Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Toru Takeshita Last modified date:2021.10.07

Associate Professor / Section of Oral Health, Growth and Development / Department of Dental Science / Faculty of Dental Science

1. Furuta, Michiko; Takeuchi, Kenji; Takeshita, Toru; Tanaka, Akihiko; Suma, Shino; Shinagawa, Takashi; Shimazaki, Yoshihiro; Yamashita, Yoshihisa , Longitudinal Associations of Toothbrushing With Obesity and Hyperglycemia, JOURNAL OF EPIDEMIOLOGY, 10.2188/jea.JE20190165, 30, 12, 556-565, 2020.12.
2. Rei Nagao, Daisuke Esaki, Yukie Shibata, Satoshi Ikawa, Katsuhisa Kitano, Yasunori Ayukawa, Yasuyuki Matsushita, Toru Takeshita, Yoshihisa Yamashita, Masaaki Matsuzaki, Kiyoshi Koyano, Investigation of a novel sterilization method for biofilms formed on titanium surfaces, dental materials journal, 10.4012/dmj.2018-274, 38, 4, 654-662, 2019.01, The development of effective methods to disinfect biofilms on dental materials is medically important. This study evaluated the bactericidal effects of peroxynitric acid (HOONO2; PNA) on biofilms formed on titanium surfaces. Streptococcus gordonii was cultured on either machined or rough titanium discs that were then used to evaluate the bactericidal effects of seven reagents, i.e., normal saline, benzalkonium chloride disinfectant solution, chlorhexidine digluconate solution, three concentration types of PNA, and inactivated PNA. Using low concentration of PNA, the bacterial count based on a CFU assay reached an undetectable level within 10 s; this bactericidal effect was the strongest observed for the seven tested reagents. Thus, PNA may be more useful than other disinfectants for sterilizing biofilms on titanium surfaces that have been contaminated with bacteria..
3. Yukari Ihara, Toru Takeshita, Shinya Kageyama, Rie Matsumi, Mikari Asakawa, Yukie Shibata, Yuki Sugiura, Kunio Ishikawa, Ichiro Takahashi, Yoshihisa Yamashita, Identification of initial colonizing bacteria in dental plaques from young adults using full-length 16S rRNA gene sequencing, mSystems, 10.1128/mSystems.00360-19, 4, 5, 2019.01, Development of dental plaque begins with the adhesion of salivary bacteria to the acquired pellicle covering the tooth surface. In this study, we collected in vivo dental plaque formed on hydroxyapatite disks for 6 h from 74 young adults and identified initial colonizing taxa based on full-length 16S rRNA gene sequences. A long-read, single-molecule sequencer, PacBio Sequel, provided 100,109 high-quality full-length 16S rRNA gene sequence reads from the early plaque microbiota, which were assigned to 90 oral bacterial taxa. The microbiota obtained from every individual mostly comprised the 21 predominant taxa with the maximum relative abundance of over 10% (95.8 6.2%, mean SD), which included Streptococcus species as well as nonstreptococcal species. A hierarchical cluster analysis of their relative abundance distribution suggested three major patterns of microbiota compositions: a Streptococcus mitis/Streptococcus sp. HMT-423-dominant profile, a Neisseria sicca/Neisseria flava/Neisseria mucosadominant profile, and a complex profile with high diversity. No notable variations in the community structures were associated with the dental caries status, although the total bacterial amounts were larger in the subjects with a high number of caries-experienced teeth (8) than in those with no or a low number of caries-experienced teeth. Our results revealed the bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults. IMPORTANCE Selective attachment of salivary bacteria to the tooth surface is an initial and repetitive phase in dental plaque development. We employed full-length 16S rRNA gene sequence analysis with a high taxonomic resolution using a third-generation sequencer, PacBio Sequel, to determine the bacterial composition during early plaque formation in 74 young adults accurately and in detail. The results revealed 21 bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults, which include several streptococcal species as well as nonstreptococcal species, such as Neisseria sicca/N. flava/N. mucosa and Rothia dentocariosa. Given that no notable variations in the microbiota composition were associated with the dental caries status, the maturation process, rather than the specific bacterial species that are the initial colonizers, is likely to play an important role in the development of dysbiotic microbiota associated with dental caries..
4. Shinya Kageyama, Toru Takeshita, Kenji Takeuchi, Mikari Asakawa, Rie Matsumi, Michiko Furuta, Yukie Shibata, Kiyoshi Nagai, Masahiko Ikebe, Masaru Morita, Muneyuki Masuda, Yasushi Toh, Yutaka Kiyohara, Toshiharu Ninomiya, Yoshihisa Yamashita, Characteristics of the salivary microbiota in patients with various digestive tract cancers, Frontiers in Microbiology, 10.3389/fmicb.2019.01780, 10, 2019.01, The salivary microbiota is constantly swallowed and delivered to the digestive tract. These bacteria may be associated with gastrointestinal diseases. This case-control study examined the salivary microbiota in patients with digestive tract cancer (DTC) and evaluated their differential distribution based on the cancer sites. We collected saliva samples from 59 patients with cancer in any part of the digestive tract (tongue/pharynx, esophagus, stomach, and large intestine) and from 118 age-and sex-matched control subjects. There was no significant difference in periodontal status between DTC patients and control subjects (P = 0.72). We examined the bacterial diversity and composition in saliva by 16S ribosomal RNA gene sequencing. Salivary bacterial diversity in DTC patients was significantly higher than that in control subjects [number of operational taxonomic units (OTUs), P = 0.02; Shannon index, P < 0.01; Chao1, P = 0.04]. Eleven differentially abundant OTUs in DTC patients were identified using the linear discriminant analysis effect size (LEfSe) method. Based on the cancer sites, the diversity of salivary bacteria was especially higher in tongue/pharyngeal or esophageal cancer patients than in control subjects. Among the 11 differentially abundant OTUs in DTC patients, an OTU corresponding to Porphyromonas gingivalis was more abundant in the saliva of all groups of DTC patients compared to that in control subjects, and an OTU corresponding to Corynebacterium species was more abundant in all groups other than gastric cancer patients (P < 0.01). In addition, the relative abundances of OTUs corresponding to Fusobacterium nucleatum, Streptococcus parasanguinis II, and Neisseria species were significantly higher in tongue/pharyngeal cancer patients compared to their abundances in control subjects (P < 0.01). The relative abundance of an OTU corresponding to the Neisseria species was also significantly higher in gastric cancer patients and that of an OTU corresponding to Actinomyces odontolyticus was significantly higher in colorectal cancer patients (P < 0.01). These results suggest that the salivary microbiota might be associated with various digestive tract cancers..
5. Shinya Kageyama, Mikari Asakawa, Toru Takeshita, Yukari Ihara, Shunsuke Kanno, Toshiro Hara, Ichiro Takahashi, Yoshihisa Yamashita, Transition of Bacterial Diversity and Composition in Tongue Microbiota during the First Two Years of Life, mSphere, 10.1128/mSphere.00187-19, 4, 3, 2019.05, Newborns are constantly exposed to various microbes from birth; hence, diverse commensal bacteria colonize the oral cavity. However, how or when these bacteria construct a complex and stable ecosystem remains unclear. This prospective cohort study examined the temporal changes in bacterial diversity and composition in tongue microbiota during infancy. We longitudinally collected a total of 464 tongue swab samples from 8 infants (age of <6 months at baseline) for approximately 2 years. We also collected samples from 32 children (aged 0 to 2 years) and 73 adults (aged 20 to 29 years) cross-sectionally as control groups. Bacterial diversities and compositions were determined by 16S rRNA gene sequencing. The tongue bacterial diversity in infancy, measured as the number of observed operational taxonomic units (OTUs), rapidly increased and nearly reached the same level as that in adults by around 80 weeks. The overall tongue bacterial composition in the transitional phase, 80 to 120 weeks, was more similar to that of adults than to that of the early exponential phase (EEP), 10 to 29 weeks, according to analysis of similarities. Dominant OTUs in the EEP corresponding to Streptococcus peroris and Streptococcus lactarius exponentially decreased immediately after EEP, around 30 to 49 weeks, whereas several OTUs corresponding to Granulicatella adiacens, Actinomyces odontolyticus, and Fusobacterium periodonticum reciprocally increased during the same period. These results suggest that a drastic compositional shift of tongue microbiota occurs before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years.IMPORTANCE Evaluating the development of oral microbiota during infancy is important for understanding the subsequent colonization of bacterial species and the process of formation of mature microbiota in the oral cavity. We examined tongue microbiota longitudinally collected from 8 infants and found that drastic compositional shifts in tongue microbiota occur before the age of 1 year, and then bacterial diversity and overall bacterial composition reach levels comparable to those in adults by the age of 2 years. These results may be helpful for preventing the development of various diseases associated with oral microbiota throughout life..
6. Koji Ogata, Toru Takeshita, Yukie Shibata, Rie Matsumi, Shinya Kageyama, Mikari Asakawa, Yoshihisa Yamashita, Effect of coffee on the compositional shift of oral indigenous microbiota cultured in vitro, Journal of Oral Science, 10.2334/josnusd.18-0269, 61, 3, 418-424, 2019.09, Coffee is a widely consumed beverage containing organic compounds with antibacterial activity. To investigate its possible effect on the growth of oral indigenous microbiota, saliva samples collected from nine young adults were inoculated into brain heart infusion (BHI) medium with or without addition of coffee compounds and cultured at 37°C in 5% CO2 for 12 h. The total bacterial density and composition after cultivation for 0, 6, and 12 h were determined by quantitative PCR analysis and 16S rRNA gene sequencing, respectively. The increase in total bacterial load was significantly inhibited by addition of the coffee compounds. The microbiota was mostly composed of Streptococcus species after culture in BHI medium regardless of the addition of coffee compounds. The proportion of Streptococcus salivarius was significantly reduced after addition of coffee relative to that in untreated medium alone, whereas the proportions of Streptococcus mitis and Streptococcus infantis were increased. These results suggest that exposure to coffee affects the composition of the oral Streptococcus population, in addition to inhibiting the overall growth of salivary bacteria. Considered in the light of data from earlier epidemiological studies, it is possible to conclude that coffee consumption contributes to better health..
7. Saori Oku, Toru Takeshita, Toshiko Futatsuki, Shinya Kageyama, Mikari Asakawa, Yasuo Mori, Toshihiro Miyamoto, Jun Hata, Toshiharu Ninomiya, Haruhiko Kashiwazaki, Yoshihisa Yamashita, Disrupted tongue microbiota and detection of nonindigenous bacteria on the day of allogeneic hematopoietic stem cell transplantation, PLoS pathogens, 10.1371/journal.ppat.1008348, 16, 3, 2020.01, Disruption of the intestinal microbiota caused by intensive chemotherapy, irradiation and antibiotics can result in development of severe gut graft-versus-host disease and infectious complications, leading to poorer outcomes among allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Although the oral cavity is also densely colonized by indigenous microorganisms, the bacterial composition in allo-HSCT recipients remains unclear. We determined the tongue microbiota composition of 45 patients with hematological disorders on the day of transplantation and compared them to 164 community-dwelling adults. The V1-V2 regions of the 16S rRNA gene sequences demonstrated that the allo-HSCT recipients had less diverse and distinct microbiota from that of community-dwelling adults. The full-length 16S rRNA gene sequences identified 146 bacterial taxa in the microbiota of allo-HSCT recipients, of which 34 bacterial taxa did not correspond to bacteria primarily inhabiting the oral cavity deposited in the expanded Human Oral Microbiome Database. Notably, the detection of Staphylococcus haemolyticus and/or Ralstonia pickettii was significantly associated with a higher risk of mortality during the follow-up period. These results demonstrate that the oral cavity of allo-HSCT recipients is colonized by a disrupted microbiota on the day of transplantation and suggest that detection of specific nonindigenous taxa could be a predictor of transplant outcome..
