Unraveling antibiotic resistance mechanisms in Mycobacterium abscessus: the potential role of efflux pumps

ABSTRACT: Objectives: Mycobacterium abscessus is an opportunistic respiratory pathogen in patients with underlying lung disease. It is infamously known for its low treatment success rates because of its resistance to multiple classes of antibiotics. Further insight into M. abscessus resistance mechanisms is needed to improve treatment options. In this in vitro study, the role of efflux pumps in reaction to antibiotic stress is explored, as well as the ability of the putative efflux inhibitors, thioridazine and verapamil, to potentiate the activity of guideline-recommended antibiotics. Methods: To evaluate the effects of antibiotic stress on mycobacterial efflux pumps, M. abscessus subspecies abscessus was exposed to amikacin, cefoxitin, clarithromycin, clofazimine, and tigecycline for 24 hours. Transcriptomic responses were measured by RNA sequencing to gain insight into upregulation of efflux pump encoding genes. Subsequently, in time-kill kinetics assays, the above-mentioned antibiotics were combined with thioridazine and verapamil to evaluate their potentiating capacity. Results: All five antibiotics led to a fold change of ≥2 Log2 in expression of one or more genes encoding transporter systems. This effect was most pronounced for the ribosome-targeting antibiotics amikacin, clarithromycin, and tigecycline. Time-kill kinetics assays demonstrated synergy between amikacin, tigecycline, clofazimine, cefoxitin, and both thioridazine and verapamil. Conclusion: Antibiotic stressors induce expression of efflux pump encoding genes in M. abscessus, especially antibiotics that target the ribosome. Putative efflux inhibitors thioridazine and verapamil show synergy with various guideline-recommended antibiotics, making them interesting candidates for the improvement of M. abscessus treatment