Development of discriminative and quantitative detection method for live and dead oral bacteria

• Project/Area Number: 22592341
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Social dentistry
• Research Institution: Kyushu Dental College
• Principal Investigator: YOSHIDA Akihiro 九州歯科大学, 歯学部, 助教 (20364151)
• Co-Investigator(Kenkyū-buntansha): ANSAI Toshihiro 九州歯科大学, 歯学部, 准教授 (80244789) ; SOH Inho 九州歯科大学, 歯学部, 助教 (10285463) ; MINEOKA Tetsuro 九州歯科大学, 歯学部, その他 (50571402) ; TAKEHARA Tadamichi 九州歯科大学, 歯学部, 教授 (00038879) ; AWANO Shuji 九州歯科大学, 歯学部, 講師 (20301442)
• Project Period (FY): 2010 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2012: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2011: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2010: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: 口腔細菌 / 生菌 / 死菌 / qPCR / 定量検出 / プロピジウムモノアザイド(PMA) / リアルタイムPCR / Enterococcus faecalis / 歯内療法

Research Abstract

Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important forunderstanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR). We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves 機関番号：27102研究種目： 基盤研究(C)研究期間： 平成22年度～平成24年度 課題番号：22592341研究課題名（和文） 生菌・死菌の区別が可能な新しい定量的口腔細菌検出法の開発研究課題名（英文） Development of discriminative and quantitative detection method for live and dead o ral bacteria研究代表者 吉田明弘（YOSHIDA, AKIHIRO）九州歯科大学・歯学部・助教研究者番号：20364151 for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. In addition, we applied this assay to analyze viable cell numbers in oral specimens. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers. PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens andthe oral status.

