Kazuyo Fujita

Cariogenicity of the Probiotic Bacterium Lactobacillus salivarius in Rats

Probiotic bacteria such as lactobacilli and bifidobacteria are considered to be non-pathogenic and non-toxigenic on the basis of long years of safe usage. However, some species of lactobacilli are thought to be associated with the development of dental caries. The purpose of the present study was to examine the cariogenicity of the probiotic bacterium Lactobacillus salivarius in rats. Rats were divided into six groups, and infected with L. salivarius LS1952R and/or Streptococcus mutans MT8148R. L. salivarius LS1952R became established in the oral cavity of rats and induced significant level of dental caries even when infected for only 5 days from 18 to 22 days of age. In addition, the caries scores of rats superinfected with both Streptococcus mutans MT8148R and L. salivarius LS1952R from 18 days of age were significantly higher than those infected with either L. salivarius LS1952R or S. mutans MT8148R alone. Since strain LS1952R can adhere to saliva-coated hydroxyapatites, it is concluded that L. salivarius strain LS1952R possesses an inherent cariogenic activity following adherence to the tooth surface.

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.

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.

Bacterial profiles of oral streptococcal and periodontal bacterial species in saliva specimens from Japanese subjects

OBJECTIVE Recent developments in molecular biological techniques have increased understanding of the distribution of oral bacterial species in clinical specimens, though few investigations have been conducted to simultaneously detect oral streptococcal and periodontal species in the same specimens. The purpose of the present study was to investigate the distribution and correlation of 6 oral streptococcal and 6 periodontal species in saliva specimens taken from children and their mothers. DESIGN Seventy-four pairs of children and their mothers were approved to participate in this study. Saliva specimens were collected and bacterial DNA extracted, which was subjected to PCR analyses using species-specific sets of primers. The combinations of species able to be detected simultaneously were determined by statistical analyses. RESULTS Streptococcus sobrinus and Porphyromonas gingivalis were detected more often in the mothers than the children. Streptococcus mutans, Streptococcus sanguinis, and Streptococcus oralis were detected simultaneously in a significant number of specimens, while the presence of Campylobacter rectus was correlated with the presence of at least one of the red complex species (P. gingivalis, Treponema denticola and Tannerella forsythensis). On the other hand, no correlation was shown between the rates of detection of oral streptococcal and periodontal species. CONCLUSIONS Our results indicate that among streptococcal and periodontal species, several are able to coexist in saliva, while the presence of both does not have an influence on each other.

Distribution of periodontopathic bacterial species in Japanese children with developmental disabilities

BackgroundRecent developments in molecular biological techniques have enabled rapid detection of periodontopathic bacterial species in clinical specimens. Accumulated evidence suggests that detection of specific bacterial species enables identification of subjects at high risk for the onset of periodontitis. We investigated the distribution of 10 selected periodontopathic bacterial species in dental plaque specimens obtained from children with disabilities who were attending daycare centers.MethodsA total of 187 children (136 boys, 51 girls) aged 1-6 years old and diagnosed with such disabilities as mental retardation, cerebral palsy, and autism, participated in the study. Subgingival dental plaque specimens were collected from the buccal side of the maxillary left second primary molar after a clinical examination. Bacterial DNA was extracted from the specimens and PCR analyses were carried out to detect 10 selected periodontopathic species using specific primers for each. In addition, statistical analyses were performed to analyze the correlations among clinical parameters and the detected species.ResultsThe most frequently detected species was Capnocytophaga sputigena (28.3%), followed by Aggregatibacter actinomycetemcomitans (20.9%) and Campylobacter rectus (18.2%). Eikenella corrodens, Capnocytophaga ochracea, and Prevotella nigrescence were detected in approximately 10% of the specimens, whereas Treponema denticola, Tannerella forsythia, and Prevotella intermedia were rarely found, and Porphyromonas gingivalis was not detected in any of the subjects. The total numbers of detected species were positively correlated with the age of the subjects. There were 10 subjects with positive reactions for T. denticola and/or T. forsythia, in whom the total number of bacterial species was significantly higher as compared to the other subjects. Furthermore, subjects possessing C. rectus showed significantly greater values for periodontal pocket depth, gingival index, and total number of species.ConclusionWe found that approximately one-fourth of the present subjects with disabilities who possessed at least one of T. denticola, T. forsythia, and C. rectus were at possible risk for periodontitis. Follow-up examinations as well as preventive approaches should be utilized for such individuals.

Distribution of 10 periodontal bacterial species in children and adolescents over a 7-year period

OBJECTIVE There is scant information available regarding the distribution of periodontal bacterial species in children and adolescents over an extended period. The purpose of this study was to compare bacterial profiles in the same individuals over a period of 7 years. SUBJECT AND METHODS Twenty-six children and adolescents from whom dental plaque and saliva specimens were obtained during both the first (1999-2000) and second (2006-2007) periods, were analyzed. Bacterial DNA was extracted from each specimen and the presence of 10 periodontal bacterial species was determined using a PCR method, with a focus on the red complex species of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. RESULTS Subjects with red complex species in saliva specimens obtained during the second collection possessed a significantly higher number of total bacterial species than those without. The detection rate of the red complex species in the second collection period samples was significantly greater in subjects who had two or more species detected in samples taken during the first collection compared with the other subjects. CONCLUSION Subjects possessing red complex species may be at possible risk for infection with a high number of periodontal bacterial species during adolescent and younger adult years.

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.

Regulation of Recombination between gtfB/gtfC Genes in Streptococcus mutans by Recombinase A

Streptococcus mutans produces 3 types of glucosyltransferases (GTFs), whose cooperative action is essential for cellular adhesion. The recombinase A (RecA) protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions. S. mutans strain MT8148 was grown in the presence of recombinant RecA (rRecA) protein, after which smooth colonies were isolated. The biological functions and sequences of the gtfB and gtfC genes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of the gtfB and gtfC genes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of the recA gene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions of gtfB and gtfC genes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.

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.

Inverted maxillary second primary molar and permanent successor teeth: X-ray photographic evaluations

A 10-year-and-4-month old girl was shown to have inverted maxillary right second primary molar and permanent successor teeth, after being referred to our clinic for examination of an unerupted maxillary right second primary molar. Orthopantomograph images taken at the age of 7Y4M showed an impacted maxillary second primary molar and ambiguous tooth germ of its permanent successor, while images obtained at the age of 10Y4M exhibited the tooth germ of the permanent successor in a position on top of the impacted primary molar. Orthopantomographs were obtained twice during the next year, which showed development of the second premolar. The dental age of the entire dentition, except for the maxillary right second premolar, at 7Y4M was considered to be consistent with her chronological age, whereas it was approximately 6 months to 1 year behind of chronological age when determined at the age of 10Y4M, 11Y4M, and 12Y3M. The dental age of the permanent second premolar gradually increased, however, remained approximately 4 years behind chronological age. At 12Y3M, computed tomography examinations were performed to clarify the three-dimensional positions of the second primary molar and its permanent successor. At that time, the impacted primary molar was located close to the inferior part of the maxillary sinus, while the tooth germ of the permanent successor was located in the apex region of the adjacent first permanent premolar and first permanent molar. We decided to carry out the periodical examinations to observe the maxillary right second primary molar and premolar.