8. Takashi Hanioka, Manabu Morita, Tatsuo Yamamoto, Koji Inagaki, Pao Li Wang, Hiroshi Ito, Toshiya Morozumi, Toru Takeshita, Nao Suzuki, Hideo Shigeishi, Masaru Sugiyama, Kouji Ohta, Toru Nagao, Nobuhiro Hanada, Miki Ojima, Hiroshi Ogawa, Smoking and periodontal microorganisms, Japanese Dental Science Review, 10.1016/j.jdsr.2019.03.002, 55, 1, 88-94, 2019.11, Resolution of dysbiosis following treatment for periodontal disease and tobacco dependence has been reported in longitudinal intervention studies. In the present report, we evaluated the biological findings regarding the effect of smoking on the periodontal microbiome. A standardized electronic search was conducted using MEDLINE; overall, 1099 papers were extracted. Studies that addressed the relationship between tobacco and periodontal pathogens were included. Finally, 42 papers were deemed appropriate for the present review. Functional changes in periodontal pathogens exposed to nicotine and cigarette smoke extract support the clinical findings regarding dysbiosis of the subgingival microbiome. Dysbiosis of the periodontal microbiome was presented in smokers regardless of their periodontal condition (healthy, gingivitis, or periodontitis) and remained significant only in smokers even after the resolution of experimentally-induced gingivitis and following reduction of clinical signs of periodontitis with non-surgical periodontal treatment and over 3 months post-therapy. Based on these findings, smoking cessation in periodontitis patients is beneficial for promoting a health-compatible subgingival microbial community. To maximize the benefits of these interventions in dental settings, further studies on periodontal microbiome are needed to elucidate the impact of tobacco intervention on preventing recurrence of periodontal destruction in the susceptible subjects..
9. Nao Suzuki, Masahiro Yoneda, Toru Takeshita, Takao Hirofuji, Takashi Hanioka, Induction and inhibition of oral malodor, Molecular Oral Microbiology, 10.1111/omi.12259, 34, 3, 85-96, 2019.06, Volatile sulfur compounds (VSCs) such as hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH) are the main components of oral malodor, and are produced as the end products of the proteolytic processes of oral microorganisms. The main pathway of proteolysis is the metabolism of sulfur-containing amino acids by gram-negative anaerobic bacteria. Gram-positive bacteria may promote VSC production by gram-negative anaerobes by cleaving sugar chains from glycoproteins and thus providing proteins. A large variety of bacteria within the oral microbiota are thought to be involved in the complex phenomenon of halitosis. Oral microbiota associated with a lack of oral malodor, oral microbiota associated with severe and H 2 S-dominant oral malodor, and oral microbiota associated with severe and CH 3 SH-dominant oral malodor have been distinguished through molecular approaches using the 16S rRNA gene. Pathological halitosis may primarily be addressed through treatment of causative diseases. In all cases, plaque control is the basis of oral malodor control, and dentifrices, mouthwashes, and functional foods play a supplementary role in addition to brushing. Recently, the use of natural ingredients in products tends to be favored due to the increase in antibiotic-resistant strains and the side effects of some chemical ingredients. In addition, probiotics and vaccines are expected to offer new strategies for improving the oral conditions through mechanisms other than antibacterial agents..
10. Kenji Takeuchi, Koichiro Matsumoto, Michiko Furuta, Satoru Fukuyama, Toru Takeshita, Hisanobu Ogata, Shino Suma, Yukie Shibata, Y. Shimazaki, Jun Hata, T. Ninomiya, Yoichi Nakanishi, H. Inoue, Yoshihisa Yamashita, Periodontitis Is Associated with Chronic Obstructive Pulmonary Disease, Journal of Dental Research, 10.1177/0022034519833630, 98, 5, 534-540, 2019.05, Although they are known to share pathophysiological processes, the relationship between periodontitis and chronic obstructive pulmonary disease (COPD) is not fully understood. The aim of the present study was to test the hypothesis that periodontitis is associated with a greater risk of development of COPD, when smoking is taken into account. The analysis in a 5-y follow-up population-based cohort study was based on 900 community-dwelling Japanese adults (age: 68.8 ± 6.3 [mean ± SD], 46.0% male) without COPD aged 60 or older with at least 1 tooth. Participants were classified into 3 categories according to baseline periodontitis severity (no/mild, moderate, and severe). COPD was spirometrically determined by a fixed ratio of <0.7 for forced expiratory volume in 1 s (FEV 1 )/forced vital capacity (FVC) and by FEV 1 /FVC below the lower limit of normal. Poisson regression was used to calculate the relative risk (RR) of developing COPD according to the severity of periodontitis. The population attributable fraction (PAF) was also calculated. During follow-up, 22 (2.4%) subjects developed COPD. Compared with no/mild periodontitis subjects, a significantly increased risk of COPD occurred among severe periodontitis subjects (RR = 3.55; 95% confidence interval [CI], 1.18 to 10.67), but no significant differences were observed between the no/mild and moderate categories (RR = 1.48; 95% CI, 0.56 to 3.90). After adjustment for potential confounders, including smoking intensity, the relationship between severe periodontitis and risk of COPD remained significant (RR = 3.51; 95% CI, 1.15 to 10.74). Likewise, there was a positive association of periodontitis severity with risk of COPD (P for trend = 0.043). The PAF for COPD due to periodontitis was 22.6%. These data highlight the potential importance of periodontitis as a risk factor for COPD..
11. Kenji Takeuchi, Maya Izumi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Shinya Kageyama, Yuka Okabe, Sumio Akifusa, Seijun Ganaha, Yoshihisa Yamashita, Denture wearing moderates the association between aspiration risk and incident pneumonia in older nursing home residents
A prospective cohort study, International journal of environmental research and public health, 10.3390/ijerph16040554, 16, 4, 2019.02, Aspiration is increasingly recognized as a major risk for pneumonia, but a potential link between wearing dentures and incident pneumonia with aspiration risk is unclear. The aim of this study was to investigate whether denture wearing moderates the association between aspiration risk and incident pneumonia in older adults. We used prospective cohort data of 156 residents aged >70 years from eight nursing homes in Aso, Japan. Aspiration risk was evaluated using the modified water swallowing test. During a 1-year follow-up (2014 to 2015), information on incident pneumonia was obtained from nursing home medical records. During follow-up, pneumonia developed in 7.1% of participants. In the multivariate-adjusted Cox proportional hazards model, after adjusting for potential confounders, aspiration risk was independently associated with a 4.4-fold higher hazard ratio (HR) of incident pneumonia (95% confidence interval, CI, 1.16–16.43). The difference in the risk of incident pneumonia between subjects with aspiration risk who were wearing dentures and those not at risk of aspiration was not significant, whereas those with aspiration risk without dentures had a 7.3-fold higher HR of incident pneumonia than those not at risk of aspiration (95% CI, 1.02–52.63). Denture wearing might partially moderate the increased risk of incident pneumonia associated with aspiration risk..
12. Naoko Yatabe, Kenji Takeuchi, Maya Izumi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Shino Suma, Shinya Kageyama, Seijun Ganaha, Haruka Tohara, Yoshihisa Yamashita, Decreased cognitive function is associated with dysphagia risk in nursing home older residents, Gerodontology, 10.1111/ger.12366, 35, 4, 376-381, 2018.12, Objective: To examine the association between cognitive function and dysphagia risk among Japanese nursing home residents. Background: Reduction in cognitive function can influence the intake of food during swallowing, and may be an aggravating factor in dysphagia. Methods: This cross-sectional study included 236 residents aged ≥60 years from eight nursing homes. Screening of dysphagia, especially aspiration risk, was conducted using the Modified Water Swallow Test, whose scores from one to three were classified as at risk of dysphagia. Cognitive function was evaluated using Mini-Mental State Examination (MMSE). Number of teeth and occlusal support were evaluated by clinical examination. The participants were stratified into dentulous and edentulous groups, because the number of teeth could influence swallowing function. The odds ratio (OR) and 95% confidence interval (CI) for dysphagia risk based on the scores of MMSE were calculated using logistic regression. Demographic characteristics, activity of daily living, comorbidities, health behaviour and occlusal support were used as covariates. Results: Among the 236 participants (111 dentulous participants and 125 edentulous participants) included in our analysis, 16.9% belonged to risk of dysphagia. Dentulous participants with higher scores of MMSE tended to have significantly lower odds of dysphagia risk after adjusting for covariates (OR = 0.87, 95% CI = 0.80-0.96). Despite the lack of significant differences, edentulous participants with higher score of MMSE tended to have lower odds of dysphagia risk (OR = 0.92, 95% CI = 0.83-1.00). Conclusion: Decreased cognitive function may be an independent predictor of dysphagia among dentulous and edentulous adults..
13. Kenji Takeuchi, Mikari Asakawa, Takafumi Hashiba, Toru Takeshita, Youji Saeki, Yoshihisa Yamashita, Effects of xylitol-containing chewing gum on the oral microbiota, Journal of Oral Science, 10.2334/josnusd.17-0446, 60, 4, 588-594, 2018.12, In this interventional study, a randomized controlled trial was used to evaluate the short-term effects of xylitol-containing chewing gum on the salivary microbiota. In total, 70 healthy adult men recruited from the Japan Ground Self Defense Force participated in the study during a 2-day training at Yamaguchi camp, Yamaguchi Prefecture, Japan. The men were randomly divided into two groups: one group chewed two pieces of xylitol-containing chewing gum 7 times/day for 2 days (n = 34) and the other did not (n = 36). Baseline and follow-up stimulated saliva samples were collected and the salivary microbial composition was assessed using the 16S rRNA gene next-generation sequencing analysis. The total salivary bacterial count was quantified using a quantitative real-time PCR system. No statistically significant difference was found between the two groups regarding any parameter analyzed in the baseline samples; however, the follow-up samples of the test group showed significantly lower total salivary bacterial count than those of the control group. Conversely, no significant difference was observed in the overall composition of the salivary microbiota between the baseline and follow-up samples of the two groups. These results indicate that xylitol-containing chewing gum inhibits the increase in total salivary bacteria over a short time during which the salivary microbial composition is not affected..
14. Kenji Takeuchi, Koichiro Matsumoto, Michiko Furuta, Satoru Fukuyama, Toru Takeshita, Hiroaki Ogata, Shino Suma, Yukie Shibata, Yoshihiro Shimazaki, Jun Hata, Toshiharu Ninomiya, Yoichi Nakanishi, Hiromasa Inoue, Yoshihisa Yamashita, Periodontal status and lung function decline in the community
the Hisayama study, Scientific reports, 10.1038/s41598-018-31610-3, 8, 1, 2018.12, This study aimed to determine whether periodontal status is related to a decline in lung function in a general Japanese population. We followed a total of 1,650 community-dwelling individuals (≥40 years) without chronic obstructive pulmonary disease, with at least one teeth, for 3 years. Periodontal status was assessed at baseline by clinical attachment loss (CAL) and probing pocket depth (PPD) at two sites for each tooth, and the mean values were calculated for each subject. Lung function was measured at baseline and follow-up using spirometry, and longitudinal decline in forced expiratory volume in one second (FEV
) was calculated. Multivariate Poisson regression with robust error variance was used to estimate risk ratio (RR). After adjusting for potential confounders including smoking status, there was a tendency for the adjusted RR of developing rapid lung function decline (≥160 mL/3years, the highest quartile of the distribution of FEV
declines) to increase as mean CAL levels increased (P trend = 0.039). Likewise, a positive association was observed between mean PPD levels and RR of developing rapid lung function decline (P trend = 0.047). Our findings suggest deterioration of periodontal status could be a risk factor for rapid lung function decline in the general Japanese population..
15. Michiko Furuta, Kenji Takeuchi, Yoshihiro Shimazaki, Toru Takeshita, Yukie Shibata, Jun Hata, Daigo Yoshida, Deok Young Park, Toshiharu Ninomiya, Yoshihisa Yamashita, Comparison of the periodontal condition in Korean and Japanese adults
A cross-sectional study, BMJ open, 10.1136/bmjopen-2018-024332, 8, 11, 2018.11, Objectives: Reports from national surveys in South Korea and Japan have indicated that the prevalence of periodontal disease is lower in Korea than in Japan. However, these national surveys have not evaluated factors related to periodontal health condition, including diabetes and metabolic syndrome. This study compared periodontal conditions between Korean and Japanese adults, in the context of general health status. Design Cross-sectional study. Setting National survey in South Korea (Korean National Health and Nutrition Examination Survey, 'KNHANES') and a population-based study in Japan (Hisayama study); both were conducted in 2012. Participants: This study included 3574 Korean and 2205 Japanese adults aged 40-79 years. Outcome measures: Periodontal condition was assessed by using the Community Periodontal Index (CPI). Examiners in Japan underwent clinical calibration training for periodontal examination with a gold-standard examiner from KNHANES, prior to the Hisayama study. Results The age-adjusted prevalences of periodontal disease, defined as CPI score ≥3, were 31.4% and 42.1% in South Korea and Japan, respectively (p<0.001). The age-adjusted prevalences of diabetes (p=0.018) and metabolic syndrome (p=0.001) were higher in Korea than in Japan. The numbers of present and filled teeth and percentages of participants who visited a dental clinic in the last 12 months were higher in Japan than in Korea (all p<0.001). Logistic regression analysis showed that the Japanese participants were more likely to have periodontal disease than were the Korean participants, after adjusting for age, sex, occupation, oral health status, oral health behaviour, diabetes and metabolic syndrome. Conclusions: A higher prevalence of periodontal disease was found in Japanese participants than in Korean participants. Further studies are needed to more clearly elucidate factors underlying the difference in periodontal conditions between the two populations, including those related to the dental healthcare system and dietary intake..
