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High-throughput mutagenesis and screening approach for the identification of drug-resistant mutations in the rifampicin resistance-determining region of mycobacteria.
- Source :
-
International journal of antimicrobial agents [Int J Antimicrob Agents] 2024 Jun; Vol. 63 (6), pp. 107158. Date of Electronic Publication: 2024 Mar 26. - Publication Year :
- 2024
-
Abstract
- Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs.<br /> (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Subjects :
- Mutation
Mutagenesis
Antitubercular Agents pharmacology
Mycobacterium smegmatis genetics
Mycobacterium smegmatis drug effects
Microbial Sensitivity Tests
High-Throughput Screening Assays methods
Humans
Rifampin pharmacology
Drug Resistance, Bacterial genetics
Mycobacterium tuberculosis genetics
Mycobacterium tuberculosis drug effects
Subjects
Details
- Language :
- English
- ISSN :
- 1872-7913
- Volume :
- 63
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- International journal of antimicrobial agents
- Publication Type :
- Academic Journal
- Accession number :
- 38537722
- Full Text :
- https://doi.org/10.1016/j.ijantimicag.2024.107158