RNA-sequencing elucidates drug-specific mechanisms of antibiotic tolerance and resistance in M. abscessus

Mycobacterium abscessus is an opportunistic pathogen notorious for its resistance to most classes of antibiotics and low cure rates. M. abscessus carries an array of mostly unexplored defence mechanisms. A deeper understanding of antibiotic resistance and tolerance mechanisms is pivotal in development of targeted therapeutic regimens. We provide the first description of all major transcriptional mechanisms of tolerance to all antibiotics recommended in current guidelines, using RNA sequencing-guided experiments. M. abscessus ATCC 19977 bacteria were subjected to sub-inhibitory concentrations of clarithromycin, amikacin, tigecycline, cefoxitin and clofazimine for 4- and 24-hours, followed by RNA sequencing. To confirm key mechanisms of tolerance suggested by transcriptomic responses, we performed time-kill kinetic analysis using bacteria after pre-exposure to clarithromycin, amikacin or tigecycline for 24-hours and we constructed isogenic knockout and knockdown strains. To assess strain specificity, pan-genome analysis of 35 strains from all three subspecies was performed. Mycobacterium abscessus shows both drug-specific and common transcriptomic responses to antibiotic exposure. Ribosome-targeting antibiotics clarithromycin, amikacin and tigecycline elicit a common response characterized by upregulation of ribosome structural genes, the WhiB7 regulon and transferases, accompanied by downregulation of respiration through NuoA-N. Exposure to any of these drugs decreases susceptibility to ribosome-targeting drugs from multiple classes. The cytochrome bd-type quinol oxidase contributes to clofazimine tolerance in M. abscessus and the sigma factor sigH but not anti-sigma factor MAB_3542c is involved in tigecycline resistance. The observed transcriptomic responses are not strain-specific, as all genes involved in tolerance, except erm(41), are found in all included strains.