WS11.06Resistance to the combination of β-lactams and β-lactamase inhibitors in Mycobacterium abscessus

Abstract

Objectives

Mycobacterium abscessus is an opportunistic pathogen responsible for chronic lung infections in cystic fibrosis patients. The treatment of these infections is complex and relies on a combination of different antibiotics, including a carbapenem (imipenem (IMI)). We have previously shown that second-generation β-lactamase inhibitors belonging to the diazabicyclooctanes (DBOs) family, avibactam (AVI) and relebactam, improve β-lactams activity. Recently, we identified a synergistic effect in vitro, in vivo, and intracellularly between two β-lactams, IMI and amoxicillin (AMOX), in the presence of DBOs against M. abscessus. The current objective is to identify the targets of the triple antibiotics combination.

Methods

The targets of the triple combination IMI/AMOX/AVI were determined by selecting resistant mutants from the M. abscessus CIP104536 strain on increasing concentrations of IMI and AMOX, in the presence of a fixed concentration of AVI (4 mg/l). Antibiotic susceptibility was assessed using the disk diffusion method, and the genomes of mutants were sequenced by the Illumina® platform.

Results

In total, 23 mutants were obtained from nine independent experiments in one to four steps. Two of the 23 mutants grew on the highest tested concentrations of AMOX (4096 mg/l) and IMI (32 mg/l). Analysis of the 23 sequenced genomes revealed no mutations in β-lactam targets. Different mutations affecting efflux pumps, permeability, regulation, and metabolism were identified. The disk diffusion method revealed acquired resistance to antibiotics belonging to different families.

Conclusion

In vitro resistance to the triple combination is not associated with mutations in β-lactam targets but appears to be due to mutations in efflux pumps or mutations affecting the mycomembrane permeability. The next objective will be to evaluate the risk of the emergence of resistance associated with the administration of the triple combination in a murine model of infection.