16. Mikari Asakawa, Toru Takeshita, Michiko Furuta, Shinya Kageyama, Kenji Takeuchi, Jun Hata, Toshiharu Ninomiya, Yoshihisa Yamashita, Tongue Microbiota and Oral Health Status in Community-Dwelling Elderly Adults, mSphere, 10.1128/mSphere.00332-18, 3, 4, 2018.08, Tongue microbiota are a dominant source of oral microbial populations that are ingested with saliva, and therefore careful attention is required for the maintenance of health of elderly adults, who are susceptible to aspiration of oral contents. This study aimed to investigate the variation in tongue microbiota among community-dwelling elderly adults. Following a dental examination, tongue coating was collected from a 15-mm-diameter circular area at the center of the tongue dorsum of 506 elderly adults aged 70 to 80 years inhabiting the town of Hisayama, Japan. The microbial composition and density were determined by a 16S rRNA gene sequencing approach using a next-generation sequencer and quantitative PCR analysis, respectively. Co-occurrence network analysis identified two cohabiting groups of predominant commensals, one of which was primarily composed of Prevotella histicola, Veillonella atypica, Streptococcus salivarius, and Streptococcus parasanguinis; these organisms have been previously associated with an increased risk of mortality due to pneumonia in the frail elderly. This bacterial group was more predominant in the elderly with fewer teeth, a higher plaque index, and more dental caries experience, whereas the total bacterial density was independent of these traits. A higher density of fungi was also observed in the elderly with these traits, as well as in individuals who wore dentures. These results suggest that elderly adults with poorer oral health swallow a more dysbiotic microbiota formed on the tongue.IMPORTANCE Aspiration of oral contents can lead to pneumonia, which is a major cause of death among elderly adults susceptible to swallowing impairments. Tongue microbiota are a dominant source of oral microbial populations that are ingested with saliva. This large-scale population-based study revealed variations in the tongue microbiota among community-dwelling elderly adults. The total bacterial density was independent of the conditions of teeth surrounding the tongue, whereas the microbiota composition, especially the relative abundances of predominant commensals, showed an association with tooth conditions. Our results demonstrate that the elderly with fewer teeth, poorer dental hygiene, and more dental caries experience constantly ingest more dysbiotic microbiota, which could be harmful for their respiratory health..
17. Akihiko Tanaka, Kenji Takeuchi, Michiko Furuta, Toru Takeshita, Shino Suma, Takashi Shinagawa, Yoshihiro Shimazaki, Yoshihisa Yamashita, Relationship of toothbrushing to metabolic syndrome in middle-aged adults, Journal of Clinical Periodontology, 10.1111/jcpe.12876, 45, 5, 538-547, 2018.05, Aim: To examine the effect of toothbrushing on the development of metabolic syndrome (MetS), including assessment of periodontal status, in middle-aged adults. Methods: This 5-year follow-up retrospective study was performed in 3,722 participants (2,897 males and 825 females) aged 35–64 years who underwent both medical check-ups and dental examinations. Metabolic components included obesity, elevated triglycerides, blood pressure, fasting glucose and reduced high-density lipoprotein. Toothbrushing frequency was assessed using a questionnaire. Periodontal disease was defined as having at least one site with a pocket depth of ≥4 mm. Logistic regression analysis was performed to evaluate the relationship between toothbrushing frequency at the baseline examination and the development of MetS (≥3 components). Results: During follow-up, 11.1% of participants developed MetS. After adjusting for potential confounders including periodontal disease, participants with more frequent daily toothbrushing tended to have significantly lower odds of developing MetS (p for trend =.01). The risk of development of MetS was significantly lower in participants brushing teeth ≥3 times/day than in those brushing teeth ≤1 time/day (odds ratio = 0.64, 95% confidence interval = 0.45–0.92). Conclusions: Frequent daily toothbrushing was associated with lower risk of development of MetS..
18. Shigeyuki Yokoyama, Kenji Takeuchi, Yukie Shibata, Shinya Kageyama, Rie Matsumi, Toru Takeshita, Yoshihisa Yamashita, Characterization of oral microbiota and acetaldehyde production, Journal of Oral Microbiology, 10.1080/20002297.2018.1492316, 10, 1, 2018.01, Background: Neisseria has been reported to be a high producer of acetaldehyde (ACH), a carcinogen, from ethanol in vitro, but no information exists regarding whether the ACH production depends on oral microbiota profiles. Objective and Design: To explore the salivary microbiota profiles with respect to ACH production ability in the oral cavity using a cross-sectional design. Results: Using 16S rRNA gene amplicon sequencing, we classified 100 saliva samples into two types of communities (I and II). Salivary ACH production ability from ethanol was measured using gas chromatography and was found to vary over a 30-fold range. ACH production ability was significantly higher in the type I community, wherein the relative abundance of Neisseria species was significantly lower. Multivariate logistic regression analysis showed that the subjects with the type I community exhibited significantly higher probability of high ACH production ability than those with the type II community (P = 0.014). Moreover, the relative abundance of Neisseria species was inversely correlated with the ACH production ability (P = 0.002). Conclusion: The salivary microbiota profile with a lower relative abundance of Neisseria species was independently associated with high ACH production ability, despite Neisseria species are dominant producers of ACH in vitro..
19. Shinya Kageyama, Toru Takeshita, Michiko Furuta, Mikiko Tomioka, Mikari Asakawa, Shino Suma, Kenji Takeuchi, Yukie Shibata, Yasuyuki Iwasa, Yoshihisa Yamashita, Relationships of variations in the tongue microbiota and pneumonia mortality in nursing home residents, Journals of Gerontology - Series A Biological Sciences and Medical Sciences, 10.1093/gerona/glx205, 73, 8, 1097-1102, 2018.01, Background: Aspiration of oral debris, containing dense oral bacteria, is a major cause of pneumonia in elderly adults. This study investigated the relationship between tongue microbiota composition and incidence of pneumonia-related deaths, in nursing home residents. Methods: The subjects were assessed for health conditions, including their tongue microbiota, at baseline. We determined tongue microbiota profiles by 16S ribosomal RNA gene sequencing and clustering approach. All subjects (n = 173) were followed prospectively for a median of 19 months to assess the incidence of all-cause death, including pneumonia-related death. We evaluated risk estimates of microbiota effects on death using multivariate Cox proportional hazards regression analysis. Results: Tongue microbiota were classified into two community types: type I was dominated by Prevotella and Veillonella species, while type II was dominated by Neisseria and Fusobacterium species. The subjects with type I microbiota exhibited a significantly greater risk of all-cause death (adjusted hazard ratio [aHR] = 3.79, 95% confidence interval [CI] = 1.38-10.39) and pneumonia-related death (aHR = 13.88, 95% CI = 1.64-117.21), than those with type II microbiota. There was no significant association between microbiota type and other-cause death. Conclusions: The tongue microbiota type was significantly associated with an increased mortality risk from pneumonia in nursing home residents..
20. Kenji Takeuchi, Michiko Furuta, Y. Okabe, Shino Suma, Toru Takeshita, S. Akifusa, M. Adachi, T. Kinoshita, T. Kikutani, Yoshihisa Yamashita, Swallowing disorders and 1-year functional decline in community-dwelling older adults receiving home care, Journal of oral rehabilitation, 10.1111/joor.12577, 44, 12, 982-987, 2017.12, The purpose of this study was to clarify the effect of swallowing disorders on functional decline in community-dwelling older adults receiving home care. This was a 1-year follow-up survey of 176 individuals ≥60 years living at home and receiving homecare services, without total dependence in basic daily living activities, in two mid-sized municipalities in Fukuoka, Japan. Functional decline was measured using the Barthel index (BI), and the primary outcome was total dependence in basic daily living activities (BI ≤ 20 points). Swallowing function was assessed using cervical auscultation, and the primary predictor was swallowing disorders. Logistic regression models were used to assess univariate and multivariate associations between baseline swallowing function and functional decline during follow-up. During follow-up 16 (9.1%), the participants became totally dependent in basic daily living activities. The participants with swallowing disorders had 6.41 times higher odds of total dependence in basic daily living activities compared to participants with normal swallowing function. After adjusting for potential confounders, swallowing disorders were significantly associated with higher odds of total dependence in basic daily living activities (odds ratio = 5.21, 95% confidence interval = 1.33-20.44). Regarding swallowing disorders, the corresponding population attributable fraction (%) of the incidence of total dependence in basic daily living activities was 50.4%. The current findings demonstrated that swallowing disorders were associated with greater risk of functional decline in basic daily living activities among older adults living at home and receiving home nursing care. Maintenance and improvement of swallowing function may prevent late-life functional decline..
21. Haruna Yasunaga, Toru Takeshita, Yukie Shibata, Michiko Furuta, Yoshihiro Shimazaki, Sumio Akifusa, Toshiharu Ninomiya, Yutaka Kiyohara, Ichiro Takahashi, Yoshihisa Yamashita, Exploration of bacterial species associated with the salivary microbiome of individuals with a low susceptibility to dental caries, Clinical Oral Investigations, 10.1007/s00784-016-2035-5, 21, 8, 2399-2406, 2017.11, Objective: Dental caries is caused by acidogenic plaque microbiota formed on saliva-bathed tooth surfaces, in which multiple organisms act collectively to initiate and expand a cavity. We explored bacterial species associated with the salivary microbiome of individuals with low susceptibility to dental caries. Materials and methods: The bacterial composition of saliva from 19 young adults was analyzed using barcoded pyrosequencing of the 16S rRNA gene; we compared 10 caries-experienced (CE) and nine caries-free (CF) individuals. A quantitative PCR assay of saliva from 139 orally healthy adults aged 40–59 years was carried out to confirm the result obtained by pyrosequencing analysis. Results: The microbiomes of CF individuals showed more diverse communities with a significantly greater proportion of the genus Porphyromonas. Among operational taxonomic units (OTUs) corresponding to the genus Porphyromonas, the OTU corresponding to P. pasteri was the most predominant and its relative abundance in CF individuals was significantly greater than in CE individuals (P < 0.001, Wilcoxon rank sum test). A quantitative PCR assay of saliva confirmed that the amounts of P. pasteri were significantly higher in individuals with lower caries experience (filled teeth <15, n = 67) than in those with higher caries experience (filled teeth ≥15, n = 72) (P < 0.001, Student’s t test). Conclusion: These results revealed an association between a greater abundance of P. pasteri and lower susceptibility to dental caries. Clinical relevance: P. pasteri may be a bacterial species that could potentially be used as a marker for maintaining a healthy oral microbiome against dental caries..
22. M. N. Zakaria, Michiko Furuta, Toru Takeshita, Yukie Shibata, R. Sundari, N. Eshima, Toshiharu Ninomiya, Yoshihisa Yamashita, Oral mycobiome in community-dwelling elderly and its relation to oral and general health conditions, Oral Diseases, 10.1111/odi.12682, 23, 7, 973-982, 2017.10, Objective: Oral fungal infection is generally associated with dysbiosis related to antibiotic use, immunodeficiency, or frailty. However, fungal colonization in a typical population without apparent symptoms and its associated conditions are poorly understood. In this study, oral fungal colonization in community-dwelling and independently living elderly populations was evaluated and factors affecting fungal colonization were analyzed. Subjects and Methods: The subjects (410; 181 males and 229 females) were 75–99 years of age; those under prior antibiotic use were excluded. Fungal populations in the saliva were evaluated by PCR-based molecular techniques. Body mass index (BMI), smoking habits, and oral health conditions were examined. Results: Salivary fungal amounts exceeded 104 CFU/ml in 63 (15.4%) of 410 subjects. Candida albicans was most frequently detected (98.4%), followed by Candida glabrata (54.0%), and Candida dubliniensis (38.1%) in those subjects with fungi at 104 CFU/ml or over. Fungi at 104 CFU/ml or over in the presence of C. glabrata or C. dubliniensis was significantly associated with low BMI. Conclusions: Candida albicans, C. glabrata, and C. dubliniensis dominated the oral mycobiome in Japanese community-dwelling elderly. Lower BMI might signify compromised health status and thus could result in susceptibility to specific candidiasis by C. glabrata and C. dubliniensis..
