Nobuhiro Takahashi

Tohoku University, Japan

Are you Nobuhiro Takahashi?

Claim your profile

Publications (33)34.32 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H2S), one of the major components of oral malodor. However, there is little information on the physiological properties of H2S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H2S production. Thus, in the present study, the H2S-producing activity of growing cells, resting cells and cell extracts of oral Veillonella species and the effects of oral environmental factors including pH and lactate were investigated. Type strains of Veillonella atypica, Veilonella dispar and Veillonella parvula, were used. These Veillonella species produced H2S during growth in the presence of L-cysteine. Resting cells of these bacteria produced H2S from L-cysteine, and the cell extracts showed enzymatic activity to convert L-cysteine to H2S. H2S production by resting cells was higher at pH 6 - 7 and lower at pH 5. The presence of lactate markedly increased H2S production by resting cells (4.5 - 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H2S, ammonia was produced in cell extracts of all the strains, indicating that H2S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H2S from L-cysteine and their H2S production can be regulated by oral environmental factors, pH and lactate.
    Applied and Environmental Microbiology 05/2014; · 3.95 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Postoperative pneumonia may occur when protective reflexes in the upper respiratory tract such as cough and/or swallowing reflexes are impaired; thus, silent aspiration of oral bacteria is possibly involved in the postoperative pneumonia. This study aimed to quantify and identify bacteria existing in intraoperative bronchial fluids, and evaluate the relationship between impairment of cough/swallowing reflexes and silent aspiration of oral bacteria in elderly patients. After obtaining informed consent, cough and swallowing reflexes were measured using an ultrasonic nebuliser and a nasal catheter, respectively. The intraoperative bronchial fluids from 9 subjects with pulmonary carcinoma were collected with the micro-sampling probe, and were cultured anaerobically on blood agar plates. After 7 days, colony forming units (CFU) were counted and isolated bacteria were identified by 16S rRNA gene sequencing. Four subjects (71.0 ± 8.4 years) showed impaired swallowing reflex with normal cough reflex, while 5 subjects (73.6 ± 6.5 years) showed normal cough and swallowing reflexes. The bacterial counts (mean CFU ± SD) in intraoperative bronchial fluids of subjects with impaired swallowing reflex [(5.1 ± 7.7) × 105] were higher than those with normal one [(1.2 ± 1.9) × 105], although the differences were not statistically significant. Predominant isolates from intraoperative bronchial fluids were Streptococcus (41.8%), Veillonella (11.4%), Gemella (8.9%), Porphyromonas (7.6%), Olsenella (6.3%) and Eikenella (6.3%). These results indicate that intraoperative bronchial fluids contains bacteria, probably derived from the oral microbiota, and suggest that silent aspiration of oral bacteria occurs in elderly patients irrespective of impairment of swallowing reflex.
    Microbiology and Immunology 05/2014; · 1.55 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Titanium-silver (Ti-Ag) alloy has been improved for machinability and mechanical properties, but its anti-biofilm properties have not been elucidated yet. Thus, this study aimed to evaluate the effects of Ti-Ag alloy on biofilm formation and bacterial viability in comparison with pure Ti, pure Ag and silver-palladium (Ag-Pd) alloy. Biofilm formation on the metal plates was evaluated by growing Streptococcus mutans and Streptococcus sobrinus in the presence of metal plates. Bactericidal activity was evaluated using a film contact method. There were no significant differences in biofilm formation between pure Ti, pure Ag and Ag-Pd alloy, while biofilm amounts on Ti-20% Ag and Ti-25% Ag alloys were significantly lower (p<0.05). In addition, Ti-Ag alloys and pure Ti were not bactericidal, although pure Ag and Ag-Pd alloy killed bacteria. These results suggest that Ti-20% Ag and Ti-25% Ag alloys are suitable for dental material that suppresses biofilm formation without disturbing healthy oral microflora.
    Dental Materials Journal 04/2014; · 0.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To characterize the metabolic system of oral squamous cell carcinoma (OSCC) by metabolome analysis.
    Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 04/2014; · 1.50 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ketone bodies including acetone are disease biomarkers for diabetes that sometimes causes severe ketoacidosis. The present study was undertaken to clarify the significance of exhaled acetone and plasma ketone bodies at bedside in a clinical setting. The oral glucose tolerance test (OGTT) was performed in 10 healthy Japanese volunteers (five females and five males). Exhaled breath acetone and volatile sulfide compounds (VSCs) in mouth air were measured simultaneously with blood sampling during the OGTT using a portable gas chromatograph equipped with an In2O3 thick-film type gas sensor and a VSC monitor. Acetone, β-hydroxybutyrate (β-OHB) and acetoacetate (AcAc) in blood plasma as well as glucose and insulin were examined. Oral conditions were examined based on the Community Periodontal Index (CPI) by one dentist. In addition, the same type of analysis was applied to two uncontrolled type 2 diabetes mellitus patients hospitalized at Tohoku University Hospital. Exhaled acetone was measured at the same time as blood withdrawal in the morning before breakfast and at night before bed at the beginning, the middle, and the end of hospitalization. All volunteers showed normal OGTT patterns with no ketonuria and periodontitis; however, there were significant correlations between breath acetone and plasma β-ΟΗΒ and between breath acetone and plasma AcAc under fasting conditions. Breath acetone of the type 2 diabetes mellitus patients showed positive correlations with plasma glucose when the level of plasma glucose tended to decrease during hospitalization. In spite of a very limited number of cases, our results support the idea that exhaled breath acetone may be related to plasma β-OHB and AcAc, which reflect glucose metabolism in the body.
    Journal of Breath Research 01/2014; 8(4):046008. · 2.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Inhibition of bacterial acid production by dental restorative materials is one of the strategies for secondary caries prevention. This study aimed to evaluate the effect of fluoride-releasing restorative materials on bacteria-induced pH fall at the bacteria-material interface. Four fluoride-releasing restorative materials, glass-ionomer cement (GIC), resin-modified glass-ionomer cement (RMGIC), resin composites (RC) and flowable resin composite (FRC) were used. Each specimen was immersed in potassium phosphate buffer at pH 7.0 for 10min and 4 weeks, and in potassium acetate buffer at pH 5.5 for 4 weeks. An experimental apparatus was made of polymethyl methacrylate and had a well with restorative materials or polymethyl methacrylate (control) at the bottom. The well was packed with cells of Streptococcus mutans, and the pH at the interface between cells and materials was monitored using a miniature pH electrode after the addition of 1% glucose for 90min, and the fluoride released into the well was quantified using a fluoride ion electrode. The pH of GIC (4.98-5.18), RMGIC (4.77-4.99), RC (4.62-4.75) and FRC (4.54-4.84) at 90min were higher than that of control (4.31-4.49). The fluoride amounts released from GIC were the highest, followed by RMGIC, RC and FRC, irrespective of immersion conditions. Saliva coating on materials had no significant effect. The fluoride-releasing restorative materials inhibited pH fall at the bacteria-material interface. The degree of inhibition of pH fall seemed to correspond to the amount of fluoride detected, suggesting that the inhibition was due to the fluoride released from these materials. A little amount of fluoride actually released from the fluoride-releasing materials may have caries preventive potential for oral bacteria.
    Journal of dentistry 11/2013; · 3.20 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Actinomyces are predominant oral bacteria; however, its cariogenic potential, such as acid production and fluoride sensitivity, has not been elucidated in details and compared with the other caries-associated oral bacteria, such as Streptococcus. Therefore, this study aimed to elucidate and compare the acid production and growth of Actinomyces and Streptococcus, considering the presence of bicarbonate and fluoride, mimicking the oral cavity. The acid production from glucose was measured by pH-stat at pH 5.5 and 7.0 under anaerobic conditions. The growth rate was assessed by optical density in anaerobic culture. Although the acid production rate by Actinomyces was lower than that of Streptococcus, the acid production of Actinomyces was more tolerant to fluoride (IDacid production 50 = 110-170 ppm at pH 7.0 and 10-13 ppm at pH 5.5) than that of Streptococcus (IDacid production 50 = 36-53 ppm at pH 7.0 and 6.3-6.5 ppm at pH 5.5). Bicarbonate increased the acid production of Actinomyces with prominent succinate production, and enhanced the fluoride tolerance (IDacid production 50 = 220-320 ppm at pH 7.0 and 33-52 ppm at pH 5.5). Bicarbonate had no effect on Streptococcus. In addition, although the growth rate of Actinomyces was lower than that of Streptococcus, Actinomyces growth was more tolerant to fluoride (IDgrowth 50 = 130-160 ppm) than Streptococcus (IDgrowth 50 = 27-36 ppm). These results indicate that oral Actinomyces are more tolerant to fluoride than oral Streptococcus, and bicarbonate enhances the fluoride tolerance of oral Actinomyces. Further study is needed to generalize to the genus level due to the limited number of species tested.
