Development of a simultaneous detection for pathogenic bacteria by foam concentration and NGS analysis existing in the water environment

• Project/Area Number: 18K11680
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 64010:Environmental load and risk assessment-related
• Research Institution: University of Miyazaki
• Principal Investigator: Suzuki Yoshihiro 宮崎大学, 工学部, 教授 (90264366)
• Co-Investigator(Kenkyū-buntansha): 真砂 佳史 国際連合大学サステイナビリティ高等研究所, サステイナビリティ高等研究, その他 (50507895)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2019: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2018: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 水系感染症 / 病原細菌 / 病原遺伝子 / 一斉検出・定量 / マルチプレックスPCR / NGSシーケンシング / コピー数 / リード数 / プライマー設計 / 一斉検出 / 次世代シーケンサー / 泡沫濃縮法 / 都市河川 / 菌叢解析 / 病原性細菌属 / 感染リスク / 河川水 / 病原細菌濃縮 / 細菌同時検出

Outline of Final Research Achievements

Waterborne diseases due to pathogenic bacteria in the water environment are a serious problem all over the world. Therefore, it is necessary to develop a method that can accurately and simultaneously obtain information on pathogenic bacteria in the water environment. In this study, the development of a detection method that combines the simultaneous amplification of target genes using the Multiplex PCR method and the simultaneous analysis of base sequences by the NGS analysis were investigated for seven types of E. coli pathogenic genes. As a result of the Multiplex PCR method, it was possible to simultaneously detect 5 out of 7 target genes. The relationship between the copy number of the digital PCR method established as a quantitative analysis and the read number of the NGS analysis, five types of E. coli pathogen-ic genes showed a positive correlation. In addition, it was suggested that the copy number can be estimated from the number of reads.

Academic Significance and Societal Importance of the Research Achievements

病原性細菌の検出技術が発展しているにも関わらず，水環境において，それら病原性細菌の一斉検出法は確立されておらず，情報・知見が乏しいのが現状である．本研究では，Multiplex PCR法における多数の標的遺伝子を一斉に増幅させるプロセスとNGS法による塩基配列の一斉解析のプロセスを組み合わせて，水環境における病原性細菌の一斉検出法の開発した．この開発した方法によって，これまで得られていなかった水環境における病原遺伝子の存在実態と分布が追跡できる．