23. Kenji Takeuchi, Tomoyuki Ohara, Michiko Furuta, Toru Takeshita, Yukie Shibata, Jun Hata, Daigo Yoshida, Yoshihisa Yamashita, Toshiharu Ninomiya, Tooth Loss and Risk of Dementia in the Community
the Hisayama Study, Journal of the American Geriatrics Society, 10.1111/jgs.14791, 65, 5, e95-e100, 2017.05, Objectives: To clarify the effect of tooth loss on development of all-cause dementia and its subtypes in an elderly Japanese population. Design: Prospective cohort study. Setting: The Hisayama Study, Japan. Participants: Community-dwelling Japanese adults without dementia aged 60 and older (N = 1,566) were followed for 5 years (2007–2012). Measurements: Participants were classified into four categories according to baseline number of remaining teeth (≥20, 10–19, 1–9, 0). The risk estimates of the effect of tooth loss on the development of all-cause dementia, Alzheimer's disease (AD), and vascular dementia (VaD) were computed using a Cox proportional hazards model. Results: During follow-up, 180 (11.5%) subjects developed all-cause dementia; 127 (8.1%) had AD, and 42 (2.7%) had VaD. After adjusting for potential confounders, there was a tendency for the multivariable-adjusted hazard ratio of all-cause dementia to increase with decrease in number of remaining teeth (P for trend =.04). The risk of all-cause dementia was 1.62 times as great in subjects with 10 to 19 teeth, 1.81 times as great in those with one to nine teeth, and 1.63 times as great in those with no teeth as in those with 20 teeth or more. An inverse association was observed between number of remaining teeth and risk of AD (P for trend =.08), but no such association was observed with risk of VaD (P for trend =.20). Conclusion: Tooth loss is associated with an increased risk of all-cause dementia and AD in the Japanese population..
24. Kenji Takeuchi, Maya Izumi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Shinya Kageyama, Seijun Ganaha, Yoshihisa Yamashita, Association between posterior teeth occlusion and functional dependence among older adults in nursing homes in Japan, Geriatrics and Gerontology International, 10.1111/ggi.12762, 17, 4, 622-627, 2017.04, Aim: To examine whether posterior teeth occlusion is associated with functional dependence regardless of the number of natural teeth among Japanese nursing home residents. Methods: The present cross-sectional study included 234 residents aged >60 years from eight nursing homes in Aso City, Japan. Functional dependence in basic activities of daily living was measured using the Barthel Index, and the primary outcome was independence for essential personal care (Barthel Index ≥ 60 points). Posterior teeth occlusion was assessed by a dentist using the total number of functional tooth units, depending on the number and location of the remaining natural and artificial teeth on implant-supported, fixed, and removable prostheses. Logistic regression models were used to assess univariate and multivariate associations between the total number of functional tooth units and independence for essential personal care. Models were sequentially adjusted for the number of natural teeth, demographic characteristics, socioeconomic status, health behaviors and comorbidities. Results: Independence for essential personal care was found in 14.1% of participants. Greater total numbers of functional tooth units were significantly associated with greater odds of independence for essential personal care (odds ratio 1.12; 95% confidence interval 1.01–1.24). The association remained significant after adjustment for the number of natural teeth and other possible confounders (odds ratio 1.14, 95% confidence interval 1.01–1.29). Conclusions: Posterior teeth occlusion was independently associated with functional dependence among nursing home older residents in Japan. The maintenance and restoration of posterior teeth occlusion might be preventive factors against late-life functional decline. Geriatr Gerontol Int 2017; 17: 622–627..
25. Shinya Kageyama, Toru Takeshita, Mikari Asakawa, Yukie Shibata, Kenji Takeuchi, Wataru Yamanaka, Yoshihisa Yamashita, Relative abundance of total subgingival plaque-specific bacteria in salivary microbiota reflects the overall periodontal condition in patients with periodontitis, PloS one, 10.1371/journal.pone.0174782, 12, 4, 2017.03, Increasing attention is being focused on evaluating the salivary microbiota as a promising method for monitoring oral health; however, its bacterial composition greatly differs from that of dental plaque microbiota, which is a dominant etiologic factor of oral diseases. This study evaluated the relative abundance of subgingival plaque-specific bacteria in the salivary microbiota and examined a relationship between the abundance and severity of periodontal condition in patients with periodontitis. Four samples (subgingival and supragingival plaques, saliva, and tongue coating) per each subject were collected from 14 patients with a broad range of severity of periodontitis before periodontal therapy. The bacterial composition was analyzed by 16S rRNA gene amplicon sequencing using Ion PGM. Of the 66 species- level operational taxonomic units (OTUs) representing the mean relative abundance of 1% in any of the four niches, 12 OTUs corresponding to known periodontal pathogens, including Porphyromonas gingivalis, were characteristically predominant in the subgingival plaque and constituted 37.3 ± 22.9% of the microbiota. The total relative abundance of these OTUs occupied only 1.6 ± 1.2% of the salivary microbiota, but significantly correlated with the percentage of diseased sites (periodontal pocket depth 4 mm; r = 0.78, P < 0.001), in addition to the abundance of subgingival plaque microbiota (r = 0.61, P = 0.02). After periodontal therapy, the total relative abundance of these 12 OTUs was evaluated as well as before periodontal therapy and reductions of the abundance through periodontal therapy were strongly correlated in saliva and subgingival plaque (r = 0.81, P < 0.001). Based on these results, salivary microbiota might be a promising target for the evaluation of subgingival plaque-derived bacteria representing the present condition of periodontal health..
26. Y. Okabe, Kenji Takeuchi, M. Izumi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Shinya Kageyama, S. Ganaha, Yoshihisa Yamashita, Posterior teeth occlusion and dysphagia risk in older nursing home residents
a cross-sectional observational study, Journal of Oral Rehabilitation, 10.1111/joor.12472, 44, 2, 89-95, 2017.02, The total number of natural teeth was related to swallowing function among older adults; however, limited information is available regarding the impact of occluding pairs of teeth on swallowing function. This study aimed to examine the association between posterior teeth occlusion and dysphagia risk in older nursing home residents. This cross-sectional study included 238 residents aged ≥60 years from eight nursing homes in Aso City, Japan. Swallowing function was evaluated using the modified water swallowing test (MWST); the primary outcome was dysphagia risk (MWST score ≤3). Posterior teeth occlusion was assessed using number of functional tooth units (FTUs), determined based on number and location of the remaining natural and artificial teeth on implant-supported, fixed or removable prostheses. Univariate and multivariate logistic regression analyses were performed to examine the association between posterior teeth occlusion and dysphagia risk, adjusted for the covariates of number of natural teeth, demographic characteristics, comorbidities, physical function, body mass index and cognitive function. Of the 238 subjects, 44 (18·5%) were determined to be at risk of dysphagia based on the MWST scores. The odds ratio (OR) of dysphagia risk decreased in subjects with higher total FTUs [OR = 0·92, 95% confidence interval (CI) 0·87–0·98]. After adjusting for covariates, this association remained significant (OR = 0·90, 95% CI 0·84–0·97). Loss of posterior teeth occlusion was independently associated with dysphagia risk in older nursing home residents. Maintaining and restoring posterior teeth occlusion may be an effective measure to prevent dysphagia..
27. Yoshihiro Shimazaki, Bohan Fu, Koji Yonemoto, Sumio Akifusa, Yukie Shibata, Toru Takeshita, Toshiharu Ninomiya, Yutaka Kiyohara, Yoshihisa Yamashita, Stimulated salivary flow rate and oral health status, Journal of Oral Science, 10.2334/josnusd.16-0372, 59, 1, 55-62, 2017.01, This study examined the relationship between stimulated salivary flow rate and oral health status in an adult population. Multinomial multivariate logistic regression analysis was used to examine the associations of salivary flow rate with dental caries status and periodontal status at the individual level among 2,110 Japanese adults with ≥10 teeth. Then, a spline model was used to examine the nonlinear relationship between salivary flow rate and teeth with dental caries or periodontal disease in multilevel analysis. Odds ratios were calculated for a 1.0-mL/min reduction in salivary flow rate at a point. After adjusting for confounding variables, participants with a flow rate ≤3.5 mL/min had significantly higher odds ratios for high caries status, and participants with a flow rate ≤1.4 mL/min had a higher odds ratio for broad periodontal disease, than did those with a flow rate >3.5 mL/min. In spline models, the odds ratio for teeth with dental caries or periodontal disease increased with reduced saliva secretion. The present findings suggest that decreased saliva secretion affects both dental caries and general periodontal health status..
28. Yoshihisa Yamashita, Toru Takeshita, The oral microbiome and human health, Journal of Oral Science, 10.2334/josnusd.16-0856, 59, 2, 201-206, 2017.01, In this brief review, we discuss our previous research on the relationship between the bacterial composition of salivary microbiota and periodontal disease. Analysis using a terminal restriction fragment length polymorphism method and an international comparison suggest that the predominance of the genera Prevotella and Veillonella in the salivary microbiota is attributable to periodontal disease conditions, and that the predominance of the genus Neisseria indicates healthy periodontal conditions. Furthermore, we recently used next-generation sequencing technology to perform a detailed large-scale analysis of the salivary microbiota. An important finding of that study was that high bacterial richness in the salivary microbiota was significantly associated with poor oral health, as indicated by decayed teeth, periodontitis, and poor oral hygiene. Another important result was that relative abundance of predominant bacteria in saliva was significantly associated with oral health-related conditions. Of the two different cohabiting groups of bacteria found in the salivary microbiota, a greater relative abundance of group I bacteria, which include Prevotella and Veillonella species, was associated with poor oral health, high body mass index, and old age. These findings suggest that the salivary microbiota reflects oral and systemic conditions..
29. Xiaochen Men, Yukie Shibata, Toru Takeshita, Yoshihisa Yamashita, Identification of anion channels responsible for fluoride resistance in oral streptococci, PloS one, 10.1371/journal.pone.0165900, 11, 11, 2016.11, Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F- channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species..
30. Yumi Imabayashi, Masafumi Moriyama, Toru Takeshita, Shinsuke Ieda, Hayashida Jun-Nosuke, Akihiko Tanaka, takashi maehara, Sachiko Furukawa, Miho Ohta, Keigo Kubota, Masaki Yamauchi, Noriko Ishiguro, Yoshihisa Yamashita, Seiji Nakamura, Molecular analysis of fungal populations in patients with oral candidiasis using next-generation sequencing, Scientific reports, 10.1038/srep28110, 6, 2016.06, Oral candidiasis is closely associated with changes in oral fungal biodiversity and is caused primarily by Candida albicans. However, the widespread use of empiric and prophylactic antifungal drugs has caused a shift in fungal biodiversity towards other Candida or yeast species. Recently, next-generation sequencing (NGS) has provided an improvement over conventional culture techniques, allowing rapid comprehensive analysis of oral fungal biodiversity. In this study, we used NGS to examine the oral fungal biodiversity of 27 patients with pseudomembranous oral candidiasis (POC) and 66 healthy controls. The total number of fungal species in patients with POC and healthy controls was 67 and 86, respectively. The copy number of total PCR products and the proportion of non-C. albicans, especially C. dubliniensis, in patients with POC, were higher than those in healthy controls. The detection patterns in patients with POC were similar to those in controls after antifungal treatment. Interestingly, the number of fungal species and the copy number of total PCR products in healthy controls increased with aging. These results suggest that high fungal biodiversity and aging might be involved in the pathogenesis of oral candidiasis. We therefore conclude that NGS is a useful technique for investigating oral candida infections..
31. Michiko Furuta, An Liu, Takashi Shinagawa, Kenji Takeuchi, Toru Takeshita, Yoshihiro Shimazaki, Yoshihisa Yamashita, Tooth loss and metabolic syndrome in middle-aged Japanese adults, Journal of Clinical Periodontology, 10.1111/jcpe.12523, 43, 6, 482-491, 2016.06, Aim Metabolic syndrome is associated with periodontal disease and dental caries; however, little attention has been given to the association between metabolic syndrome and tooth loss, which is the endpoint of these two diseases. This study examined this association in middle-aged adults over a 5-year period. Methods A retrospective study was performed in 2107 participants (1718 males and 389 females) aged 35-60 years who underwent annual dental check-ups, to evaluate tooth loss and metabolic components, including obesity, elevated triglycerides, blood pressure, fasting glucose, and reduced high-density lipoprotein. Logistic regression analysis was performed to evaluate the association between metabolic syndrome (≥3 components) at the baseline examination and tooth loss. Results Over a 5-year period, 10.8% of the participants lost at least one tooth. Compared to those with no metabolic components, participants with ≥3 components had an increased risk of tooth loss (odds ratio = 1.54; 95% confidence interval: 1.01-2.37), adjusting for age, gender, dental caries experience, attachment loss, oral hygiene status, number of teeth, tooth brushing frequency, smoking, and occupational status. Conclusions Metabolic syndrome was associated with the incidence of tooth loss among middle-aged adults..
