical analysis of osteopontin, a marker of acellular cementum, revealed that Alpl-/- mice displayed impaired formation of cementum and alveolar bone, similar to the human dental phenotype. Cementum formation was clearly present in Alpl-/- mice that underwent gene therapy, but did not recover to the same level as that in wild-type (Alpl+/+ ) mice. Micro-computed tomography examination showed that gene therapy improved alveolar bone mineral density in Alpl-/- mice to a similar level to that in Alpl+/+ mice. CONCLUSIONS: Our results suggest that gene therapy can improve the general condition of Alpl-/- mice, and induce significant alveolar bone formation and moderate improvement of cementum formation, which may contribute to inhibition of early spontaneous tooth exfoliation.

A specific Streptococcus mutans strain aggravates non-alcoholic fatty liver disease.

OBJECTIVE: Streptococcus mutans, a major dental caries pathogen, has shown to be associated with the aggravation of cerebral hemorrhage and inflammatory bowel diseases. In this study, we evaluated the effects ofS. mutans on the development of non-alcoholic steatohepatitis (NASH) in a mouse model. MATERIALS AND METHODS: Streptococcus mutans oral strain MT8148 (serotype c) and a blood isolate TW871 (k) were used. C57BL/6J mice (6 weeks old)were fed a high-fat diet for 4 weeks; the test strains or phosphate-buffered saline was then intravenously administered. Mice were euthanized after 8 or 12 weeks. Whole body, extirpated liver, and visceral fat weights were determined, and histopathological evaluations of the liver specimens were performed. RESULTS: Mice infected with TW871 showed significantly greater body and liver weights than those administered MT8148 or phosphate-buffered saline. Histopathological analyses revealed prominent infiltration of inflammatory cells and adipocellular deposition in livers extirpated 8 weeks after an infection with TW871; fibrosis was also observed in livers extirpated after 12 weeks. CONCLUSION: These results suggest that a specific strain of S. mutans could induce NASH.

Aggravation of inflammatory bowel diseases by oral streptococci.

OBJECTIVES: Streptococcus mutans can aggravate colitis in mice. We evaluated the virulence of colitis using type strains as well as blood isolates of several oral streptococcal species. MATERIALS AND METHODS: We investigated the susceptibility of blood isolates of several oral streptococci to phagocytosis, adhesion to and invasion of hepatic cells and interferon-γ secretion. A mouse model of dextran sodium sulphate-induced colitis was used to evaluate bacterial aggravation of colitis. In addition, interferon-γ antibody was administered to mice with prominent aggravation of colitis. RESULTS: In vitro analyses showed that Streptococcus sanguinis ATCC 10556 was a possible virulent strain among type strains of several oral streptococci, and that analysis of blood isolates of S. sanguinis TW289 revealed a potential virulent strain. Intravenous administration of ATCC 10556 and TW289 caused prominent aggravation of dextran sodium sulphate-induced colitis, and histopathological examinations showed that interferon-γ secretion due to infection of hepatic cells caused colitis aggravation. Administration of interferon-γ antibody suppressed TW289-induced colitis. CONCLUSION: These results suggest that some virulent oral streptococcal strains are associated with the aggravation of colitis induced by enhanced secretion of interferon-γ when they invade the bloodstream.

Potential high virulence for infective endocarditis in Streptococcus mutans strains with collagen-binding proteins but lacking PA expression.

