Evolution toward extremely high imipenem resistance in <i>Mycobacterium abscessus</i> outbreak strains

Treatment of <i>Mycobacterium abscessus</i> pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem). <i>M. abscessus</i> produces a β-lactamase (Bla_Mab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in <i>M. abscessus</i> and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected <i>M. abscessus</i> subsp. <i>massiliense</i> isolates from the index case of a cystic fibrosis center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC > 512 µg/mL). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased <i>bla</i>_Mab mRNA level. Concurrent increase in transcription of the preceding <i>ppiA</i> gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin <i>mspA</i> coincided with the first increase in MIC (from 8 to 32 µg/mL). A frameshift mutation in <i>msp2</i> responsible for the rough colony morphology and a SNP in ATP-dependent helicase <i>hrpA</i> cooccurred with the second increase in MIC (from 32 to 256 µg/mL). Increased Bla_Mab expression and enzymatic activity may have been due to altered regulation of the <i>ppiA-bla</i>_Mab operon by the mutated HrpA alone or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating <i>M. abscessus</i>, particularly imipenem-resistant strains.