Research Products

• [Journal Article] The level of antimicrobial resistance of sewage isolates is higher than that of river isolates in different Escherichia coli lineages (2020)
  • Author(s): Ogura Yoshitoshi、Ueda Takuya、Nukazawa Kei、Hiroki Hayate、Xie Hui、Arimizu Yoko、Hayashi Tetsuya、Suzuki Yoshihiro
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 17880-17880
  • DOI: 10.1038/s41598-020-75065-x
  • Peer Reviewed
• [Journal Article] Are bacteria potential sources of fish environmental DNA ? (2020)
  • Author(s): Nukazawa K., Akahoshi K., Suzuki Y.
  • Journal Title: PLoS ONE Volume: 15 Issue: 3 Pages: e0230174-e0230174
  • DOI: 10.1371/journal.pone.0230174
  • Peer Reviewed / Open Access
• [Journal Article] Preliminary assessment of the impacts of sediment sluicing events on stream insects in the Mimi River, Japan. (2020)
  • Author(s): Nukazawa, K., Kajiwara, S., Saito, T., Suzuki, Y.
  • Journal Title: Ecological Engineering Volume: 145 Pages: 105726-105726
  • DOI: 10.1016/j.ecoleng.2020.105726
  • Peer Reviewed / Open Access
• [Journal Article] Fast, sensitive, and reliable detection of waterborne pathogens by digital PCR after coagulation and foam concentration. (2019)
  • Author(s): Jikumaru, A., Ishii, S., Fukudome, T., Kawahara, Y., Iguchi, A., Masago, Y., Nukazawa, K., Suzuki, Y.
  • Journal Title: Journal of Bioscience and Bioengineering Volume: - Issue: 1 Pages: 76-81
  • DOI: 10.1016/j.jbiosc.2020.02.004
  • NAID: 40022293623
  • Peer Reviewed
• [Journal Article] Growth and antibiotic resistance acquisition of Escherichia coli in a river that receives treated sewage effluent. (2019)
  • Author(s): Suzuki, Y., Hashimoto, R., Xie, H., Nishimura, E., Nishiyama, M., Nukazawa, K., Ishii, S.
  • Journal Title: Science of the Total Environment Volume: 690 Pages: 696-704
  • DOI: 10.1016/j.scitotenv.2019.07.050
  • Peer Reviewed
• [Journal Article] 下水消化汚泥の遠心脱水プロセスにおける高分子凝集剤の最適薬注率の設定 (2019)
  • Author(s): 鈴木祥広, 遠藤圭吾, 大幸和佳奈, 糠澤桂, 古橋勇一, 仲元寺宣明
  • Journal Title: 水環境学会誌 Volume: 42 Pages: 269-275
  • NAID: 130007743154
  • Peer Reviewed
• [Journal Article] A highly efficient method for concentrating DNA from river water by combined coagulation and foam separation (2019)
  • Author(s): Suzuki, Y., Imafuku, Y., Nishiyama, M., Teranishi, K., Nukazawa, K., Ogura, Y.
  • Journal Title: Separation Science and Technology Volume: - Issue: 18 Pages: 3128-3134
  • DOI: 10.1080/01496395.2019.1565774
  • Peer Reviewed
• [Journal Article] Effects of bacterial pollution caused by a strong typhoon event and the restoration of a recreational beach: Transitions of fecal bacterial counts and bacterial flora in beach sand (2018)
  • Author(s): Suzuki, Y., Teranishi, K., Matsuwaki, T., Nukazawa, K., Ogura, Y.
  • Journal Title: Science of the Total Environment Volume: 640-641 Pages: 52-61
  • DOI: 10.1016/j.scitotenv.2018.05.265
  • Peer Reviewed
• [Presentation] Multiplex PCR 法による病原大腸菌 7 種の一斉検出系の確立 (2021)
  • Author(s): 水宏樹, 真砂佳史, 糠澤桂, 鈴木祥広
  • Organizer: 第54回日本水環境学会年会講演
• [Presentation] Establishment of simultaneous detection system for seven Escherichia coli pathogenic genes from water environment (2020)
  • Author(s): Shimizu, H., Masago, Y., Ogura, Y., Nukazawa, K. and Suzuki, Y.
  • Organizer: Water and Environment Technology Conference (WET2020)
  • Int'l Joint Research