OBJECTIVE: Streptococcus mutans, an aetiologic agent of dental caries, is a pathogen for infective endocarditis (IE). We investigated strains that express collagen-binding proteins (CBPs) with further classification based on expression of the 190-kDa protein antigen (PA). METHOD: Zeta-potential values of strains TW871 (CBP+/PA+) and MT8148 (CBP-/PA+), and their respective PA-defective mutant strains TW871PD (CBP+/PA-) and MT8148PD (CBP-/PA-), were analysed, as were their adhesion to and invasion of human umbilical vein endothelial cells (HUVECs). The distribution of strains from the oral cavities of 200 healthy individuals was analysed for CBP and/or PA expression and the strains were characterised for their adhesion and invasion properties. RESULTS: TW871PD and MT8148PD showed significantly lower zeta-potential values than TW871 and MT8148, respectively. Collagen-binding rates were significantly higher for TW871PD than for TW871 but nearly negligible for MT8148 and MT8148PD. The adhesion and invasion rates of HUVECs were significantly higher for TW871PD than for TW871 and significantly higher for TW871 than for MT8148 and MT8148PD. The prevalence of CBP+ strains was ~10% and ~3% in the case of CBP+/PA- strains. Analyses of 200 clinical strains showed the CBP+/PA- group to have higher adhesion and invasion rates than other groups. CONCLUSIONS: CBP+/PA- S. mutans strains, despite their low distribution frequency, may be highly virulent for infective endocarditis.

Potential involvement of collagen-binding proteins of Streptococcus mutans in infective endocarditis.

OBJECTIVE: Streptococcus mutans, a major pathogen of dental caries, is considered to be one of the causative agents of infective endocarditis (IE). Two types of cell surface collagen-binding proteins, Cnm and Cbm, have been identified in the organism. The aim of the present study was to analyze these proteins as possible etiologic factors for IE. MATERIALS AND METHODS: The binding activities of S. mutans strains to collagen types I, III, and IV were analyzed relative to the presence of Cnm and Cbm, as were their adhesion and invasion properties with human umbilical vein endothelial cells (HUVEC). In addition, distributions of the genes encoding Cnm and Cbm in S. mutans-positive heart valve specimens extirpated from IE and non-IE patients were analyzed by PCR. RESULTS: Most of the Cbm-positive strains showed higher levels of binding to type I collagen as well as higher rates of adhesion and invasion with HUVEC as compared to the Cnm-positive strains. Furthermore, the gene encoding Cbm was detected significantly more frequently in heart valve specimens from IE patients than from non-IE patients. CONCLUSIONS: These results suggest that the collagen-binding protein Cbm of S. mutans may be one of the potential important factor associated with the pathogenesis of IE.

Identification and characterization of a collagen-binding protein, Cbm, in Streptococcus mutans.

Streptococcus mutans, a major pathogen of dental caries, is occasionally isolated from the blood of patients with infective endocarditis. Bacterial attachment of exposed collagen tissue in the impaired endothelium is an important step in the onset of infective endocarditis. In our previous studies, some S. mutans strains were shown to possess collagen-binding activities and most of them had an approximately 120-kDa cell-surface collagen-binding protein called Cnm. However, several strains without Cnm proteins show collagen-binding properties. In the present study, another collagen-binding protein, Cbm, was characterized and its coding gene cbm was sequenced in these strains. The amino acid alignment in the putative collagen-binding domain of Cbm was shown to have approximately 80% identity and 90% similarity to the comparable region of Cnm. Cbm-deficient isogenic mutant strains constructed by insertional inactivation of the cbm gene, lacked collagen-binding properties, which were recovered in the complemented mutant. Analyses of a large number of clinical isolates from Japan, Thailand and Finland revealed that cbm-positive strains were present in all of these countries and that cnm-positive and cbm-positive strains were detected in the oral cavity of approximately 10 and 2% of systemically healthy subjects, respectively. In addition, cnm-positive strains were predominantly identified in the serotype f group, whereas cbm-positive strains were frequently detected in serotype k. These results suggest that Cbm as well as Cnm are major cell surface proteins of S. mutans associated with binding to type I collagen and predominantly identified in serotype k strains.

Distribution of periodontopathic bacterial species in dogs and their owners.

OBJECTIVE: Presently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners. DESIGN: Dental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis, Porphyromonas gulae, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens were performed. RESULTS: P. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them. CONCLUSIONS: These results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.

