Development of new enteropathogen detection system target at IgA

• Project/Area Number: 25670272
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Laboratory medicine
• Research Institution: Okayama University (2014); Osaka University (2013)
• Principal Investigator: GOTOH Kazuyoshi 岡山大学, 医歯(薬)学総合研究科, 助教 (20626593)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: IgA / 腸内細菌叢 / 感染症診断 / メタゲノム解析 / 腸管粘膜免疫 / 腸内フローラ / 分泌型IgA / セルソーティング / 次世代シークエンサー

Outline of Final Research Achievements

When we detect nucleic acids of enteric pathogens from diarrheal stool normal gut microbiota, a dominant in fecal DNA pools, masks the low abundance of pathogens. To solve this problem we employed IgA-coated bacterial sorting technologies. First, we examined that IgA-coated cells were separated by a cell sorter from healthy human stool. DNA was extracted from sorting fraction. However, undetectable level DNA by PCR was obtained from low sorted cell count with our equipment. Because it was difficult to find other optimal conditions, I tried magnetic beads-based separation against IgA. Analysis of 16S rDNA deep sequencing using beads bound showed that no any significant differences was revealed between total and IgA-positive fractions despite the direct observation of IgA-positive bacteria by fluorescent microscopy.
We proposed that IgA-targeted separation should be carefully validated using microscopy although that technique has been published as successful experiences.

Research Products

• [Journal Article] Generation of colonic IgA-secreting cells in the cecal patch (2014)
  • Author(s): Masahata, K., Umemoto, E., Kayama, H., Kotani, M., Nakamura, S., Kurakawa, T., Kikuta, J., Gotoh, K., Motooka, D., Sato, S., Higuchi, T., Baba, Y., Kurosaki, T., Kinoshita, M., Shimada, Y., Kimura, T., Okumura, R., Takeda, A., Tajima, M., Yoshie, O., Fukuzawa, M., Kiyono, H., Fagarasan, S., Iida, T., Ishii, M., Takeda, K
  • Journal Title: Nature Comm Volume: 5 Issue: 1 Pages: 3704-3704
  • DOI: 10.1038/ncomms4704
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive metagenomic approach for detecting causative microorganisms in culture-negative infective endocarditis. (2014)
  • Author(s): Imai A, Gotoh K, Asano Y* (corresponding author), Yamada N, Motooka D, Fukushima M, Kanzaki M, Ohtani T, Sakata Y, Nishi H, Toda K, Sawa Y, Komuro I, Horii T, Iida T, Nakamura S, Takashima S.
  • Journal Title: Int J Cardiol. Volume: 172 Issue: 2 Pages: 288-9
  • DOI: 10.1016/j.ijcard.2013.12.197
  • Peer Reviewed
• [Journal Article] Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system. (2013)
  • Author(s): Seki M., et. al.
  • Journal Title: Journal of Medical Microbiology Volume: (印刷中) Issue: 5 Pages: 801-803
  • DOI: 10.1099/jmm.0.051334-0
  • Peer Reviewed
• [Journal Article] Physiologic and metagenomic attributes of the rhodoliths forming the largest CaCO3 bed in the South Atlantic Ocean. (2013)
  • Author(s): Cavalcanti GS, Gregoracci GB, dos Santos EO, Silveira CB, Meirelles PM, Longo L, Gotoh K, Nakamura S, Iida T, Sawabe T, Rezende CE, Francini-Filho RB, Moura RL, Amado-Filho GM, Thompson FL.
  • Journal Title: ISME J Volume: 8 Issue: 1 Pages: 52-62
  • DOI: 10.1038/ismej.2013.133
  • Peer Reviewed
• [Presentation] 虫垂が腸内細菌叢の形成に果たす役割 (2014)
  • Author(s): 後藤和義、倉川尚、飯田哲也
  • Organizer: 第67回日本細菌学会中国・四国支部総会
  • Place of Presentation: 徳島文理大学
  • Year and Date: 2014-10-05
• [Presentation] Dynamics of Fecal Microbiota in Bangladeshi Children (2014)
  • Author(s): K., Gotoh, D., Motooka, S., Nakamura, S., Monira, M., Alam, T., Iida
  • Organizer: 48th US-Japan Cooperative Medical Sciences Program Conference on Cholera and Other Bacterial Infections
  • Place of Presentation: Dhaka, Bangladesh
• [Presentation] Dysbiosis of gut microbiota during cholera and recovery (2013)
  • Author(s): Gotoh, K., Monira, S., Nakamura, S., Alam, N.H., Horii, T., Alam, M., Iida, T.
  • Organizer: The Annual Medical Sciences Conference 21st
  • Place of Presentation: Nonthaburi, Thai
• [Presentation] 次世代シークエンサーを用いた病原体の網羅的診断法 (2013)
  • Author(s): 後藤 和義。元岡 大祐、中村 昇太、飯田 哲也、堀井 俊宏
  • Organizer: 第25回臨床微生物迅速診断研究会総会
  • Place of Presentation: 東京
• [Presentation] バングラデシュにおける小児糞便細菌叢のダイナミクス (2013)
  • Author(s): 後藤 和義、元岡 大祐、中村 昇太、飯田 哲也、堀井 俊宏
  • Organizer: 第47回腸炎ビブリオシンポジウム
  • Place of Presentation: 広島