• [Presentation] Antibiotic resistant coliform bacteria survived in treated sewage transfer antibiotic resistance to susceptible Escherichia coli (2020)
  • Author(s): Xie, H., Nukazawa, K. and Suzuki, Y.
  • Organizer: Water and Environment Technology Conference (WET2020)
  • Int'l Joint Research
• [Presentation] Multiplex PCR 法による病原大腸菌 7 種の一斉検出系の確立. (2020)
  • Author(s): 清水宏樹, 真砂佳史, 糠澤桂, 鈴木祥広
  • Organizer: 54回日本水環境学会年会
• [Presentation] 凝集・泡沫濃縮法を利用した河川水からの病原大腸菌の検出・単離手法の開発 (2020)
  • Author(s): 片渕真人, 清水宏樹, 糠澤桂, 鈴木祥広
  • Organizer: 54回日本水環境学会年会
• [Presentation] Development of Comprehensive Detection Method of Pathogenic Bacteria from River Water Combining Foam Concentration and Bacterial Flora Analysis. (2019)
  • Author(s): Shimizu, H., Jikumaru, A., Nukazawa, K., Masago, Y., Ogura, Y., Ishii, S., Suzuki, Y.
  • Organizer: Water Environment Technology Conference 2019 (WET2019)
• [Presentation] A Proposal of Source Tracking of Fecal Pollution in Recreational Waters by Applying Pulsed-field Gel Electrophoresis (PFGE) (2019)
  • Author(s): uzuki, Y., Furukawa, T., Nukazawa, K.
  • Organizer: AOGS2019 in Singapore
  • Invited
• [Presentation] Resistant Escherichia Coli Isolated from Livestock, Wild Animals and Wastewater in a Stock Farm. (2019)
  • Author(s): Hiroki, H., Kobayashi, I., Sakamoto, S.H., Uemura, R., Nukazawa, K., Suzuki, Y.
  • Organizer: Water Environment Technology Conference 2019 (WET2019)
• [Presentation] 自然河川における大腸菌と腸球菌の薬剤耐性率の比較および汚染源の推定 (2019)
  • Author(s): 鈴木祥広, 西村恵美, 糠澤桂
  • Organizer: 第22回日本水環境学会シンポジウム
• [Presentation] 自然河川の最上流域から検出される薬剤耐性菌の供給源の推定 (2019)
  • Author(s): 西村恵美, 糠澤桂, 鈴木祥広
  • Organizer: 第53回日本水環境学会年会
• [Presentation] 泡沫濃縮と菌叢解析を組み合わせた河川水からの病原性細菌の網羅的検出法の開発 (2019)
  • Author(s): 清水宏樹, 軸丸淳史, 糠澤桂, 真砂佳史, 小椋義俊, 石井聡, 鈴木祥広
  • Organizer: 第53回日本水環境学会年会
• [Presentation] 畜産場のウシ，ネズミ，および畜産排水における薬剤耐性大腸菌の実態調査 (2019)
  • Author(s): 廣木颯, 畔柳聴, 坂本信介, 小林郁雄, 上村涼子, 糠澤桂, 鈴木祥広
  • Organizer: 第53回日本水環境学会年会
• [Presentation] Antibiotic resistance profiles of regrowth Escherichia coli in urban rivers inflowing treated wastewater (2019)
  • Author(s): Xie, H., Hashimoto, R., Nukazawa, K., Suzuki, Y.
  • Organizer: 第53回日本水環境学会年会
• [Presentation] 河川上流から河口域に至るDNA濃度の変化 (2019)
  • Author(s): 軸丸淳史, 糠澤桂, 鈴木祥広
  • Organizer: 第53回日本水環境学会年会
• [Presentation] 自然河川の最上流域における大腸菌と腸球菌の薬剤耐性株の存在実態 (2018)
  • Author(s): 西村恵美, 糠澤桂, 鈴木祥広
  • Organizer: 第55回環境工学研究フォーラム
• [Presentation] Changes in phylogroups and antibiotic-resistant profiles of Escherichia coli in municipal wastewater under aerobic condition (2018)
  • Author(s): Xie, H., Nukazawa, K., Suzuki, Y.
  • Organizer: 第55回環境工学研究フォーラム
• [Presentation] Development of high efficiency concentration method of DNA from river water by combined process of coagulation and form separation (2018)
  • Author(s): Jikumaru, A., Imafuku, Y., Teranishi, K., Nukazawa, K., Ogura, Y., Suzuki, Y.
  • Organizer: Water Environment Technology Conference 2018
  • Int'l Joint Research
• [Presentation] Comparison of antibiotic-resistant Escherichia coli collected from uninhabited island and urban ponds in Miyazaki, Japan (2018)
  • Author(s): Nishimura, E., Sugita, K., Sakamoto, S.H., Nukazawa, K., Suzuki, Y.
  • Organizer: Water Environment Technology Conference 2018
  • Int'l Joint Research
• [Remarks] 宮崎大学社会環境システム工学科 水環境研究室
  • URL: http://www.suzuki-labo.com/
• [Remarks] 宮崎大学工学部社会環境システム工学科水環境研究室（鈴木・糠澤研究室）
  • URL: http://www.suzuki-labo.com/