Y. TAKASHIMA

Okayama University, Okayama, Okayama, Japan

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Publications (16)11.8 Total impact

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    ABSTRACT: Black tooth staining is an extrinsic discoloration found in both primary and permanent dentition, and seen as dark pigmented lines extending to the gingival margin or an incomplete coalescence of dark dots that rarely extend beyond the cervical third of the crown. An association between black tooth staining and Actinomyces bacterial strains has been reported, while black-pigmented bacteria associated with such staining are known to be harbored in the oral cavity. Prevotella intermedia and Prevotella nigrescens are black-pigmented bacteria known to be dependent on the heme portion of hemoglobin as an iron source required for their growth. Recently, developments in molecular biological techniques have enabled rapid and easy detection of periodontopathic bacterial species using bacterial DNA extracted from oral specimens, such as plaque and saliva. Here, we report a case of black pigmentary deposition identified on all teeth of a 2-year-old girl, as well as the results of analysis of the distribution of oral bacteria in saliva and plaque specimens obtained from the patient using a molecular biological technique. In addition, a literature search found a case of disease related to the oral bacteria detected in our patient. We concluded that the bacteria detected in this case may have a strong relationship with black pigmentation, although the route of bacterial infection and cause of staining remain to be elucidated.
    Pediatric Dental Journal. 11/2014;
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    ABSTRACT: Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by the action of glucosyltransferases (GTFs) encoded by gtfB, gtfC, and gtfD. Several stress proteins, including DnaK and GroEL encoded by dnaK and groEL, are related to environmental stress tolerance. The contribution of Gbp expression to biofilm formation was analyzed by focusing on the expression levels of genes encoding GTFs and stress proteins. Biofilm forming assays were performed using GbpA-, GbpB-, and GbpC-deficient mutant strains and the parental strain MT8148. The expression levels of gtfB, gtfC, gtfD, dnaK, and groEL were evaluated by reverse transcription-qPCR (RT-qPCR). Furthermore, the structure of biofilms formed by these Gbp-deficient mutant strains was observed using confocal laser scanning microscopy (CLSM). Biofilm forming assay findings demonstrated that the amount formed by the GbpA-deficient mutant strain (AD1) was nearly the same as that by the parental strain, while the GbpB- and GbpC-deficient mutant strains produced lower amounts than MT8148. Furthermore, RT-qPCR assay results showed that the expressions of gtfB, dnaK, and groEL in AD1 were elevated as compared to MT8148. CLSM also revealed that the structure of biofilm formed by AD1 was prominently different as compared to that by the parental strain. These results suggest that a defect of GbpA influences the expression of genes controlling biofilm formation, indicating its importance as a protein for firm and stable biofilm formation.This article is protected by copyright. All rights reserved.
    Molecular Oral Microbiology. 09/2014;
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    ABSTRACT: Objective: Streptococcus mutans, implicated as a primary causative agent of dental caries in humans, produces multiple glucan-binding proteins (Gbps), which are thought to promote its adhesion to tooth surfaces. Among them, GbpC encoded by the gbpC gene is responsible for dextran-dependent aggregation in S. mutans when cultured under defined stressful conditions. We previously analyzed the GbpC dextran-binding domain using bioinformatics and reported that the possible region encoding the glucan-binding domain was located in the middle of the gbpC gene. Here, we examined the effects of peptides encoded by this region on dextran-dependent aggregation and biofilm formation. Method: We used S. mutans MT8148 (serotype c) and 2 mutant strains with isogenic regions deleted (GB4: GDPTKTIF、GB5: IMSLSSLNHWT). Biofilm formation was examined after addition ofpre-grown bacterial cells (1 μl) from each strain to Todd-Hewitt broth (100 μl) along with peptides encoding the 2 deleted regions various concentrations in 96-well polystyrene microtiter plates. The plates were incubated at 37°C with 5% CO2 for 48 hours, then stained with 1% crystal violet, washed, fixed with 95% ethanol, and air dried. Stained biofilms were quantified by measuring absorbance at 570 nm with a microplate reader. Cell-aggregation by the strains was observed in brain heart infusion broth containing 0.25% sucrose after adding MT8148 and the peptides. Result: The amount of biofilm formed by each strain was increased by adding the GB4 peptide, while those formed by MT8148 and the GB4-deletion mutant did not change by adding the GB5 peptide. In addition, cell aggregation was observed with addition of both peptides, though that by the GB4 peptide was greater than that by the GB5 peptide. Conclusion: Our results suggest that the GB4 region has a strong relationship with cell aggregation and biofilm formation. This study was supported by Grant-in-Aid for Young Scientists (B) 25862013.
