2022 Fiscal Year Research-status Report
Establishment of a novel identification system for Enterotoxigenic Bacteroides fragilis using CRISPR-Cas13 and bacteriophage technology
| Project/Area Number |
22K20894
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| Research Institution | Jichi Medical University |
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Principal Investigator |
Nguyen・Minh・Thuy 自治医科大学, 医学部, ポスト・ドクター (20964191)
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| Project Period (FY) |
2022-08-31 – 2024-03-31
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| Keywords | CRISPR-Cas13a / Bacteriophage / gut microbe / Bacteroides fragilis / Identification |
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| Outline of Annual Research Achievements |
Enterotoxigenic Bacteroides fragilis (ETBF) is the cause of inflammatory diarrhea of the colon and intestinal cancer. 5-20% of the world's population carries this strain asymptomatically. Therefore, rapid and easy detection methods are needed to properly prevent and treat the disease. Our laboratory has independently developed an antibacterial capsid using the CRISPR-Cas13a system. This application aims to establish a simple identification system using the bft gene of Bacteroides fragilis. This study culminates in the development of new therapeutic strategies for controlling gut microbiota in the gut microbiota community.
In the first year, I have achieved some results: I collected 215 Bacteroides fragilis (BF) strains, including 12 bft1-positive, 8 bft-2-positive, and 1 bft-3-positive strain. I performed whole-genome sequencing on 50 strains, completing a comprehensive survey of their genomes and found out 11 strains revealed prophage sequences in their genome. Based on the results of MIC, six strains that susceptible to antibiotics and not carry prophage in their genome, were suitable to carry Capsid.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
(Research plan 2022): Delay
A.Isolation and identification of phage infected with B. fragilis
B.Characteristic analysis of B. fragilis phage
The reason for delay: Prophage induction was performed using mitomycin C from Bacteroides fragilis. We have used an already established method for another strain, but it was not successful for Bacteroides fragilis. In order to deal with this problem, we reexamined the phage induction method and established a method. We have improved methods of prophage induction by incorporating different antibiotics such as nalidixic acid, hydromycin B, norfloxacin, oxfloxacin, ciprofloxacin, gatifloxacin, lomefloxacin and mitomycin C.
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| Strategy for Future Research Activity |
Continuing the first year, the research plan involves the enhancement of prophage induction from Bacteroides fragilis by incorporating various antibiotics. we will measure the infection rate of the phage using clinical isolate B. fragilis strains. We will also observe the phage morphology with a transmission electron microscope. In particular, we will Select the optimal phage for Cas13a loading. In this year, I will generate the Capsid-Cas13a follow Laboratory's published protocol (Kiga's publication in Nat. Comm., 2020). We have been searched and optimized of CRISPR-cas sequences that recognize target genes 16 spacers based on bft gene sequence. We will search and optimize the spacer to regconize and kill ETBF. After construct the capsid, we will demonstrate the usefulness of the genotype method using clinical isolates.
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| Causes of Carryover |
In last year I plan to prophage identification using whole genome sequence. However, I cannot get any prophage from Bacteroides fragilis, it is remaining the budget of doing whole genome sequence of phage. This year, it is necessary to use the budget for whole genome sequence of prophage, and other more budget in the research plan. For example: sequence check spacer and in vivo experiment with mice. It is possible to totally using the budget.
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