Inhibitory effects of Oenothera biennis (evening primrose) seed extract on Streptococcus mutans and S. mutans-induced dental caries in rats.

BACKGROUND: Oenothera biennis (evening primrose) seed extract (OBSE) is known to contain polyphenols, which may possess antioxidant activities. Polyphenols extracted from several plants are reported to exhibit cariostatic activities by inhibiting mutans streptococcus growth and glucosyltransferase activities. The purpose of the present study was to examine the inhibitory effects of OBSE on the development of dental caries, both in vitro and in vivo. METHODS: OBSE was investigated for its inhibitory effects on cellular aggregation, hydrophobicity, sucrose-dependent adherence and insoluble glucan synthesis. Furthermore, biofilm formation was examined in the presence of OBSE, using confocal microscopic imaging. An animal experiment was also performed to examine the in vivo effects. RESULTS: OBSE induced a strong aggregation of Streptococcus mutans MT8148 cells, while cell surface hydrophobicity was decreased by approximately 90% at a concentration of 0.25 mg/ml. The sucrose-dependent adherence of the MT8148 cells was also reduced by addition of OBSE, with a reduction rate of 73% seen at a concentration of 1.00 mg/ml. Additionally, confocal microscopic observations revealed the biofilm development phase to be remarkably changed in the presence of OBSE. Furthermore, insoluble glucan synthesis was significantly reduced when OBSE was present at concentrations greater than 0.03 mg/ml. In an animal experiment, the caries scores in rats given OBSE (0.05 mg/ml in drinking water) were significantly lower than those in rats given water without OBSE. CONCLUSION: Our results indicate that OBSE has inhibitory activity on dental caries.

Characterization of aortic aneurysms in cardiovascular disease patients harboring Porphyromonas gingivalis.

OBJECTIVE: Porphyromonas gingivalis was recently shown to cause intimal hyperplasia in a mouse model by a novel cholesterol-independent mechanism, suggesting to be a pathogen-specific feature of cardiovascular diseases. The aim of this study was to characterize the clinical and histopathological features of aortic aneurysms in cardiovascular disease patients harboring oral P. gingivalis. SUBJECT AND METHODS: Aortic aneurysm specimens were collected from 76 Japanese patients who underwent surgery, of whom dental plaque specimens were also collected from 31 patients. Bacterial DNA was extracted from each specimen to detect P. gingivalis by polymerase chain reaction. Histopathological analyses of the aortic aneurysm specimens, including immunohistochemical staining for embryonic myosin heavy chain isoform (SMemb) and S100 calcium-binding protein A9 (S100A9), were also performed. RESULTS: The number of aneurysms occurring in the distal aorta was significantly higher in subjects positive for P. gingivalis in dental plaque compared with those who were negative. The expressions of S100A9 and SMemb were also significantly greater in the subjects positive for P. gingivalis in dental plaque. On the other hand, there were no significant differences in adipocellular accumulation between the groups. CONCLUSIONS: These results suggest that aortic aneurysms in patients harboring oral P. gingivalis have greater expression of S100A9 and proliferative smooth muscle cells, which was different from the present patients without oral P. gingivalis.

Correlation of biological properties with glucan-binding protein B expression profile in Streptococcus mutans clinical isolates.

OBJECTIVE: Streptococcus mutans is known to be a primary causative agent of dental caries and its surface proteins have been investigated to specify their association with its virulence. Amongst those, 4 glucan-binding proteins (Gbps) are considered to be important factors due to their glucan-binding properties, of which GbpB has been shown to participate in cell-wall construction and cell separation. DESIGN: We examined clinical isolates of S. mutans collected from the oral cavities of Japanese and Finnish subjects, and focused on the association of their GbpB expression profiles and biological properties related to virulence. RESULTS: Western blot analysis of GbpB expression by the isolates revealed a variety of patterns. Strains that showed single and multiple bands were used to designate S and M type strains, respectively, whilst those with no GbpB expression were classified as N type. The distribution of GbpB expression patterns was shown to be quite different between the Japanese and Finnish isolates. Furthermore, the chain length and doubling time of the N type in both populations were significantly longer than those of the other types. CONCLUSION: Our results suggest variations in S. mutans GbpB expression patterns, which may have relationships with the virulence of S. mutans.