    Microbiology and Immunology 09/2013; · 1.55 Impact Factor
  • Yoko Sakuma, Jumpei Washio, Keiichi Sasaki, Nobuhiro Takahashi
    [Show abstract] [Hide abstract]
    ABSTRACT: Oral bacteria adhered to dental material surfaces are known to cause various oral diseases. This study aimed to develop a highsensitive and non-radioisotopic fluorescence dye method for quantification of oral bacteria (Streptococcus, Actinomyces and Veillonella) adhered to denture material surfaces. The amount of adhered bacteria was estimated from the fluorescence intensity derived from resazurin, which is reduced by bacterial metabolic reactions. The addition of bacterial metabolic substrates (glucose for Streptococcus and Actinomyces and sodium lactate for Veillonella) to the reaction mixture increased the fluorescence intensity by 2.3-110 times, subsequently improved the sensitivity. Furthermore, an experimental device having silicon wells containing test material was carefully designed for accurate quantification of bacteria adhered to test materials. The improved resazurin method using a new experimental device successfully enabled the quantification of bacterial adhesion to polymethyl methacrylate and other three conventional denture materials.
    Dental Materials Journal 01/2013; 32(4):585-91. · 0.81 Impact Factor
  • Source
    Nobuhiro Takahashi, Jumpei Washio, Gen Mayanagi
    [Show abstract] [Hide abstract]
    ABSTRACT: Research approaches to biofilm are stratified by a series of analyses: (1) microbial number and species; (2) microbial proteins, such as enzymes; and (3) microbial activity, such as metabolic activity. On the other hand, the hierarchical structure of biology includes the genome, proteome, and metabolome, in which the metabolome is the final output of biological function. Metabolome analysis is the comprehensive analysis of the metabolome, a new strategy for biological research in the 21st century. The stratified structure of biofilm research corresponds to the biological hierarchy, and the analysis of microbial activity, especially metabolic activity, is comparable to metabolome analysis; however, oral biofilm samples are too small to analyze the metabolome by conventional methods. Recently, a new device involving capillary electrophoresis (CE) and time-of-flight mass spectrometry (MS) has been developed, facilitating metabolomic investigation of the central carbon metabolic pathways (i.e., the EMP pathway, pentose phosphate pathway, and TCA cycle) in oral biofilm. Using CE–MS, we analyzed metabolome profiles of oral biofilm after oral rinsing with glucose in vivo and evaluated the effects of oral rinsing with fluoride and xylitol on the metabolome profiles of oral biofilm. The results were somewhat consistent with previous in vitro data obtained from single bacterial strains, namely, Streptococcus and Actinomyces; however, new information describing the metabolic properties of oral biofilm was also obtained. This metabolomic approach will reveal the functional characteristics of oral biofilm in vivo, potentially providing new insights into the nature of oral biofilm in health and disease.
    Journal of Oral Biosciences 08/2012; 54(3):138–143.
  • [Show abstract] [Hide abstract]
    ABSTRACT: To quantify and identify bacteria detected in acrylic resin dentures and dento-maxillary obturator-prostheses after long-term use. The internal layer of denture bases from 13 daily-use removable acrylic resin dentures was sampled, while the inner fluid samples/no-fluid samples of obturators were collected from 11 in-use acrylic resin dento-maxillary obturator-prostheses. Samples were cultured, and isolated bacteria were counted and identified by molecular biological methods. Bacteria were detected in five (38.5%) acrylic resin dentures and six (54.5%) acrylic resin obturators. Four Lactobacillus species and one Propionibacterium species were isolated from three repaired denture bases, and from two non-repaired dentures, two Actinomyces species and Streptococcus mutans were isolated. On the other hand, 17 bacterial species, belonging to the family and genera of Olsenella, Bacillus, Citrobacter, Enterobacteriaceae, Lactobacillus, Pantoea, Peptoniphilus, Klebsiella and Pseudomonas, were isolated from obturators. Several species of viable bacteria were detected in acrylic resin denture bases and obturators.
