{Reference Type}: Journal Article
{Title}: High-throughput mutagenesis and screening approach for the identification of drug-resistant mutations in the rifampicin resistance-determining region of mycobacteria.
{Author}: Zhao H;Li J;Feng S;Xu L;Yan B;Li C;Li M;Wang Y;Li Y;Liang L;Zhou D;Wan J;Wang W;Tian GB;Gu B;Huang X;
{Journal}: Int J Antimicrob Agents
{Volume}: 63
{Issue}: 6
{Year}: 2024 Jun 26
{Factor}: 15.441
{DOI}: 10.1016/j.ijantimicag.2024.107158
{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.