Establishment of a novel identification system for Enterotoxigenic Bacteroides fragilis using CRISPR-Cas13 and bacteriophage technology

2023 Fiscal Year Final Research Report

• Project/Area Number: 22K20894
• Research Category: Grant-in-Aid for Research Activity Start-up
• Allocation Type: Multi-year Fund
• Review Section: 0902:General internal medicine and related fields
• Research Institution: Jichi Medical University
• Principal Investigator: Nguyen Thuy 自治医科大学, 医学部, ポスト・ドクター (20964191)
• Project Period (FY): 2022-08-31 – 2024-03-31
• Keywords: CRISPR-Cas13a / Bacteriophage / gut microbe / Bacteroides fragilis / Identification

Outline of Final Research Achievements

In this study, we aimed to establish a simple method for detecting enterotoxin-producing Bacteroides fragilis (ETBF) positive for the target gene bft gene by using an antibacterial capsid with sequence-specific bactericidal activity. We performed a lysogenic phage induction test using mitomycin C on 310 clinically isolated B. fragilis strains. We isolated five strains with a host infection range of 1.36% to 5.47% against 73 clinically isolated B. fragilis strains. To identify the phages obtained, we performed morphological observation by TEM and determined the whole genome sequence. In addition, we designed a total of 39 guide RNAs targeting the bft gene and compared their bactericidal activities. We are currently working on optimizing the antibacterial capsid construction method and the evaluation method for distinguishing the target bacteria.

Free Research Field

細菌学

Academic Significance and Societal Importance of the Research Achievements

本研究の成果は、配列特異的な抗菌カプシドを用いて標的遺伝子であるbft遺伝子を持つETBFのみを、迅速かつ安価に鑑別できる簡易検出法の確立である。申請者は、配列特異的な抗菌カプシドを構築するために、臨床分離Bacteroides fragilisの細菌ゲノム上から効率的にプロファージ（溶原性ファージ）の誘発法を確立した。本技術は、核酸の増幅を必要としない検体細菌に構築した抗菌カプシドを共培養することで細菌の生死を黙示確認するのみで鑑別できる。この技術は、特別な装置や煩雑な手技が不要であるため、技術導入の負担が少なく幅広い医療機関で導入することができると期待される。