    IADR General Session and Exhibition 2014; 06/2014
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    ABSTRACT: Streptococcus mutans possesses a number of ATP-binding cassette (ABC) transporters, which function as multiple sugar metabolism transporters and are promising targets for antimicrobial strategies. In the present study, we performed functional analyses of SMu0836 and SMu0837 products, which are possible ABC transporters. Isogenic mutant strains Δ0836 and Δ0837 were generated by insertional inactivation of SMu0836, and SMu0837, respectively, of strain MT8148 and found to be more sensitive to antibiotics as compared to MT8148. In addition, assays of membrane transport functions using a fluorescent probe showed that export pumps did not function properly in strain Δ0836. Expression of those genes was elevated when strain MT8148 was cultured with a sublethal concentration of tetracycline, as well as when exposed to heat shock, osmotic stress, and oxidative stress conditions. Furthermore, the expressions of other genes including SMu0374, SMu0215, SMu0475, SMu0986, and SMu1051, which encode possible ABC transporters, were also elevated when strain MT8148 was cultured with a sublethal concentration of tetracycline. Together, these results suggest that the products of SMu0836 and SMu0837 are ABC transporters, which may function in stress response.
    Oral health and dental management. 06/2014; 13(2):359-65.
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    ABSTRACT: Streptococcus mutans, a Gram-positive bacterium, is considered to be a major etiologic agent of human dental caries and reported to form biofilms known as dental plaque on tooth surfaces. This organism is also known to possess a large number of transport proteins in the cell membrane for export and import of molecules. Nitrogen is an essential nutrient for Gram-positive bacteria, though alternative sources such as ammonium can also be utilized. In order to obtain nitrogen for macromolecular synthesis, nitrogen-containing compounds must be transported into the cell. However, the ammonium transporter in S. mutans remains to be characterized. The present study focused on characterizing the ammonium transporter gene of S. mutans and its operon, while related regulatory genes were also analyzed. The SMU.1658 gene corresponding to nrgA in S. mutans is homologous to the ammonium transporter gene in Bacillus subtilis and SMU.1657, located upstream of the nrgA gene and predicted to be glnB, is a member of the PII protein family. Using a nrgA-deficient mutant strain (NRGD), we examined bacterial growth in the presence of ammonium, calcium chloride, and manganese sulfate. Fluorescent efflux assays were also performed to reveal export molecules associated with the ammonium transporter. The growth rate of NRGD was lower, while its fluorescent intensity was much higher as compared to the parental strain. In addition, confocal laser scanning microscopy revealed that the structure of biofilms formed by NRGD was drastically different than that of the parental strain. Furthermore, transcriptional analysis showed that the nrgA gene was co-transcribed with the glnB gene. These results suggest that the nrgA gene in S. mutans is essential for export of molecules and biofilm formation.
    PLoS ONE 01/2014; 9(9):e107569. · 3.53 Impact Factor
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    ABSTRACT: 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 of S. mutans on the development of non-alcoholic steatohepatitis (NASH) in a mouse model. Materials and methodsStreptococcus 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. ResultsMice 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.
    Oral Diseases 10/2013; · 2.38 Impact Factor
  • Y. TAKASHIMA, K. FUJITA, A.C. ARDIN, M. NAKANO
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    ABSTRACT: Objective: Streptococcus mutans, implicated as a primary causative agent of dental caries in humans, produces multiple glucan-binding proteins (Gbps) including GbpA, GbpB, GbpC, and GbpD, which are thought to promote its adhesion to tooth surfaces. Of those, GbpC is considered to have strong cariogenic properties, though its functional biological domains remain to be elucidated. The purpose of the present study was to examine the cariogenic and biological properties of S. mutans GbpC to identify its functional domains. Method: S. mutans MT8148 (serotype c) and its isogenic mutant strain (CD1) with the gbpC gene inactivated by insertion of an erythromycin resistant gene were used. The nucleotide alignment of gbpC encoding GbpC in the database indicated that 5 regions were possible functional domains. Thus, 5 shuttle vectors were constructed by insertion of each region in gbpC, then transformed to MT8148 (CDGB1, CDGB2, CDGB3, CDGB4, and CDGB5, respectively). Thereafter, sucrose-dependent adherence, acid tolerance, and phagocytosis assay were performed with these strains. Result: The sucrose-dependent adherence rates of CDGB2 and CDGB3 were similar to that of CD1, while acid tolerance assay findings revealed that CDGB3, CDGB4, and CDGB5 were more sensitive to low pH as compared to the other strains, among which CDGB4 had an extremely lower survival in an acid environment. Furthermore, the phagocytosis rates after 10 min of incubation for strains CD1, CDGB1, CDGB2, CDGB3, CDGB4, and CDGB5 were significantly lower than that of the parental strain MT8148, with that rate for CDGB5 the highest among the transformant strains. Conclusion: Our results suggest that the various GbpC domains may have different biological functions for biofilm formation, indicating that the functional cariogenic domain may be located in the upstream region of the gbpC gene, while other functional biological domains are located downstream.
    IADR Asia/Pacific Region (APR) Regional Meeting and Co-Annual Scientific Meeting of IADR Divisions 2013; 08/2013
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    ABSTRACT: 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. 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 and 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 and genes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of the gene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions of and genes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.
    The Scientific World Journal 01/2013; 2013:405075. · 1.73 Impact Factor
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    ABSTRACT: We used the Isolite system for treatment of dental caries identified in a submerged mandibular right primary second molar. A 5-year-6-month-old girl was referred to our clinic for close examination of an impacted mandibular right second primary molar. An intraoral examination showed a slight pit extending inside the gingiva and on the occlusal surface of the tooth. X-ray photographic examination revealed that the affected tooth was severely submerged and had a radiolucent area on the occlusal surface, which extended close to the pulp cavity. Most of the periodontal ligament space could not be clearly identified except for the distal side of the distal root. We considered that the area of the tooth was partially ankylosed and consulted with oral surgeons, who decided to postpone extraction, due to the presence of the permanent successor close to the affected tooth. Thus, we treated the dental caries, which appeared to be technically difficult because of the deep location of the tooth. The Isolite system was utilized in this case, as we considered that adjacent soft tissue and saliva could be excluded with its use. Under infiltration anesthesia, gingival tissue covering the occlusal surface was removed with an electric knife, and the carious lesion was removed, which resulted in pulp exposure. Severe inflammation of the pulp was revealed and pulpectomy was performed. There were no signs and symptoms after the treatment. At 1 year after treatment, the occlusal surface remained exposed and no inflammatory findings were observed in adjacent gingival tissue.
    Pediatric Dental Journal. 01/2012; 22(1):78–83.
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    ABSTRACT: Objective: Streptococcus mutans is known to be a primary causative agent of dental caries, with its acidogenicity and biofilm formation properties considered to be the most important factors for their development. S. mutans possesses ATP-binding cassette (ABC) transporters, which are involved in the uptake of carbohydrates and sugar metabolism. We recently demonstrated that the SMu0835, SMu0836, and SMu0837 genes, presumed to encode ABC transporters, are associated with the antibiotic susceptibility of S. mutans. In the present study, we performed functional analyses of these ABC transporters in regard to biofilm formation by S. mutans. Methods: S. mutans MT8148 (serotype c) and its isogenic mutant strains with inactivation of the SMu0835, SMu0836, SMu0837 genes, produced by inserting an erythromycin resistant gene (termed ΔSMu0835, ΔSMu0836, and ΔSMu0837, respectively), were used. Biofilm formation by each mutant strain was quantitatively measured at an optimal density of 595 nm (OD595) using crystal violet staining. Next, planktonic and biofilm cells were separated from Todd-Hewitt broth by incubation with strain MT8148 for 16 hours. Thereafter, extraction of total RNA from both cell types was performed, and expressions of the SMu0835, SMu0836, and SMu0837 genes were determined by RT-PCR. Results: In biofilm formation assays, the OD595 values of strains ΔSMu0835, ΔSMu0836, and ΔSMu0837 were significantly lower than that of MT8148. As for the levels of SMu0835, SMu0836, and SMu0837 gene expression, those in planktonic cells were significantly lower than those in biofilm cells. Among those 3 genes, the level of SMu0837 expression was the lowest followed by SMu0836 and SMu0835. Conclusion: These results suggest that ABC transporters, such as the products of SMu0835, SMu0836, and SMu0837 genes, may be related to biofilm formation.
    IADR General Session 2011; 03/2011
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    ABSTRACT: Objective: Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. The bacteria produce multiple glucan-binding proteins (GBPs), which presumably promote adhesion. Four types of GBPs (GbpA, GbpB, GbpC, GbpD) have been identified, among which GbpC belongs to the PAc family of streptococcal proteins and is expressed under conditions of stress. However, the glucan-binding domain in the gbpC gene encoding GbpC, remains to be elucidated. In this study, we performed specification of the functional domain for glucan-binding in GbpC. Methods: S. mutans MT8148 (serotype c) and its isogenic mutant strains with inactivation of the gbpC gene, produced by inserting an erythromycin resistant genes (termed CD1) were used. Five regions (termed GB1, GB2, GB3, GB4, and GB5) were determined to be possible glucan-binding domains based on the complete UA159 genome sequence database. Next, 5 shuttle vectors were constructed by inserting gbpC with deletion of each region (GB1, GB2, GB3, GB4, and GB5) and transformed to GbpC-defective mutant strain CD1, which resulted in generation of strains CDGB1, CDGB2, CDGB3, CDGB4, and CDGB5. The expression of GbpC in each of these strains was confirmed by western-blotting analysis using anti-GbpC serum. Finally, the dextran-binding activities of these 5 strains were determined and compared with those of strains MT8148 and CD1. Results: Western blotting revealed that the expression of GbpC in the 5 strains with the complement gbpC gene inserted into GbpC-defective strain CD1 was similar to that in MT8148. In addition, dextran-binding assay results showed that the activities of strains CDGB1, CDGB2, CDGB3, and CDGB5 were quite similar to that of MT8148. In contrast, those of CDGB4 and CD1 were similar, and significantly lower than the other strains. Conclusion: These results suggest that the GB4 region may be a glucan-binding domain of GbpC in S. mutans.
    IADR General Session 2011; 03/2011
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    ABSTRACT: Objectives: Streptococcus mutans synthesizes adhesive glucans from sucrose by the actions of 3 types of glucosyltransferases (GTFs), which are encoded by the gtfB, gtfC, and gtfD genes. In our previous study, inactivation of those genes resulted in colony morphology changes from rough to smooth on Mitis-salivarius agar plates. Recently, the expression of recA, which encodes RecA protein and is associated with quorum sensing, was demonstrated to influence alterations of GTF activity. In the present study, the relationship among GTF activities and recA expression was analyzed in S. mutans clinical isolates following recombination with gtfB and gtfC. Methods: S. mutans MT8148 (serotype c) and 4 clinical strains subjected to recombination of the gtfB and gtfC genes (termed gtfB/gtfC-fusion strains) were used. The sucrose-dependent adherence and GTF activities of these strains in vitro were determined using a glass-tube assay and PAS staining, respectively. Next, the entire regions of gtfB and gtfC in these strains were amplified by PCR, and the nucleotide alignment was determined and compared to that of MT8148. In addition, total RNA of the gtfB/gtfC-fusion strains was extracted and the expressions of recA were determined by RT-PCR. Results: Both the sucrose-dependent adherence rates and GTF activities of the gtfB/gtfC-fusion strains were significantly lower than those of MT8148. Sequence analyses revealed the gtfB and gtfC recombination occurred at the same position in all of the gtfB/gtfC-fusion strains. Furthermore, the expression recA in these strains was elevated as compared to MT8148. Conclusion: Our results suggest that recombination of gtfB and gtfC in clinical strains may be caused by overexpression of recA, as the recombination occurs at the same position. In addition, gtfB/gtfC-fusion strains are possibly less cariogenic because of low GTF activities derived from the recombination of gtfB and gtfC.
    IADR General Session 2011; 03/2011
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    ABSTRACT: 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. 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. 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. Our results indicate that OBSE has inhibitory activity on dental caries.
    Caries Research 02/2011; 45(1):56-63. · 2.51 Impact Factor
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    ABSTRACT: 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. 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. 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. Our results suggest variations in S. mutans GbpB expression patterns, which may have relationships with the virulence of S. mutans.
    Archives of oral biology 10/2010; 56(3):258-63. · 1.65 Impact Factor
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    ABSTRACT: Objectives: Streptococcus mutans has been implicated as a primary causative agent of dental caries and is known to synthesize adhesive glucans from sucrose by the action of glucosyltransferases (GTFs), which mediate firm adherence of the bacterial cells to tooth surfaces, leading to biofilm formation. In our previous study, the function of RecA protein was examined using a molecular biological method, which showed that expression levels of the recA gene were significantly increased under low pH conditions. The purpose of the present study was to analyze gtf expression and biofilm formation of S. mutans in the same low pH condition that leads to overexpression of RecA protein. Methods: The recA gene was cloned into expression vector pET42a (+) and purified recombinant RecA (rRecA) protein was produced. Next, S. mutans MT8148 was cultured in Brain Heart Infusion broth in the presence or absence of rRecA, then inoculated onto Mitis-salivarius agar plates. Colonies showing a smooth morphology were collected from the plates, after which they were examined for sucrose-dependent adherence activity and western blotting analysis was performed to assess GTFB activity. Results: Approximately 5% of the colonies displayed a smooth morphology in the presence of rRecA and the sucrose-dependent adherence activity of those isolates was lower than that of MT8148. In addition, western blot analysis revealed only a single band between the positions of GTFB and GTFC, whereas two bands corresponding to GTFB and GTFC were identified at their original positions in MT8148. Conclusion: Our results suggest that overexpression of RecA causes recombination of the gtfB and C genes, and RecA protein may have a role in biofilm formation in acidic conditions.
    IADR General Session 2010; 07/2010
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    ABSTRACT: Objective: Streptococcus mutans is known to be a primary causative agent of dental caries, with its glucan-binding proteins (Gbps: GbpA, GbpB, GbpC, and GbpD) regarded as important cell surface proteins related to its glucan-binding ability. The purpose of present study was to analyze GbpB expression by clinical isolates of S. mutans and its relationship to the glucan-binding properties of this pathogen. Methods: One hundred strains of S. mutans isolates from Japanese children at our clinic and 100 strains isolated from Finish children, provided by Helsinki University Hospital, were analyzed, GbpB expression was investigated by western blotting using GbpB-specific antisera, with glucan-binding properties determined by dextran-binding assay. In addition, the growth time of the strains was also determined. Results: Three types of GbpB expression patterns were identified, among which single band (S-type) and multiple band (M-type) types were noted, while strains with no visible bands were also seen (N-type). More than 80% of the strains isolated in Japan were classified as S-type, while the distribution frequencies of M-type (15%) and N-type (4%) were extremely low. In contrast, the distribution frequency of S-type in the strains isolated in Finland was approximately 40% and that of N-type was 20%. The doubling time of N-type in both populations was significantly longer as compared to the other types, whereas the dextran-binding activities were not significantly different among all types. Conclusion: Our results suggest geographical differences in regard to the GbpB expression patterns of S. mutans clinical strains. Furthermore, strains without GbpB expression may be associated with a low rate of dental caries due to their low levels of cell cycling and cell maintenance.
    IADR General Session 2010; 07/2010

Publication Stats

3 Citations
11.80 Total Impact Points

Institutions

  • 2013–2014
    • Okayama University
      Okayama, Okayama, Japan
  • 2011–2013
    • Osaka City University
      Ōsaka, Ōsaka, Japan
  • 2010
    • Osaka University
      • Department of Pediatric Dentistry
      Suika, Ōsaka, Japan