Research Products

• [Journal Article] Quantitative discrimination of Aggregatibacter actinomycetemicomitans highly leukotoxic JP2 clone form non-JP2 clones in diagnosis of aggressive periodontitis. (2012)
  • Author(s): Yoshida, A., Ennibi, O-K., Miyazaki, H., Hoshino, T., Hayashida, H., Nishihara, T., Awano, S. and Ansai, T
  • Journal Title: BMC Infectious Dis. Volume: 12 Pages: 253-253
• [Journal Article] 歯周病と細菌検査 (2012)
  • Author(s): 吉田明弘
  • Journal Title: 九州歯会誌 Volume: 65 Pages: 169-178
  • NAID: 10030022160
• [Journal Article] Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis. (2012)
  • Author(s): Yoshida A, Ennibi OK, Miyazaki H, Hoshino T, Hayashida H, Nishihara T, Awano S, Ansai T.
  • Journal Title: BMC Infectious diseases Volume: 12 Issue: 1 Pages: 253-253
  • DOI: 10.1186/1471-2334-12-253
  • Peer Reviewed
• [Journal Article] Streptococcus anginosusL-cysteine desulfhydrase gene expression is associated with abscess formation in BALB/c mice. (2011)
  • Author(s): Takahashi, Y., Yoshida, A., Nagata, E., Hoshino, T., Oho, T., Awano, S., Takehara, T. and Ansai, T.
  • Journal Title: Molecular Oral Microbiol. Volume: 26 Pages: 221-227
• [Journal Article] Enumeration of visible Enterococcus faecalis, a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time Polymerase chain reaction. (2011)
  • Author(s): Takahashi, Y., Yoshida, A., Nagayoshi, M.,Kitamura, C., Nishihara, T., Awano, S.and Ansai, T
  • Journal Title: Microbiol. Immunol. Volume: 55 Pages: 889-892
• [Journal Article] Enumeration of viable Enterococcus faecalis, a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time polymerase chain reaction (2011)
  • Author(s): Takahashi Y, Yoshida A, Nagayoshi M, Kitamura C Nishihara T, Awano S, Ansai T
  • Journal Title: Microbiol Immunol Volume: 55(12) Issue: 12 Pages: 889-92
  • DOI: 10.1111/j.1348-0421.2011.00390.x
  • NAID: 10031121546
  • Peer Reviewed
• [Journal Article] Correlation between health status and oralChroma-determined volatile sulfide levels in mouth air of the elderly (2011)
  • Author(s): Awano, S., et al
  • Journal Title: J Breath Res Volume: 5 Issue: 4 Pages: 46007-46007
  • DOI: 10.1088/1752-7155/5/4/046007
  • Peer Reviewed
• [Journal Article] 若年層の女性を対象とした口臭に関する意識調査口臭への一般認識と対人行動についてのパイロット研究 (2011)
  • Author(s): 峰岡哲郎、粟野秀慈、吉田明弘、邵仁浩、濱嵜朋子、安細敏弘
  • Journal Title: 口腔衛生会誌 Volume: 61 Pages: 282-287
  • Peer Reviewed
• [Journal Article] The identification of genes specific to Prevotella intermedia and Prevotella nigrescens using genomic subtractive hybridization. (2010)
  • Author(s): Masakiyo, Y., Yoshida, A., Shintani, Y., Takahashi, Y., Ansai, T. and Takehara, T.
  • Journal Title: Anaerobe Volume: 16 Pages: 265-269
• [Journal Article] Rapid LED-based fluorescence microscopy distinguishes between live and dead bacteria in oral clinical samples. (2010)
  • Author(s): Masakiyo, Y., Yoshida, A., Takahashi, Y., Shintani, Y., Awano, S., Ansai, T., Sawayama, S., Shimakita, T. and Takehara, T
  • Journal Title: Biomed Res. Volume: 31 Pages: 21-26
  • NAID: 130004903779
• [Journal Article] The identification of genes specific to Prevotella intermedia and Prevotella nigrescens using genomic subtractive hybridization (2010)
  • Author(s): Masakiyo, Y., Yoshida, A., Shintani, Y., Takahashi, Y., Ansai, t., Takehara, T
  • Journal Title: Anaerobe Volume: 16 Pages: 265-269
  • Peer Reviewed
• [Presentation] Streptococcus gordoniiと共培養時にStreptococcus mutansに発現するタンパク質のプロテオーム解析 (2012)
  • Author(s): 吉田明弘、安永愛、仁木満美子、山本裕司、西原達次、中村卓、安細敏弘
  • Organizer: 第85回日本細菌学会総会
  • Place of Presentation: 長崎
• [Presentation] 生菌選択的なう蝕細菌定量法の開発と小児口腔内試料の解析. (2012)
  • Author(s): 安永 愛、吉田明弘、正清義朗、森川和政、牧憲司、邵 仁浩、粟野秀慈、安細敏弘
  • Organizer: 第61回日本口腔衛生学会総会
  • Place of Presentation: 横須賀
• [Presentation] Aggregatibacter actinomycetemcomitansロイコトキシン産生/非産生株の選択的定量検出法の開発. (2012)
  • Author(s): 吉田明弘、宮さき秀夫、林田秀明、粟野秀慈、安細敏弘
  • Organizer: 第61回日本口腔衛生学会総会
  • Place of Presentation: 横須賀
• [Presentation] 歯面初期定着菌群との共培養時におけるStreptococcus mutans抗酸化タンパク質の機能解析. (2012)
  • Author(s): 安永 愛、吉田明弘、西原達次、安細敏弘
  • Organizer: 第54回日本歯科基礎医学会
  • Place of Presentation: 郡山
• [Presentation] Proteome analysis of Streptococcus mutansbiofilms co-cultured with Streptococcus gordonii (2012)
  • Author(s): Yoshida, A., Yasunaga, A., Niki, M., Yamamoto, Y., Nishihara, T. and Ansai, T.
  • Organizer: 6th ASM Conference on Biofilms.
  • Place of Presentation: Miami, Florida
• [Presentation] Aggregatibacter actinomycetemcomitansロイコトキシン産生/非産生株の選択的定量検出法の開発 (2012)