Defect of glucosyltransferases reduces platelet aggregation activity of Streptococcus mutans: analysis of clinical strains isolated from oral cavities.

OBJECTIVE: Streptococcus mutans is a major pathogen of dental caries and occasionally isolated from the blood of patients with infective endocarditis, though the association of its cell-surface glucosyltransferases (GTFB, GTFC, and GTFD) with pathogenicity for infective endocarditis remains to be elucidated. In this study, we investigated the contribution of S. mutans GTFs to platelet aggregation and analysed GTF expression profiles in a large number of clinical oral isolates. DESIGN: The platelet aggregation properties of GTF-defective isogenic mutant strains constructed from S. mutans reference strain MT8148 were evaluated using whole blood and platelet-rich plasma (PRP) taken from mice, as well as human PRP. In addition, GTF expression profiles for 396 S. mutans strains isolated from the oral cavities of 396 subjects were analysed by western blotting using antisera specific for each GTF. RESULTS: The platelet aggregation activities of the GTF-defective isogenic mutants were significantly lower than that of MT8148 when added to a large number of cells. Western blotting revealed no strains without GTF expression, though six strains had alterations of GTFB and GTFC as compared to MT8148. PCR analyses indicated that the gtfB-gtfC region length was approximately 4.5 kb shorter in those strains as compared to MT8148. These were designated as "GTFBC-fusion" strains and they demonstrated lower levels of platelet aggregation. CONCLUSIONS: Our findings indicate that GTFs are associated with platelet aggregation. Although the clinical detection frequency of S. mutans strains with altered expressions is extremely low, GTFBC-fusion strains have activities similar to GTF-defective mutant strains.

Molecular characterization of Streptococcus mutans strains containing the cnm gene encoding a collagen-binding adhesin.

OBJECTIVE: Streptococcus mutans, known to be a major pathogen of dental caries, is also considered to cause infective endocarditis. Its 120-kDa Cnm protein binds to type I collagen, which may be a potential virulence factor. In this study, we characterized S. mutans clinical strains focusing on the cnm gene encoding Cnm. DESIGN: A total of 528 S. mutans strains isolated from Japanese, Finnish, and Thai subjects were investigated. Using molecular techniques, the distribution frequency of cnm-positive strains and location of the inserted cnm were analyzed. Furthermore, isogenic mutant strains were constructed by inactivation of the cnm gene, then their biological properties of collagen-binding and glucan-binding were evaluated. Southern hybridization of the genes encoding glucan-binding proteins was also performed. RESULTS: The distribution frequency of cnm-positive strains from Thai subjects was 12%, similar to that previously reported for Japanese and Finnish subjects. Furthermore, the location of insertion of cnm was the same in all cnm-positive clinical isolates. As for the cnm-inactivated mutant strains constructed from 28 clinical isolates, their collagen-binding activity was negligible. In addition, glucan-binding activity in the cnm-positive clinical isolates was significantly reduced and corresponded to a lack of gbpA encoding glucan-binding protein A. CONCLUSIONS: Our results indicate that strains with cnm genes, the most crucial factor for the collagen-binding property of S. mutans, are detectable at similar frequencies over several different geographic locations. In addition, the common properties of these strains are a high level of collagen-binding activity and tendency for a low level of glucan-binding activity.