    American journal of dentistry 06/2012; 25(3):171-5. · 1.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: PurposeIn order to elucidate the characteristics of orthodontic appliance-associated dental plaque, this study aimed to profile the microflora and to estimate the acidogenic potential of supragingival plaque on first molars with orthodontic bands or brackets or without appliances.Material and methodsSupragingival plaque samples from the surface of upper and lower first molars with orthodontic bands or brackets or without appliances in 6 subjects (age, 11–30 years) were cultured anaerobically on blood agar plates. Isolated bacteria were identified by 16S rRNA sequencing. The acidogenicity of isolated bacteria was examined using fastidious anaerobe agar plates containing bromocresol.ResultsBacterial growth (log CFU/mg) was 6.6±6.5, 6.9±7.1, and 7.4±7.6 in samples obtained from molars with bands, brackets, and without appliances, respectively. Actinomyces (43.5 and 40.0%) and Streptococcus (23.5 and 34.7%) were the predominant species present on molars with orthodontic brackets and without appliances, respectively. In contrast, the proportion of Streptococcus (44.4%) was higher than that of Actinomyces (17.6%) on molars with orthodontic bands (P<0.01). The proportions of acidogenic bacteria in plaque samples from molars with bands, brackets, and without appliances were 74.5%, 71.3%, and 81.6%, respectively, although these differences were not statistically significant.Discussion and conclusionThese results indicate that there are differences in the microbial composition and acidogenic potential of supragingival plaque from first molars with bands, brackets, or without appliances, and suggest that supragingival plaque on teeth with brackets carries predominantly periodontitis-associated bacteria but less caries-associated bacteria.
    Journal of Oral Biosciences 05/2012; 54(2):107–112.
  • [Show abstract] [Hide abstract]
    ABSTRACT: This study aimed to profile the microflora in infected root canals before and after root canal treatment using culture-independent methods. Six infected root canals in single-rooted teeth with periapical lesions from five subjects were included. Quantification of total bacteria was performed by real-time PCR with primers targeting 16S rRNA genes. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species level was performed by comparative analysis with the GenBank database. The concentration of extracted DNA before treatment was higher than that after root canal treatment, although the difference was not statistically significant. Sequence analysis revealed that oral bacteria such as Fusobacterium, Streptococcus, Olsenella, and Pseudoramibacter detected in cases before root canal treatment disappeared after treatment. These results suggest that the root canal microflora are distinct before and after root canal treatment, and that treatment changes the microflora in both quantity and quality.
    The Journal of Microbiology 02/2012; 50(1):58-62. · 1.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Objective. Periapical periodontitis is an infectious and inflammatory disease of the periapical tissues caused by oral bacteria invading the root canal. In the present study, profiling of the microbiota in infected root canals was performed using anaerobic culture and molecular biological techniques for bacterial identification. Methods. Informed consent was obtained from all subjects (age ranges, 34-71 years). Nine infected root canals with periapical lesions from 7 subjects were included. Samples from infected root canals were collected, followed by anaerobic culture on CDC blood agar plates. After 7 days, colony forming units (CFU) were counted and isolated bacteria were identified by 16S rRNA gene sequencing. Results. The mean bacterial count (CFU) in root canals was (0.5 ± 1.1) × 10(6) (range 8.0 × 10(1)-3.1 × 10(6)), and anaerobic bacteria were predominant (89.8%). The predominant isolates were Olsenella (25.4%), Mogibacterium (17.7%), Pseudoramibacter (17.7%), Propionibacterium (11.9%) and Parvimonas (5.9%). Conclusion. The combination of anaerobic culture and molecular biological techniques makes it possible to analyze rapidly the microbiota in infected root canals. The overwhelming majority of the isolates from infected root canals were found to be anaerobic bacteria, suggesting that the environment in root canals is anaerobic and therefore support the growth of anaerobes.
    International Journal of Dentistry 01/2012; 2012:609689.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23-79 years). Samples from infected root canals were collected at the beginning of the treatment (termed #25 First), the end of the first day of treatment (termed #55 First), and the next appointment day (termed #55 Second). Then, the bacterial count (CFU) was measured using fluorescence reagents (4',6'-diamidino-2-phenylindole and propidium iodide) and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of "#25 First" was (1.0 ± 1.4) × 10(5). As the root canal treatment progressed, the CFU decreased as 7.9 × 10(3) (#55 First) and 4.3 × 10(2) (#55 Second). Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis).
    International Journal of Dentistry 01/2012; 2012:172935.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to evaluate the ability of a coating material containing the surface pre-reacted glass-ionomer (S-PRG) filler to protect the root from demineralization in vitro. The proprietary coating resin containing the S-PRG filler (PRG Barrier Coat) was applied to human root dentin and immersed in acid buffer at pH 4.5 for 3 d. Demineralization was evaluated by micro-CT scanning and the dentin-material interface observed by scanning electron microscopy. The ability of the coating resin to modify acid production by Streptococcus mutans was investigated by monitoring pH using an ion-sensitive field-effect transistor pH electrode. Application of PRG Barrier Coat produced a coating layer with the thickness of approximately 200 µm and completely inhibited demineralization. The bacteria-induced pH fall at the material surface was significantly inhibited. We conclude that S-PRG fillercontaining coating resin may be an effective material for protecting exposed root from both chemical and biological challenges.
    Dental Materials Journal 01/2012; 31(6):909-15. · 0.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To profile plaque microflora on root-caries lesions, and to examine the protein-denaturing activity as a pilot study. Six subjects with root-caries were investigated. Plaque samples on root caries lesions (R), as well as from healthy supragingival sites (S) and periodontal pockets (> or = 5 mm) (P) were collected and cultured anaerobically on blood agar plates. The isolated bacteria were identified by 16S rRNA sequencing analysis, and examined for the protein-denaturing activity using the skim-milk plates and the SDS-PAGE, and for the acidogenicity using the FAB broth containing 1% glucose. Propionibacterium, Actinomyces, Streptococcus, Lactobacillus and Bifidobacterium were predominant in R, while Actinomyces, Streptococcus, Veillonella and Capnocytophaga in S, and Actinomyces, Prevotella, Actinobaculum, Streptococcus, Olsenella and Eubacterium were predominant in P. Proteolytic bacteria comprised 40%, 26% and 57% of microflora in R, S and P, respectively. The skim-milk plates distinguished between protein-degrading and protein-coagulating bacteria, which comprised 7 and 33%, 0 and 26%, and 17 and 40% of microflora, in R, S and P, respectively. The SDS-PAGE analysis revealed that protein-degrading isolates were capable of degrading collagen molecules. Furthermore, the final culture pHs of protein-degrading and -coagulating bacteria were 5.0-5.4 and 3.8-3.9, respectively. The latter pH was low enough to denature proteins in skim milk. The microbial composition of R was distinct from those of S and P.
    American journal of dentistry 10/2011; 24(5):295-9. · 1.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 μg ml(-1) of MDPB for 60 s. At a concentration of 4-8 μg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 μg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.
    European Journal Of Oral Sciences 04/2011; 119(2):175-81. · 1.42 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Candida species were detected and identified in samples from the buccal mucosa, dorsal surface of the tongue and supragingival plaque of subjects with oral lichen planus (OLP). The Candida in the samples were cultured on selection agars, and identified by sequence analyses of 18S, 5.8S and 25/28S rRNA. The isolation frequency of Candida was higher in subjects with OLP than in those with healthy oral mucosa. Non-C. albicans were only isolated from people with OLP. These results support the notion that subjects with OLP are more likely to have oral colonization with Candida, and that non-C. albicans are specifically present in subjects with this condition.
    Microbiology and Immunology 01/2011; 55(1):66-70. · 1.55 Impact Factor
  • Jumpei Washio, Gen Mayanagi, Nobuhiro Takahashi
    Journal of Oral Biosciences 01/2010; 52(3):225-232.
  • [Show abstract] [Hide abstract]
    ABSTRACT: This study aimed to profile plaque microflora on first molars with orthodontic bands (Ba), brackets (Br), and without appliances (C). The mean bacterial numbers of plaque (logCFUs/mg) on the molars with Ba, Br, and C were 6.6, 6.7, and 6.9, respectively. Actinomyces, Streptococcus and Veillonella were predominant in Br and C, while the proportions of Actinomyces and Veillonella were low in Ba. Periodontitis-associated bacteria including Eubacterium, Fusobacterium, Porphyromonas, and Prevotella were isolated in Br, but virtually not detected in Ba and C, suggesting that supragingival plaque biofilm of teeth with Br carries bacteria related to periodontitis. These findings may provide a helpful suggestion for self-care and regular professional plaque control in clinical orthodontics. Key wordsmicroflora-orthodontic appliances-plaque biofilm-polymerase chain reaction
    12/2009: pages 248-249;