  • Author(s): 吉田明弘、宮﨑秀夫、林田秀明、粟野秀慈、安細敏弘
  • Organizer: 第61回日本口腔衛生学会総会
  • Place of Presentation: 横須賀
• [Presentation] 歯周病と細菌検査 (2011)
  • Author(s): 吉田明弘
  • Organizer: 九州歯科学会
  • Place of Presentation: 北九州
  • Year and Date: 2011-05-29
• [Presentation] 歯周病と細菌検査 (2011)
  • Author(s): 吉田明弘
  • Organizer: 九州歯科学会総会(北九州)シンポジウム
  • Place of Presentation: 北九州
• [Presentation] Streptococcus gordoniiと共培養時にStreptococcus mutansに発現するタンパク質の解析. (2011)
  • Author(s): 安永 愛、吉田明弘、仁木満美子、山本裕司、安細敏弘
  • Organizer: 日本細菌学会地方会
  • Place of Presentation: 門司
• [Presentation] Enumeration of viable cariogenic pathogens assessed by PMA-based real-time PCR (2011)
  • Author(s): Yasunaga, A., Yoshida, A., Masakiyo, Y., Morikawa, K., Maki, K., Soh, I., Awano, S. and Ansai, T.
  • Organizer: JADR
  • Place of Presentation: 広島
• [Presentation] Streptococcus gordoniiと共培養時にStreptococcus mutansに発現するタンパク質の解析 (2011)
  • Author(s): 安永愛、吉田明弘、仁木満美子、山本裕司、安細敏弘
  • Organizer: 日本細菌学会九州地方会
  • Place of Presentation: 北九州
• [Presentation] マウス膿瘍形成におけるStreptococcus anginosus βC-S liase遺伝子の発現解析. (2010)
  • Author(s): 高橋優介、吉田明弘、新谷泰之、粟野秀慈、安細敏弘
  • Organizer: 第70回九州歯科学会総会
  • Place of Presentation: 北九州
• [Presentation] 生菌・死菌を区別した根管内細菌の定量検出法の開発. (2010)
  • Author(s): 高橋優介、吉田明弘、粟野秀慈、安細敏弘
  • Organizer: 第59回日本口腔衛生学会・総会
  • Place of Presentation: 新潟
• [Presentation] Streptococcus mutansの生菌と死菌/損傷菌を区別した定量検出法の開発 (2010)
  • Author(s): 正清義朗、吉田明弘、高橋優介、安細敏弘
  • Organizer: 第59回日本口腔衛生学会・総会
  • Place of Presentation: 新潟
• [Presentation] マウス実験膿瘍におけるStreptococcus anginosus lcd遺伝子の発現解析 (2010)
  • Author(s): 吉田明弘、高橋優介、長田恵美、於保孝彦、粟野秀慈、安細敏弘
  • Organizer: 第59回日本口腔衛生学会・総会
  • Place of Presentation: 新潟
• [Presentation] Streptococcus anginosus βC-S lyase gene expression and experimental abscess formation. (2010)
  • Author(s): Yoshida, A., Takahashi, Y., Nagata, E., Hoshino, T., Oho, T., Awano, S. and Ansai, T.
  • Organizer: 第88回IADR
  • Place of Presentation: Centre Convencions Internacional Barcelona
• [Presentation] Novel qualitative detection method for Aggregatibacter actinomycetemcomitans JP2. (2010)
  • Author(s): Dewake, N., Yoshida, A., Takahashi, Y., Awano, S., Miyazaki, H. and Ansai, T.
  • Organizer: 58th JADR
• [Presentation] Streptococcus anginosus βC-S lyase gene expression and experimental abscess formation (2010)
  • Author(s): Yoshida, A., Takahashi, Y., Nagata, E., Hoshino, T., Oho, T., Awano, S., Ansai, T.
  • Organizer: 第88回International Association for Dental Research
  • Place of Presentation: Centre Convencions Internacional Barcelona
• [Presentation] マウス実験膿瘍におけるStreptococcus anginosus Icd遺伝子の発現解析 (2010)
  • Author(s): 吉田明弘、高橋優介、長田恵美、於保孝彦、粟野秀慈、安細敏弘
  • Organizer: 第59回日本口腔衛生学会・総会
  • Place of Presentation: 新潟
• [Book] Oral disease and hydrogen sulfideproduction by oral bacteria (eds. Derin Adebayo and Aramide Okafor) (2013)
  • Author(s): Yoshida, A., Niki, M., Ansai, T. and Nakayama, K.
  • Publisher: Nova SciencePublishers, New York, USA
• [Book] DHEAS and periodontal status in older Japanese. In: DHEA in Human Health and Aging, ed (2012)
  • Author(s): Yoshida, A. and Ansai, T.
  • Publisher: Ronald Ross Watson, CRC Press, Taylor & Francis Group LLC.
• [Book] Microbiological Diagnosis for Periodontal Diseases. In: Periodontal Diseases - A Clinician's Guide (Ed. by Jane Manakil) (2012)
  • Author(s): Yoshida, A. and Ansai, T.
• [Book] DHEAs and periodontal status in Older Japanese. In: DHEA in Human Health and Aging. (2012)
  • Author(s): Yoshida, A. and Ansai, T. (Akihiro Yoshida)
  • Total Pages: 14
  • Publisher: CRC Press, Taylor & Francis Group LLC.,
• [Book] Microbiological Diagnosis for Periodontal Diseases. In: Periodontal Diseases - A Clinician's Guide (2012)
  • Author(s): Yoshida, A. and Ansai, T. (Akihiro Yoshida)
  • Total Pages: 12
  • Publisher: InTech
• [Book] DHEA in Human Health and Aging (2012)
  • Author(s): Yoshida, A., Ansai, T.
  • Total Pages: 452
  • Publisher: CRC Press, Taylor & Francis Group LLC
• [Book] 必修 臨床研修歯科医ハンドブック (2010)
  • Author(s): 粟野秀慈、吉田明弘、邵 仁浩、安細敏弘
  • Publisher: 医歯薬出版
• [Remarks]
  • URL: http://www2.kyu-dent.ac.jp/dept/oral-health/
• [Remarks] 九州歯科大学地域健康開発歯学分野
  • URL: http://www2.kyu-dent.ac.jp/dept/oral-health/index.html
• [Remarks]
  • URL: http://www.kenkoyobou.net/college/index.html
• [Remarks]
  • URL: http://www.kenkoyobou.net/college/index.html
• [Patent(Industrial Property Rights)] 口腔細菌の迅速検出 (2011)
  • Inventor(s): 吉村満美子、中山浩次、大原直也、吉田明弘、竹原直道
  • Industrial Property Rights Holder: 吉田明弘
  • Acquisition Date: 2011
  • Overseas