Molecular analysis of aortic intimal hyperplasia caused by Porphyromonas gingivalis infection in mice with endothelial damage.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection is thought to be a significant etiological factor in the development of cardiovascular diseases. However, scant definitive evidence has been presented concerning the pathological molecular mechanisms of these disorders. In the present study, we performed a molecular analysis of the developmental mechanisms of aortic intimal hyperplasia induced by P. gingivalis. MATERIAL AND METHODS: The effects of P. gingivalis-induced bacteremia on intimal hyperplasia were evaluated using a mouse model of aortic hyperplasia created by photochemical-induced endothelial cell injury. Alterations of gene expression profiles in injured blood vessels of the mice were extensively analyzed using DNA microarray assays to identify the key molecules involved in P. gingivalis-induced hyperplasia. In addition, human aneurismal specimens from patients with or without P. gingivalis infection were analyzed histochemically. RESULTS: Intravenous administration of P. gingivalis dramatically induced intimal hyperplasia in the mouse model. Concomitantly, S100 calcium-binding protein A9 (S100A9) and embryonic isoform of myosin heavy chain (SMemb), a proliferative phenotypic marker of smooth muscle cells, were significantly overexpressed on the surfaces of smooth muscle cells present in the injured blood vessels. Similarly, increased expressions of S100A9 and SMemb proteins were observed in aneurismal specimens obtained from P. gingivalis-infected patients. CONCLUSION: We found that bacteremia induced by P. gingivalis leads to intimal hyperplasia associated with overexpressions of S100A9 and SMemb. Our results strongly suggest that oral-hematogenous spreading of P. gingivalis is a causative event in the development of aortic hyperplasia in periodontitis patients.

Contribution of cell surface protein antigen c of Streptococcus mutans to platelet aggregation.

INTRODUCTION: Streptococcus mutans is considered to be one of the pathogens that cause infective endocarditis. The purpose of the present study was to examine the properties of S. mutans with regard to platelet aggregation by focusing on its high molecular protein antigen c (PAc). METHODS: The platelet aggregation properties of six clinical strains and one isogenic mutant strain of S. mutans were analysed using an aggregometer and confocal microscopy, as well as with an inhibition assay of platelet aggregation using anti-PAc serum. RESULTS: S. mutans strains with PAc expression induced platelet aggregation, while a PAc-deficient mutant and two clinical isolates with no PAc expression did not. When platelets were pretreated with higher amounts of anti-PAc serum, the platelet aggregation rate was reduced in a dose-dependent manner, indicating that PAc binds directly to platelets. CONCLUSION: S. mutans PAc is involved in human platelet aggregation and may be one of the virulence factors in the pathogenesis of infective endocarditis.

Detection of serotype k Streptococcus mutans in Thai subjects.

INTRODUCTION: Streptococcus mutans, known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c, e, f and k, based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present. METHODS: A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains. RESULTS: Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE, which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan. CONCLUSION: Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.

Effects of recombinase A deficiency on biofilm formation by Streptococcus mutans.

BACKGROUND/AIM: Recombinase A (RecA) is essential for the transformation of both plasmid and chromosomal DNA in Streptococcus pneumoniae and is considered to be related to the SOS-response in Streptococcus mutans. METHODS: In the present study, a RecA-deficient mutant strain (RAD) was constructed by insertional inactivation of the recA gene encoding the RecA protein in strain MT8148 of S. mutans, after which the biological functions of acid tolerance and biofilm formation were investigated. RESULTS: RAD showed reduced acid tolerance and produced lower density biofilm compared with the wild-type strain. In addition, confocal microscopic observation indicated that the biofilm produced by RAD was composed of cells with significantly lower viability compared with that produced by strain MT8148. CONCLUSION: These results suggest that RecA has a relationship with biofilm formation.

Molecular analyses of bacterial DNA in extirpated heart valves from patients with infective endocarditis.