32. Toru Takeshita, Shinya Kageyama, Michiko Furuta, Hidenori Tsuboi, Kenji Takeuchi, Yukie Shibata, Yoshihiro Shimazaki, Sumio Akifusa, Toshiharu Ninomiya, Yutaka Kiyohara, Yoshihisa Yamashita, Bacterial diversity in saliva and oral health-related conditions
The Hisayama Study, Scientific reports, 10.1038/srep22164, 6, 2016.02, This population-based study determined the salivary microbiota composition of 2,343 adult residents of Hisayama town, Japan, using 16S rRNA gene next-generation high-throughput sequencing. Of 550 identified species-level operational taxonomic units (OTUs), 72 were common, in ≥75% of all individuals, as well as in ≥75% of the individuals in the lowest quintile of phylogenetic diversity (PD). These "core" OTUs constituted 90.9 ± 6.1% of each microbiome. The relative abundance profiles of 22 of the core OTUs with mean relative abundances ≥1% were stratified into community type I and community type II by partitioning around medoids clustering. Multiple regression analysis revealed that a lower PD was associated with better conditions for oral health, including a lower plaque index, absence of decayed teeth, less gingival bleeding, shallower periodontal pockets and not smoking, and was also associated with tooth loss. By contrast, multiple Poisson regression analysis demonstrated that community type II, as characterized by a higher ratio of the nine dominant core OTUs, including Neisseria flavescens, was implicated in younger age, lower body mass index, fewer teeth with caries experience, and not smoking. Our large-scale data analyses reveal variation in the salivary microbiome among Japanese adults and oral health-related conditions associated with the salivary microbiome..
33. Yoshihisa Yamashita, Toru Takeshita, Clinical importance of oral microbiome evaluation, Japanese Journal of Clinical Chemistry, 44, 4, 290-297, 2015.10.
34. Kenji Takeuchi, Maya Izumi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Shinya Kageyama, Seijun Ganaha, Yoshihisa Yamashita, Posterior teeth occlusion associated with cognitive function in nursing home older residents
A cross-sectional observational study, PloS one, 10.1371/journal.pone.0141737, 10, 10, 2015.10, Early detection and subsequent reduction of modifiable risk factors for cognitive decline is important for extending healthy life expectancy in the currently aging society. Although a recent increase in studies on the state or number of the teeth and cognitive function, few studies have focused on the association between posterior teeth occlusion necessary to maintain chewing function and cognitive function among older adults. This study examined the association between posterior teeth occlusion and cognitive function in nursing home older residents. In this cross-sectional study, 279 residents aged ≤60 years from eight nursing homes in Aso City, Japan participated in cognitive function and dental status assessments and completed a comprehensive questionnaire survey in 2014. Cognitive function was measured using a Mini-Mental State Examination (MMSE). Posterior teeth occlusion was assessed using a total number of functional tooth units (total-FTUs), depending on the number and location of the remaining natural and artificial teeth on implant-supported, fixed, and removable prostheses. Linear regression models were used to assess univariate and multivariate associations between total-FTUs and MMSE scores. Models were sequentially adjusted for demographic characteristics, number of natural teeth, socioeconomic status, health behaviors, comorbidities, physical function, and nutritional status. Among the 200 residents included in our analysis, mean MMSE scores and total-FTUs were 11.0 ± 8.6 and 9.3 ± 4.6, respectively. Higher total-FTUs were significantly associated with higher MMSE scores after adjustment for demographics and teeth number (B = 0.48, 95% confidence interval [CI] = 0.22-0.74). The association remained significant even after adjustment for all covariates (B = 0.25, 95% CI = 0.01-0.49). The current findings demonstrated that loss of posterior teeth occlusion was independently associated with cognitive decline in nursing home older residents in Japan. Maintenance and restoration of posterior teeth occlusion may be a preventive factor against cognitive decline in aged populations..
35. K. Moritani, Toru Takeshita, Yukie Shibata, Toshiharu Ninomiya, Y. Kiyohara, Yoshihisa Yamashita, Acetaldehyde production by major oral microbes, Oral Diseases, 10.1111/odi.12341, 21, 6, 748-754, 2015.01, Objectives: To assess acetaldehyde (ACH) production by bacteria constituting the oral microbiota and the inhibitory effects of sugar alcohols on ACH production. Materials and Methods: The predominant bacterial components of the salivary microbiota of 166 orally healthy subjects were determined by barcoded pyrosequencing analysis of the 16S rRNA gene. Bacterial ACH production from ethanol or glucose was measured using gas chromatography. In addition, inhibition by four sugars and five sugar alcohols of ACH production was assayed. Results: Forty-one species from 16 genera were selected as predominant and prevalent bacteria based on the following criteria: identification in ≥95% of the subjects, ≥1% of mean relative abundance or ≥5% of maximum relative abundance. All Neisseria species tested produced conspicuous amounts of ACH from ethanol, as did Rothia mucilaginosa, Streptococcus mitis and Prevotella histicola exhibited the ability to produce ACH. In addition, xylitol and sorbitol inhibited ACH production by Neisseria mucosa by more than 90%. Conclusions: The oral microbiota of orally healthy subjects comprises considerable amounts of bacteria possessing the ability to produce ACH, an oral carcinogen. Consumption of sugar alcohols may regulate ACH production by oral microbes..
36. Aisyah Ahmad, Michiko Furuta, Takashi Shinagawa, Kenji Takeuchi, Toru Takeshita, Yoshihiro Shimazaki, Yoshihisa Yamashita, Association of periodontal status with liver abnormalities and metabolic syndrome, Journal of oral science, 10.2334/josnusd.57.335, 57, 4, 335-343, 2015.01, Although an association between periodontal status and liver abnormalities has been reported, it has not been described in relation to metabolic syndrome (MetS), which often coexists with non-alcoholic fatty liver disease. We examined the association of a combination of liver abnormality and MetS with periodontal condition in Japanese adults, based on the level of alcohol consumption. In 2008, 4,207 males aged 45.4 ± 8.9 years and 1,270 females aged 45.9 ± 9.7 years had annual workplace health check-ups at a company in Japan. Periodontal status was represented as periodontal pocket depth at the mesio-buccal and mid-buccal sites for all teeth. Alanine aminotransferase (ALT), and metabolic components were examined. Multiple linear regression analysis showed a significant association between deep pocket depth and the coexistence of elevated ALT and MetS in males with low alcohol consumption. Females showed no such relationship. In conclusion, the association between periodontal condition and the combination of elevated ALT and MetS was confirmed in males. That is, a clear association between liver abnormalities and periodontal condition was seen in male subjects with no or low alcohol consumption and MetS, providing new insights into the connection between liver function and periodontal health..
37. Takafumi Hashiba, Kenji Takeuchi, Yoshihiro Shimazaki, Toru Takeshita, Yoshihisa Yamashita, Chewing xylitol gum improves self-rated and objective indicators of oral health status under conditions interrupting regular oral hygiene, Tohoku Journal of Experimental Medicine, 10.1620/tjem.235.39, 235, 1, 39-46, 2015.01, Chewing xylitol gum provides oral health benefits including inhibiting Streptococcus mutans plaque. It is thought to be especially effective in conditions where it is difficult to perform daily oral cleaning. Our study aim was to determine the effects of chewing xylitol gum on self-rated and objective oral health status under a condition interfering with oral hygiene maintenance. A randomized controlled intervention trial was conducted on 55 healthy ≥ 20-year-old men recruited from the Japan Ground Self Defense Force who were undergoing field training. Participants were randomly assigned to a test group (chewing gum; n = 27) or a control group (no gum; n = 28) and the researchers were blinded to the group assignments. The Visual Analog Scale (VAS) scores of oral conditions subjectively evaluated oral health, and the stimulated salivary bacteria quantity objectively evaluated oral health 1 day before field training (baseline) and 4 days after the beginning of field training (follow-up). VAS scores of all three oral conditions significantly increased in the control group (malodor: p < 0.001; discomfort: p < 0.001; dryness: p < 0.001), but only two VAS scores increased in the test group (malodor: p = 0.021; discomfort: p = 0.002). The number of salivary total bacteria significantly increased in the control group (p < 0.01), while no significant change was observed in the test group (p = 0.668). Chewing xylitol gum positively affects self-rated and objective oral health status by controlling oral hygiene under conditions that interfere with oral hygiene maintenance..
38. Toru Takeshita, Masaki Yasui, Yukie Shibata, Michiko Furuta, Yoji Saeki, Nobuoki Eshima, Yoshihisa Yamashita, Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach, Scientific reports, 10.1038/srep08136, 5, 2015.01, Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5, and 7 days with saliva on day 0 were collected from 19 young adults using a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a time-related manner, which became increasingly similar to the salivary microbiota. Facultative anaerobic bacteria such as Streptococcus, Neisseria, Abiotrophia, Gemella, and Rothia were predominant in the plaque bacterial community in the earlier days, whereas obligate anaerobes, such as Porphyromonas, Fusobacterium, Prevotella, and Capnocytophaga showed increased dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the plaque bacterial community as well as the inter-individual differences associated with dental caries experience..
39. Michiko Furuta, Yoshihiro Shimazaki, Shunichi Tanaka, Kenji Takeuchi, Yukie Shibata, Toru Takeshita, Fusanori Nishimura, Yoshihisa Yamashita, Gender-specific associations of serum antibody to Porphyromonas gingivalis and inflammatory markers, BioMed Research International, 10.1155/2015/897971, 2015, 2015.01, It remains unclear whether serum antibody titer against Porphyromonas gingivalis (Pg) and inflammatory components lead to periodontal deterioration in each gender, as periodontal and systemic status is influenced by gender. The present study investigates the gender-specific probable effects of titer against Pg and inflammatory markers on periodontal health status in a longitudinal study. A retrospective study design was used. At two time points over an 8-year period (in 2003 and 2011), 411 individuals (295 males with a mean age of 57.6 ± 11.2 years and 116 females with a mean age of 59.2 ± 10.3 years) were surveyed. Periodontal status, serum antibody titer against Pg, and high-sensitive C-reactive protein (hsCRP) were evaluated. Poisson regression analyses revealed that the elevated titer against Pg and hsCRP significantly predicted the persistence of periodontal disease 8 years later in females with periodontal disease in 2003. Elevated hsCRP was significantly associated with the incidence of periodontal disease 8 years later in females who were periodontally healthy in 2003. Males had a weaker association among titer against Pg, inflammatory markers, and periodontal disease. These findings suggest that immune response to Pg infection in addition to inflammatory components affects periodontal deterioration in females..
40. M. N. Zakaria, Toru Takeshita, Yukie Shibata, Hidefumi Maeda, Naohisa Wada, A. Akamine, Yoshihisa Yamashita, Microbial community in persistent apical periodontitis
A 16S rRNA gene clone library analysis, International Endodontic Journal, 10.1111/iej.12361, 48, 8, 717-728, 2015.01, Aim: To characterize the microbial composition of persistent periapical lesions of root filled teeth using a molecular genetics approach. Methodology: Apical lesion samples were collected from 12 patients (23-80 years old) who visited the Kyushu University Hospital for apicectomy with persistent periapical lesions associated with root filled teeth. DNA was directly extracted from each sample and the microbial composition was comprehensively analysed using clone library analysis of the 16S rRNA gene. Enterococcus faecalis, Candida albicans and specific fimA genotypes of Porphyromonas gingivalis were confirmed using polymerase chain reaction (PCR) analysis with specific primers. Results: Bacteria were detected in all samples, and the dominant findings were P. gingivalis (19.9%), Fusobacterium nucleatum (11.2%) and Propionibacterium acnes (9%). Bacterial diversity was greater in symptomatic lesions than in asymptomatic ones. In addition, the following bacteria or bacterial combinations were characteristic to symptomatic lesions: Prevotella spp., Treponema spp., Peptostreptococcaceae sp. HOT-113, Olsenella uli, Slackia exigua, Selemonas infelix, P. gingivalis with type IV fimA, and a combination of P. gingivalis, F. nucleatum, and Peptostreptococcaceae sp. HOT-113 and predominance of Streptococcus spp. On the other hand, neither Enterococcus faecalis nor C. albicans were detected in any of the samples. Conclusion: Whilst a diverse bacterial species were observed in the persistent apical lesions, some characteristic patterns of bacterial community were found in the symptomatic lesions. The diverse variation of community indicates that bacterial combinations as a community may cause persistent inflammation in periapical tissues rather than specific bacterial species..
41. Mitsuo Sakamoto, Dan Li, Yukie Shibata, Toru Takeshita, Yoshihisa Yamashita, Moriya Ohkuma, Porphyromonas pasteri sp. Nov., isolated from human saliva, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijs.0.000294, 65, 8, 2511-2515, 2015.01, A bacterial strain, designated KUFDS01T, isolated from human saliva was characterized using a polyphasic taxonomic approach that included analysis of physiological and biochemical features, cellular fatty acid profiles and phylogenetic position based on 16S rRNA gene sequence analysis. Cells of the strain were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-negative rods. Growth of the strain was inhibited on medium containing 20% bile. The 16S rRNA gene sequence analysis showed that the strain was a member of the genus Porphyromonas. Strain KUFDS01T was closely related to Porphyromonas catoniae JCM 13863T (96.6% sequence similarity). An hsp60 gene sequence analysis indicated that strain KUFDS01T was different from P. catoniae JCM 13863T, with a sequence similarity value of 87.8%. The major cellular fatty acids of strain KUFDS01T were C16:0, iso-C15:0, anteiso-C15:0,C18: 2ω6, 9c and C18: 1ω9c. The DNA G + C content of strain KUFDS01T was 57.7 + 0.66 mol%. On the basis of these data, strain KUFDS01T represents a novel species of the genus Porphyromonas, for which the name Porphyromonas pasteri sp. nov. is proposed. The type strain of P. pasteri is KUFDS01T (=JCM 30531T=CCUG 66735T)..
42. Mikio Shoji, Toru Takeshita, Fumito Maruyama, Hiroaki Inaba, Kenichi Imai, Miki Kawada-Matsuo, Recent advances in the field of oral bacteriology, Nihon saikingaku zasshi. Japanese journal of bacteriology, 10.3412/jsb.70.333, 70, 2, 333-338, 2015.01, The oral cavity is inhabited by more than 600 bacterial species; these species compete for nutrients or coexist in order to survive along with the indigenous population. Extreme conditions are prevalent in the oral cavity, and these conditions are influenced by our immunity and variations in nutrition, temperature, and pH. Pathogens that cause dental caries or periodontal disease can survive in these extreme environments; these pathogens are virulent and can cause several diseases. Therefore, research on oral bacteriology is warranted to analyze the virulence factors of these bacteria as well as to ascertain environmental stress responses, interactions between bacteria and human immunity, comparisons of bacterial genomes, and oral microflora. In this review, we provide new data in the fields of bacteriology, immunology, and genomics and describe recent advances in the field of oral bacteriology..
43. Kenji Takeuchi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Yoshihiro Shimazaki, Sumio Akifusa, Toshiharu Ninomiya, Yutaka Kiyohara, Yoshihisa Yamashita, Risk factors for reduced salivary flow rate in a Japanese population
The Hisayama study, BioMed Research International, 10.1155/2015/381821, 2015, 2015.01, The purpose of this study was to determine distinct risk factors causing reduced salivary flow rate in a community-dwelling population using a prospective cohort study design. This was a 5-year follow-up survey of 1,377 community-dwelling Japanese individuals aged ≥40 years. The salivary flow rate was evaluated at baseline and follow-up by collecting stimulated saliva. Data on demographic characteristics, use of medication, and general and oral health status were obtained at baseline. The relationship between reduced salivary flow rate during the follow-up period and its predictors was evaluated after adjustment for confounding factors. In a multivariate logistic regression model, higher age and plaque score and lower serum albumin levels were significantly associated with greater odds of an obvious reduction in salivary flow rate (age per decade, odds ratio [OR] = 1.25, 95% confidence interval [CI] = 1.03-1.51; serum albumin levels <4 g/dL, OR = 1.60, 95% CI = 1.04-2.46; plaque score ≥1, OR = 1.53, 95% CI = 1.04-2.24). In a multivariate linear regression model, age and plaque score remained independently associated with the increased rate of reduced salivary flow. These results suggest that aging and plaque score are important predictors of reduced salivary flow rate in Japanese adults..
44. Kenji Takeuchi, Michiko Furuta, Toru Takeshita, Yukie Shibata, Yoshihiro Shimazaki, Sumio Akifusa, Toshiharu Ninomiya, Yutaka Kiyohara, Yoshihisa Yamashita, Serum antibody to Porphyromonas gingivalis and periodontitis progression
The Hisayama Study, Journal of Clinical Periodontology, 10.1111/jcpe.12431, 42, 8, 719-725, 2015.01, Aim To assess the effectiveness of serum titres of antibody to Porphyromonas gingivalis in the prediction of periodontitis progression in a community-dwelling adult population. Materials and Methods This 4-year follow-up cohort study included 183 Japanese individuals (75 men and 108 women) aged 40-49 years. The clinical attachment level was measured at two sites on all teeth present at baseline and follow-up, and periodontitis progression was defined as progression in attachment loss of ≥3 mm at ≥2 measurement sites. Serum titres of antibody to Porphyromonas gingivalis were evaluated at baseline and served as the primary predictor variable for periodontitis progression. Results Periodontitis progression was found in 8.7% subjects. In a multivariate logistic regression model, higher titres of antibody to Porphyromonas gingivalis and male gender were significantly associated with greater odds of periodontitis progression, even after adjustment for other confounding variables (titres of antibody to Porphyromonas gingivalis per 10 units: odds ratio, 1.66; 95% confidence interval, 1.16-2.36; male gender: odds ratio, 4.13; 95% confidence interval, 1.20-14.27). Conclusions Elevated serum titres of antibody to Porphyromonas gingivalis may be a risk factor for periodontitis progression..
45. Toru Takeshita, Distinct composition of the oral indigenous microbiota in South Korean and Japanese adults, SCIENTIFIC REPORTS, 10.1038/srep06990, 4, 2014.11.
46. Junko Obata, Toru Takeshita, Yukie Shibata, Wataru Yamanaka, Masako Unemori, Akifumi Akamine, Yoshihisa Yamashita, Identification of the microbiota in carious dentin lesions using 16S rRNA gene sequencing, PloS one, 10.1371/journal.pone.0103712, 9, 8, 2014.08, While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4-76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella , or Propionibacterium with Streptococcus or Actinomyces . Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium , were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating..
47. Yoshio Nakano, Toru Takeshita, Noriaki Kamio, Susumu Shiota, Yukie Shibata, Nao Suzuki, Masahiro Yoneda, Takao Hirofuji, Yoshihisa Yamashita, Supervised machine learning-based classification of oral malodor based on the microbiota in saliva samples, Artificial Intelligence in Medicine, 10.1016/j.artmed.2013.12.001, 60, 2, 97-101, 2014.02, Objective: This study presents an effective method of classifying oral malodor from oral microbiota in saliva by using a support vector machine (SVM), an artificial neural network (ANN), and a decision tree. This approach uses concentrations of methyl mercaptan in mouth air as an indicator of oral malodor, and peak areas of terminal restriction fragment (T-RF) length polymorphisms (T-RFLPs) of the 16S rRNA gene as data for supervised machine-learning methods, without identifying specific species producing oral malodorous compounds. Methods: 16S rRNA genes were amplified from saliva samples from 309 subjects, and T-RFLP analysis was carried out with the DNA fragments. T-RFLP analysis provides information on microbiota consisting of fragment lengths and peak areas corresponding to bacterial strains. The peak area is equivalent to the frequency of a specific fragment when one molecule is selected from terminal fragments. Another frequency is obtained by dividing the number of species-containing samples by the total number of samples. An SVM, an ANN, and a decision tree were trained based on these two frequencies in 308 samples and classified the presence or absence of methyl mercaptan in mouth air from the remaining subject. Results: The proportion that trained SVM expressed as entropy achieved the highest classification accuracy, with a sensitivity of 51.1% and specificity of 95.0%. The ANN and decision tree provided lower classification accuracies, and only classification by the ANN was improved by weighting with entropy from the frequency of appearance in samples, which increased the accuracy to 81.9% with a sensitivity of 60.2% and a specificity of 90.5%. The decision tree showed low classification accuracy under all conditions. Conclusions: Using T-RF proportions and frequencies, models to classify the presence of methyl mercaptan, a volatile sulfur-containing compound that causes oral malodor, were developed. SVM classifiers successfully classified the presence of methyl mercaptan with high specificity, and this classification is expected to be useful for screening saliva for oral malodor before visits to specialist clinics. Classification by a SVM and an ANN does not require the identification of the oral microbiota species responsible for the malodor, and the ANN also does not require the proportions of T-RFs..
48. Dan Li, Yukie Shibata, Toru Takeshita, Yoshihisa Yamashita, A novel gene involved in the survival of streptococcus mutans under stress conditions, Applied and Environmental Microbiology, 10.1128/AEM.02549-13, 80, 1, 97-103, 2014.01, A Streptococcus mutans mutant defective in aciduricity was constructed by random-insertion mutagenesis. Sequence analysis of the mutant revealed a mutation in gidA, which is known to be involved in tRNA modification in Streptococcus pyogenes. Complementation of gidA by S. pyogenes gidA recovered the acid tolerance of S. mutans. Although the gidA-inactivated S. pyogenes mutant exhibited significantly reduced expression of multiple extracellular virulence proteins, the S. mutans mutant did not. On the other hand, the gidA mutant of S. mutans showed reduced ability to withstand exposure to other stress conditions (high osmotic pressure, high temperature, and bacitracin stress) besides an acidic environment. In addition, loss of GidA decreased the capacity for glucose-dependent biofilm formation by over 50%. This study revealed that gidA plays critical roles in the survival of S. mutans under stress conditions, including lower pH..
49. Koichi Yamane, Yasunori Ayukawa, Toru Takeshita, Akihiro Furuhashi, Yoshihisa Yamashita, Kiyoshi Koyano, Bacterial adhesion affinities of various implant abutment materials, Clinical Oral Implants Research, 10.1111/j.1600-0501.2012.02574.x, 24, 12, 1310-1315, 2013.12, Objectives: To investigate bacterial adhesion to various abutment materials. Material and methods: Thirty volunteers participated in this study. Resin splints were fabricated, and five types of disks were fabricated from pure titanium, gold-platinum alloy, zirconia, alumina, and hydroxyapatite with uniform surface roughness and attached to the buccal surface of each splint. After 4 days of use by the subjects, the plaque accumulated on the disk surfaces was analyzed. The bacterial community structure was evaluated using 16S rRNA gene profiling with terminal restriction fragment length polymorphism analysis. The total bacterial count on each disk was estimated using quantitative polymerase chain reaction. Results: Terminal restriction fragment length polymorphism profiles were more similar between tested materials than between subjects, suggesting that the bacterial community structures on the abutment material were influenced more by the individuals than by the type of material. However, the total number of bacteria attached to a disk was significantly different among five materials (P < 0.001, Brunner-Langer test for longitudinal data). Fewer bacteria were attached to the gold-platinum alloy than to the other materials. Conclusions: Gold-platinum alloy appears to be useful material for abutments when considering the accumulation of plaque. However, alternative properties of the abutment material, such as effects on soft tissue healing, should also be taken into consideration when choosing an abutment material..
50. Kosaku Iha, Nao Suzuki, Masahiro Yoneda, Toru Takeshita, Takao Hirofuji, Effect of mouth cleaning with hinokitiol-containing gel on oral malodor
A randomized, open-label pilot study, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology, 10.1016/j.oooo.2013.05.021, 116, 4, 433-439, 2013.10, Aim The aim of the study was to evaluate the effect of mouth cleaning with hinokitiol-containing gel on oral malodor. Methods An open-label, randomized, controlled trial was conducted to assess oral malodor and clinical parameters related to oral malodor before and after mouth cleaning with hinokitiol-containing gel (n = 9) or with gel not including hinokitiol (n = 9). Mouth cleaning included the teeth, gingiva, and tongue and was carried out 3 times per day for 4 weeks. Results Organoleptic test (OLT) scores (P =.021), levels of hydrogen sulfide (P =.008) and methyl mercaptan (P =.020), frequency of bleeding on probing, average probing pocket depth, and plaque index significantly improved in the group using hinokitiol. In contrast, only the OLT score (P =.031) significantly improved in the control group after the treatment regimen. Conclusion Mouth cleaning with hinokitiol-containing gel may be effective for reduction of oral malodor..
51. Michiko Furuta, Yoshihiro Shimazaki, Toru Takeshita, Yoshihisa Yamashita, Gender differences in the association between metabolic syndrome and periodontal disease: the Hisayama Study, JOURNAL OF CLINICAL PERIODONTOLOGY, 10.1111/jcpe.12119, 40, 8, 743-752, 2013.08.
52. Toru Takeshita, Relationship of periodontal clinical parameters with bacterial composition in human dental plaque, ARCHIVES OF MICROBIOLOGY, 10.1007/s00203-013-0883-9, 195, 6, 371-383, 2013.06.
53. Hong Li, Toru Takeshita, Michiko Furuta, Mikiko Tomioka, Yukie Shibata, Yoshihiro Shimazaki, Kouichi Makimura, Yoshihisa Yamashita, Molecular characterization of fungal populations on the tongue dorsum of institutionalized elderly adults, ORAL DISEASES, 10.1111/j.1601-0825.2012.01944.x, 18, 8, 771-777, 2012.11.
54. Wataru Yamanaka, Toru Takeshita, Yukie Shibata, Kazuki Matsuo, Nobuoki Eshima, Takeshi Yokoyama, Yoshihisa Yamashita, Compositional Stability of a Salivary Bacterial Population against Supragingival Microbiota Shift following Periodontal Therapy, PLOS ONE, 10.1371/journal.pone.0042806, 7, 8, 2012.08.
55. Toru Takeshita, Nao Suzuki, Yoshio Nakano, Masaki Yasui, Masahiro Yoneda, Yoshihiro Shimazaki, Takao Hirofuji, Yoshihisa Yamashita, Discrimination of the oral microbiota associated with high hydrogen sulfide and methyl mercaptan production, Scientific reports, 10.1038/srep00215, 2, 2012.04, Both hydrogen sulfide (H 2S) and methyl mercaptan (CH 3SH) are frequently detected in large amounts in malodorous mouth air. We investigated the bacterial composition of saliva of 30 subjects with severe oral malodor exhibiting extreme CH 3SH/H 2S ratios (high H 2S but low CH 3SH concentrations, n = 14; high CH 3SH but low H 2S concentrations, n = 16) and 13 subjects without malodor, using barcoded pyrosequencing analysis of the 16S rRNA gene. Phylogenetic community analysis with the UniFrac distance metric revealed a distinct bacterial community structure in each malodor group. The H 2S group showed higher proportions of the genera Neisseria, Fusobacterium, Porphyromonas and SR1 than the other two groups, whereas the CH 3SH group had higher proportions of the genera Prevotella, Veillonella, Atopobium, Megasphaera, and Selenomonas. Our results suggested that distinct bacterial populations in the oral microbiota are involved in production of high levels of H 2S and CH 3SH in the oral cavity..
56. Nao Suzuki, Kazunari Tanabe, Toru Takeshita, Masahiro Yoneda, Tomoyuki Iwamoto, Sueko Oshiro, Yoshihisa Yamashita, Takao Hirofuji, Effects of oil drops containing Lactobacillus salivarius WB21 on periodontal health and oral microbiota producing volatile sulfur compounds, Journal of Breath Research, 10.1088/1752-7155/6/1/017106, 6, 1, 2012.03, The objective of this paper is to evaluate the effects of oil drops containing Lactobacillus salivarius WB21 on periodontal health and oral microbiota producing volatile sulfur compounds (VSCs). For this study, 42 subjects were randomly assigned to receive oil samples containing L. salivarius WB21 or a placebo for two weeks. Oral assessment and saliva collection were performed on days 1 and 15. Bacterial analysis was performed using the real-time polymerase chain reaction and terminal restriction fragment length polymorphism (T-RFLP). In both the experimental and placebo groups, the average probing depth, number of periodontal pockets, and the percentage of bleeding on probing (BOP) decreased while stimulated salivary flow increased on day 15. BOP was reduced in the experimental group compared with the placebo group (P = 0.010). In the experimental group, total bacterial numbers decreased, and the number of L. salivarius increased. The number of Prevotella intermedia, which is correlated with hydrogen sulfide concentration in mouth air, increased in the placebo group and did not change in the experimental group. T-RFLP analysis found that the peak area proportions representing Porphyromonas gingivalis, P. intermedia, Tannerella forsythensis, and Fusobacterium nucleatum decreased in the experimental group, although there was no significant change in the bacterial composition. Thus we observed oil drops containing L. salivarius WB21 improved BOP and inhibited the reproduction of total and VSC-producing periodontopathic bacteria compared with the placebo group, but also showed the limit of its efficacy in controlling VSCs producing and periodontal pathogens..
57. Toru Takeshita, Yoshihisa Yamashita, Evaluation of salivary microbiota from an ecological perspective, Journal of Oral Biosciences, 10.1016/j.job.2012.04.003, 54, 3, 128-131, 2012.01, A variety of bacteria densely colonize the human oral cavity not as individuals, but as members of an indigenous microbiota that exhibit extensive intracellular interactions. To prevent the onset of oral diseases caused by the members of this microbiota, regulation of the total oral microbiota, including the surrounding bacterial environment, is required. In this review, we highlight current knowledge on the global composition of the salivary bacterial population associated with oral conditions through a molecular ecological approach using the 16S rRNA gene. The salivary bacterial populations of Japanese subjects were commonly dominated by bacterial genera such as Streptococcus, Prevotella, and Neisseria, and their relative abundances differed according to the specific periodontal condition of the patient. This suggests that manipulation of the predominant oral microbiota may assist in the maintenance of periodontal health. A broad view of the relationship of oral health with oral microbes would provide novel insights into oral health and disease..
58. Toru Takeshita, Masaki Yasui, Mikiko Tomioka, Yoshio Nakano, Yoshihiro Shimazaki, Yoshihisa Yamashita, Enteral tube feeding alters the oral indigenous microbiota in elderly adults, Applied and Environmental Microbiology, 10.1128/AEM.00651-11, 77, 19, 6739-6745, 2011.10, Enteral tube feeding is widely used to maintain nutrition for elderly adults with eating difficulties, but its long-term use alters the environment of the oral ecosystem. This study characterized the tongue microbiota of tube-fed elderly adults by analyzing the 16S rRNA gene. The terminal restriction fragment length polymorphism (T-RFLP) profiles of 44 tube-fed subjects were compared with those of 54 subjects fed orally (average age, 86.4 ± 6.9 years). Bar-coded pyrosequencing data were also obtained for a subset of the subjects from each group (15 tube-fed subjects and 16 subjects fed orally). The T-RFLP profiles demonstrated that the microbiota of the tube-fed subjects was distinct from that of the subjects fed orally (permutational multivariate analysis of variance [perMANOVA], P < 0.001). The pyrosequencing data revealed that 22 bacterial genera, including Corynebacterium, Peptostreptococcus, and Fusobacterium, were significantly more predominant in tube-fed subjects, whereas the dominant genera in the subjects fed orally, such as Streptococcus and Veillonella, were present in much lower proportions. Opportunistic pathogens rarely detected in the normal oral microbiota, such as Corynebacterium striatum and Streptococcus agalactiae, were often found in high proportions in tube-fed subjects. The oral indigenous microbiota is disrupted by the use of enteral feeding, allowing health-threatening bacteria to thrive..
59. Norio Kitagawa, Susumu Shiota, Yukie Shibata, Toru Takeshita, Yoshihisa Yamashita, Characterization of MbrC involved in bacitracin resistance in Streptococcus mutans, FEMS Microbiology Letters, 10.1111/j.1574-6968.2011.02238.x, 318, 1, 61-67, 2011.05, Streptococcus mutans, a major etiological agent of dental caries, is resistant to bacitracin. Microarray analysis revealed that mbrA and mbrB, encoding a putative ATP-binding cassette transporter, are prominently induced in the presence of bacitracin. On the basis of the latest report that MbrC, a putative response regulator in a two-component signaling system, binds the promoter region of mbrA and thus regulates its transcription, we cut into the mechanism by generating a mutant MbrC (D54N-MbrC) that substituted asparagine for aspartate at position 54, the predicted phosphorylation site. MbrC, but not the mutant D54N-MbrC, showed affinity for a DNA probe that contained the hypothetical mbrA promoter sequence. Furthermore, we introduced a point mutation (D54N-MbrC) into UA159; this mutant strain exhibited neither mbrA induction nor resistance in the presence of bacitracin. These data suggest that the aspartate residue at position 54 of MbrC is a promising candidate for phosphorylation in a bacitracin-sensing system and indispensable for S. mutans bacitracin resistance..
60. Yoshihiro Shimazaki, Sumio Akifusa, Toru Takeshita, Yukie Shibata, Yasufumi Doi, Jun Hata, Toshiharu Ninomiya, Yoichiro Hirakawa, Yutaka Kiyohara, Yoshihisa Yamashita, Effectiveness of the salivary occult blood test as a screening method for periodontal status, Journal of Periodontology, 10.1902/jop.2010.100304, 82, 4, 581-587, 2011.04, Background: Community-based periodontal examinations are not popular despite the high prevalence of periodontal disease among adults. This study examines the effectiveness of a novel salivary occult blood test (SOBT) as a screening method for periodontal status. Methods: Comprehensive health examinations were conducted in adult residents aged ≥40 years in Hisayama, Fukuoka, Japan; 1, 998 subjects, each with ≥20 teeth, were analyzed. A paper test strip was used to perform the SOBT and was followed by a periodontal examination. Results were ranked as negative or positive. Subjects with ≥15% of teeth with bleeding on probing (BOP) or ≥1 tooth with a probing depth (PD) ≥4 mm were defined as having a poor periodontal status. The relationship between the results of the SOBT and periodontal parameters and among other variables was examined. Results: The sensitivity and specificity of the SOBT in screening for poor periodontal status were 0.72 and 0.52, respectively. In a multivariate logistic regression analysis, the results of the SOBT were significantly associated with the proportion of teeth with BOP and the proportion of teeth with PD ≥4 mm, independent of age, sex, use of antihypertensive medication, use of antidiabetic medication or insulin therapy, and the number of decayed or filled teeth. Conclusion: The SOBT may offer a simple screening method for periodontal status when athorough periodontal examination is not possible, although it is not sufficiently specific to be a reasonable substitute for a periodontal examination..
61. Yoshihisa Yamashita, Toru Takeshita, Oral flora composition and its connection to oral health, journal of oral biosciences, 10.2330/joralbiosci.53.206, 53, 3, 206-212, 2011.01, More than 700 species of commensal bacteria inhabit the human oral cavity, of which many have been of keen interest due to their pathogenicity in oral diseases (e.g., dental caries and periodontal diseases); however, the interactions between the pathogens and the remaining commensal bacteria are not well known, thus preventing us from understanding the genuine etiologies of oral diseases. To overcome this challenge, it is essential to comprehensively identify the species compositions of individual oral flora in order to associate them with various conditions of oral health and understand the virulence derived from the oral flora community. In this review, we refer to modern molecular genetic technologies, such as terminal restriction fragment length polymorphism, DNA microarray and pyrosequencing analyses using bioinformatics. We also discuss their potential to further our comprehension of the complexities of floral composition..
62. Yoshihisa Yamashita, Toru Takeshita, A comprehensive evaluation of the virulence of oral flora, Informatics in Oral Medicine Advanced Techniques in Clinical and Diagnostic Technologies, 10.4018/978-1-60566-733-1.ch015, 251-267, 2010.12, The oral cavity is inhabited by more than 700 species of commensal bacteria. Several have been identified as pathogens of oral diseases, dental caries, and periodontal diseases. However, the lack of information concerning the interaction between pathogens and the remaining commensal bacteria has made it difficult to understand the precise etiologies of oral diseases. We need to identify the comprehensive species found in individual oral flora and compare these results with corresponding oral health conditions. In this chapter, the authors delineate the previous attempts to identify oral commensal bacterial flora and discuss the potential capability of modern molecular genetic technologies such as terminal restriction fragment length polymorphism, DNA microarray and pyrosequencing analyses utilizing bioinformatics..
63. Iwamoto T, Suzuki N, Tanabe K, Takeshita T, Hirofuji T, Effects of probiotic Lactobacillus salivarius WB21 on halitosis and oral health: an open-label pilot trial, Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2010.08.
64. Tomoyuki Iwamoto, Nao Suzuki, Kazunari Tanabe, Toru Takeshita, Takao Hirofuji, Effects of probiotic Lactobacillus salivarius WB21 on halitosis and oral health
An open-label pilot trial, Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology, 10.1016/j.tripleo.2010.03.032, 110, 2, 201-208, 2010.08, Objective: The objective of this study was to evaluate whether oral administration of lactobacilli alters the degree of halitosis and clinical conditions associated with halitosis. Study design: Twenty patients with genuine halitosis were given 2.0 × 109 Lactobacillus salivarius WB21 and xylitol in tablet form daily. Oral malodor and clinical parameters were evaluated at the same time of day for each patient after 2 and 4 weeks. Results: All 20 patients were positive for L. salivarius DNA in their saliva at 2 weeks, although 12 patients were negative for this organism at baseline. Oral malodor parameters significantly decreased at 2 weeks in the subjects with physiologic halitosis. The scores of an organoleptic test and bleeding on probing significantly decreased at 4 weeks in the subjects with oral pathologic halitosis. Conclusions: Oral administration of probiotic lactobacilli primarily improved physiologic halitosis and also showed beneficial effects on bleeding on probing from the periodontal pocket..
65. Toru Takeshita, Nao Suzuki, Yoshio Nakano, Yoshihiro Shimazaki, Masahiro Yoneda, Takao Hirofuji, Yoshihisa Yamashita, Relationship between oral malodor and the global composition of indigenous bacterial populations in saliva, Applied and Environmental Microbiology, 10.1128/AEM.02304-09, 76, 9, 2806-2814, 2010.05, Oral malodor develops mostly from the metabolic activities of indigenous bacterial populations within the oral cavity, but whether healthy or oral malodor-related patterns of the global bacterial composition exist remains unclear. In this study, the bacterial compositions in the saliva of 240 subjects complaining of oral malodor were divided into groups based on terminal-restriction fragment length polymorphism (T-RFLP) profiles using hierarchical cluster analysis, and the patterns of the microbial community composition of those exhibiting higher and lower malodor were explored. Four types of bacterial community compositions were detected (clusters I, II, III, and IV). Two parameters for measuring oral malodor intensity (the concentration of volatile sulfur compounds in mouth air and the organoleptic score) were noticeably lower in cluster I than in the other clusters. Using multivariate analysis, the differences in the levels of oral malodor were significant after adjustment for potential confounding factors such as total bacterial count, mean periodontal pocket depth, and tongue coating score (P < 0.001). Among the four clusters with different proportions of Indigenous members, the T-RFLP profiles of cluster I were implicated as the bacterial populations with higher proportions of Streptococcus, Granulicatella, Rothia, and Treponema species than those of the other clusters. These results clearly correlate the global composition of indigenous bacterial populations with the severity of oral malodor..
66. Toru Takeshita, Mikiko Tomioka, Yoshihiro Shimazaki, Miwa Matsuyama, Kiyoshi Koyano, Koichi Matsuda, Yoshihisa Yamashita, Microfloral characterization of the tongue coating and associated risk for pneumonia-related health problems in institutionalized older adults, Journal of the American Geriatrics Society, 10.1111/j.1532-5415.2010.02867.x, 58, 6, 1050-1057, 2010.01, Objectives: To characterize a comprehensive outline of the oral microflora related to pneumonia in older adults. Design: Prospective and retrospective longitudinal study. Setting: Community. Participants: Long-term hospitalized patients and people in nursing homes (343 subjects, aged ≥65). Measurements: Subjects were assessed at baseline for pneumonia-related health problems. The flora of the tongue coating was characterized according to terminal restriction fragment length polymorphism (T-RFLP) analysis. All subjects were followed prospectively for 6 months for a diagnosis of pneumonia. The number of febrile days (>37.5°C) was assessed in 299 subjects who were observed for 12 months (retrospectively for 6 months and prospectively for 6 months). The follow-up data were analyzed using multivariate regression analyses in relation to the baseline data, including T-RFLP patterns. RESUTS: T-RFLP patterns outlining the floral composition of the tongue coating were grouped into Clusters A, B, C, and D. According to Cox regression analysis, the subjects in Clusters C (hazard ratio (HR)=4.0, 95% confidence interval (CI)=1.1-15.1) and D (HR=4.9, 95% CI=1.2-21.1) were at a significantly greater risk of pneumonia than those in Cluster A, independent of other confounding factors. Logistic regression analysis adjusting for the same covariates indicated that the number of subjects with more than 9 febrile days per year was significantly lower in Cluster A than in the other clusters. Conclusion: The comprehensive microfloral profile of the tongue coating is closely related to pneumonia-related health problems in institutionalized older adults..
67. Yoshio Nakano, Toru Takeshita, Masaki Yasui, Yoshihisa Yamashita, Prediction of Plausible Bacterial Composition Based on Terminal Restriction Fragment Length Polymorphisms using a Monte Carlo Method, Microbial Ecology, 10.1007/s00248-010-9703-9, 60, 2, 364-372, 2010, We have developed a new approach for the estimation of bacterial proportional compositions in microbiota based on terminal restriction length polymorphism (T-RFLP) data and a Monte Carlo algorithm. This program estimates proportional compositions by minimizing distances between peak values and the relative abundance of each group, containing several species, estimated from peak areas of capillary electrophoresis for T-RFLP analysis. Oral bacteria in 36 saliva samples obtained from three individuals were analyzed using the program. Upon comparison, the estimated proportional composition obtained from one of the samples matched that from a clone library. Additionally, comparisons among the bacterial proportional compositions of saliva samples obtained from three individuals four times per day for 3 days revealed that the types of microbiota present in each individual did not change within each 24-h time period and were distinguishable from those in other individuals..
68. Yukie Shibata, Jan R. van der Ploeg, Takeshi Kozuki, Yasuhito Shirai, Naoaki Saito, Miki Kawada-Matsuo, Toru Takeshita, Yoshihisa Yamashita, Kinase activity of the dgk gene product is involved in the virulence of Streptococcus mutans, Microbiology, 10.1099/mic.0.023812-0, 155, 2, 557-565, 2009.04, C-terminal deletion of the diacylglycerol kinase (Dgk) homologue of the cariogenic oral bacterium Streptococcus mutans resulted in loss of aciduricity. To confirm the role of the C terminus of the Dgk homologue in aciduricity, various mutants of S. mutans UA159 with a C-terminally truncated Dgk homologue were constructed. The deletion of one or two amino acid residues at the C terminus had no effect on the acid-tolerance properties of mutants. When further amino acid residues at the C terminus were removed, mutants became more acid-sensitive. The mutant with deletion of eight amino acid residues at the C terminus did not grow at pH 5.5, suggesting that the C-terminal tail of the Dgk homologue was indispensable for tolerance to acid stress in S. mutans. Kinase activity assays revealed that deletion of the C-terminal amino acids of Dgk led to a reduction of kinase activity for undecaprenol. A truncated mutant that had completely lost kinase activity was unable to grow at pH 5.5. These results suggest that the acid tolerance of S. mutans is closely related to kinase activity of the Dgk homologue. Additionally, the dgk deletion mutant exhibited markedly reduced levels of smooth-surface carious lesions in pathogen-free rats, despite there being no difference between the mutant and the parental organism in the extent of total smooth surface plaque. The results suggest that Dgk activity may play a direct role in the virulence of S. mutans..
69. Toru Takeshita, Yoshio Nakano, Takashi Kumagai, Masaki Yasui, Noriaki Kamio, Yukie Shibata, Susumu Shiota, Yoshihisa Yamashita, The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status, ISME Journal, 10.1038/ismej.2008.91, 3, 1, 65-78, 2009.01, To obtain deeper insights into the etiology of oral disease, an understanding of the composition of the surrounding bacterial environments that lead to health or disease is required, which is attracting increasing attention. In this study, the bacterial compositions in the saliva of 200 subjects aged 15-40 years were depicted as peak patterns by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. The subjects were classified into three clusters by partitioning around medoids clustering based on their T-RFLP profiles, and the clinical oral health parameters of the clusters were compared. The clustering of the T-RFLP profiles in this study was mainly based on differences in the abundance distribution of the dominant terminal restriction fragments (TRFs) detected in most of the subjects. Predicted from the sizes of the TRFs, the characteristically more predominant members of each were Prevotella and Veillonella species in cluster I; Streptococcus species in cluster II and Neisseria, Haemophilus or Aggregatibacter species and Porphyromonas species in cluster III. The parameters associated with periodontal disease were significantly different among the clusters. Clusters I and II had a higher percentage of sites of periodontal pockets greater than 4 mm than cluster III, and cluster I contained sites exhibiting bleeding on probing more often than cluster II or III; no significant differences were observed in other parameters. These results suggest that the abundance distribution of commensal bacteria in saliva is correlated with periodontal health, and might be involved in the susceptibility of an individual to periodontal disease..
70. Yoshio Nakano, Toru Takeshita, Noriaki Kamio, Susumu Shiota, Yukie Shibata, Masaki Yasui, Yoshihisa Yamashita, Development and application of a T-RFLP data analysis method using correlation coefficient matrices, Journal of Microbiological Methods, 10.1016/j.mimet.2008.08.002, 75, 3, 501-505, 2008.12, Environmental microbiology studies commonly use terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, for example, to analyze changes in community structure in relation to changing physicochemical and biological conditions over space and time. Although T-RFLP is most useful for comparing samples from different environments, a large number of samples makes effective analysis difficult using the Web-based tools that are currently available. To resolve this dilemma, we used a new approach for calculating data from multiple T-RFLP samples by estimating terminal fragment combinations, then applying a correlation analysis using two different fluorescent dyes generated simultaneously from all samples. This calculation was based on the expectation that the proportions of two terminal fragments from one full-length polymerase chain reaction fragment would be nearly the same in each analysis. Using this program, the oral microflora in 73 human saliva samples were analyzed, and 24 bacterial groups, with peak areas of at least 0.5% and correlation coefficients of 0.55 or greater, were identified from the T-RFs within 40 s..
71. Toru Takeshita, Y. Nakano, Yoshihisa Yamashita, Improved accuracy in terminal restriction fragment length polymorphism phylogenetic analysis using a novel internal size standard definition, Oral Microbiology and Immunology, 10.1111/j.1399-302X.2007.00384.x, 22, 6, 419-428, 2007.12, Background: Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used to analyze microbial communities, including oral microflora. However, accurate identification of terminal restriction fragment (T-RF) origins is prevented by unpredictable errors in sizing, thus necessitating the clone library analysis. To minimize sizing errors, we proposed optimizing the size definition of internal standards. Methods: GeneScan-1000 ROX was regenerated as an internal standard by redefining the fragment sizes in terms of molecular weight (MW) based on their mobility relative to 6-carboxyfluorescein (FAM) -labeled restriction fragments derived from the 16S recombinant RNA gene of Porphyromonas gingivalis. Using the new size definition, the average sizing error among eight oral bacteria from six phyla was estimated and compared with that of the conventional method. Microbial communities isolated from saliva were analyzed using the new MW size definition. Bacterial species were assigned to peaks using TRFMA, a Web-based tool for T-RFLP analysis, and compared with those identified in a clone library analysis. Results: Using the new size definition, the average sizing error for 40 T-RFs was drastically reduced from 2.42 to 0.62 bases, and large sizing errors (more than two bases) were eliminated. More than 90% of the total bacterial clones detected by the clone library analysis were assigned by T-RFLP. Conclusion: The size definition of the newly constructed internal standards reduced fragment sizing errors and allowed for accurate assignment of bacteria to peaks by the T-RFLP analysis. This provided a more effective means for studying microbial communities, including the oral microflora..
72. Yoshio Nakano, Toru Takeshita, Yoshihisa Yamashita, TRFMA
A web-based tool for terminal restriction fragment length polymorphism analysis based on molecular weight, Bioinformatics, 10.1093/bioinformatics/btl186, 22, 14, 1788-1789, 2006.07, Summary: TRFMA provides a Web environment for analyzing T-RFLP results based on molecular weights of the fragments, rather than the numbers of nucleotides, to increase accuracy. The 16S rRNA data are saved as an XML file containing around 650 sequences (light version) and a MySQL database containing around 50 000 sequences (full version), which are connected to Web server via PHP5 and manipulated on an Internet browser..