A single upstream mutation of whiB7 underlies amikacin and clarithromycin resistance in Mycobacterium abscessus

Aims: We aimed to investigate the molecular mechanisms underlying the survival of Mycobacterium abscessus when faced with antibiotic combination therapy. By conducting evolution experiments and whole-genome sequencing (WGS), we sought to identify genetic variants associated with stress response mechanisms, with a particular focus on drug survival and resistance. Methods and results: We conducted evolution experiments on M. abscessus, exposing the bacteria to a combination therapy of amikacin and rifabutin. Genetic mutations associated with increased antibiotic survival and altered susceptibility were subsequently identified by WGS. We focused on mutations that contribute to stress response mechanisms and tolerance. Of particular interest was a novel frameshift mutation in MAB_3509c, a gene of unknown function within the upstream open reading frame of whiB7. A MAB_3509c knockout mutant was constructed, and expression of downstream drug resistance genes was assessed by RT-qPCR. Mutation of MAB_3509c results in increased RNA levels of whiB7and downstream stress response genes such as eis2, which is responsible for aminoglycoside resistance. Conclusion: Our findings demonstrate the importance of whiB7in the adaptive stress response in M. abscessus. Moreover, our results highlight the complexity of M. abscessus adapting to drug stress and underscore the need for further research. Impact Statement Interventional therapy options for Mycobacterium abscessus infections are severely limited, and antimicrobial resistance further restricts available treatments, leading to prolonged, poorly tolerated regimens with suboptimal outcomes. Here, we investigate a polymorphism in the upstream open reading frame of whiB7, which contributes to increased resistance in M. abscessus. Understanding the mechanisms behind WhiB7 induction offers novel insights into intrinsic resistance and may serve as a valuable tool in drug discovery.