BACKGROUND/AIMS: Infective endocarditis (IE) is caused by a microbial infection of the endothelial surface of the heart. Although blood culture examinations are commonly used to determine the associated bacterial species, molecular techniques, which enable rapid identification of targeted bacterial species, have recently been applied in clinical cases. METHODS: Nine heart valve specimens from IE patients (six subacute cases and three acute cases) were extirpated and collected, then bacterial DNA was extracted. Bacterial species in the specimens were determined by two different molecular methods and the results were compared with those from a conventional blood culture technique. In addition, a comparison between the two molecular methods was carried out using known numbers of six streptococcal species. RESULTS: The conventional blood culture method revealed the bacterial species in eight cases, while one was found to be negative. Multiple species were identified in most of the cases by both molecular methods; however, those specified by one method were not always consistent with those specified by the other. Furthermore, the species determined by the blood culture technique were not always identified by the molecular methods. We also found that the two molecular methods used in the present study were extremely sensitive to detect from 1 to 100 cells of individual oral streptococcal species. CONCLUSION: Our results suggest that species specified by molecular methods may have disseminated incidentally into the bloodstream, so interpretation of such results should be carefully undertaken in clinical situations.

Detection of oral bacteria in cardiovascular specimens.

BACKGROUND/AIMS: Oral bacteria, including cariogenic and periodontal pathogens, are thought to be etiological factors in the development of cardiovascular diseases. To define this relationship, we analyzed the distribution of oral bacterial species in cardiovascular specimens. METHOD: Following acceptance into the study, 203 consecutive patients were analyzed, from whom 82 aortic valve specimens, 35 mitral valve specimens, and 86 aortic aneurysmal wall specimens, of which 16 contained aneurysmal thrombus tissues, were obtained. In addition, a total of 58 dental plaque specimens were collected from the same group of patients who underwent heart valve replacement or removal of aortic aneurysms. Bacterial DNA was extracted from both cardiovascular tissues and dental plaque in those cases and then species-specific polymerase chain reaction assays were used to analyze the occurrences of six oral streptococcal and six periodontal bacterial species. RESULTS: Streptococcus mutans was the most frequently detected species in the cardiovascular specimens, followed by Aggregatibacter actinomycetemcomitans. As for dental plaque specimens from patients who underwent cardiovascular operations, most of the tested periodontitis-related species as well as oral streptococci were detected at high frequencies. Furthermore, the positive rate of S. mutans in cardiovascular specimens from patients whose dental plaque specimens were also positive for S. mutans was 78%, which was significantly higher than any other tested species when the same analysis was performed. CONCLUSION: Our results suggest that specific oral bacterial species, such as S. mutans and A. actinomycetemcomitans, are related to bacteremia and may be etiologic factors for the development of cardiovascular diseases.

Demonstration of mother-to-child transmission of Streptococcus mutans using multilocus sequence typing.

A new reliable genotyping method, multilocus sequence typing (MLST), was used to evaluate vertical transmission of the cariogenic pathogen Streptococcus mutans. A total of 136 S. mutans strains were isolated from saliva samples of 20 Japanese mother-child pairs, including 5 girls and 5 boys with primary dentition, and 5 girls and 5 boys with mixed dentition. The nucleotide sequences of 8 partial housekeeping genes, aroE, murI, gltA, glnA, glk, tkt, lepC, and gyrA, were analyzed and a similarity for all of those sequences between strains from a mother-child pair was regarded as indicating transmission, which was shown in 70% of the pairs. Interestingly, the rate of transmitted strains from mothers was significantly higher in the girls (90%) than in the boys (p = 0.001). Furthermore, the S. mutans sequence type (ST) with the highest distribution percentage in each maternal saliva sample was found to be transferred to their children. In addition, variations in two large conjugative-transfer associated regions, TnSmu1 and TnSmu2, were determined and compared with the STs defined by MLST. No variations in those two regions shown by PCR patterns were present in any of the strains isolated from the same families with the same STs, though isolates of some STs from different families showed distinct patterns for TnSmu2. Our results indicate that mothers are the main source for transmission of S. mutans to their children, while the present MLST method was also shown to be useful for investigating bacterial transmission.

Protein antigen in serotype k Streptococcus mutans clinical isolates.

Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis.