No abstract

Mycobacterium abscessus is a highly drug-resistant nontuberculous mycobacterium (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating, with a focus on cell wall synthesis proteins (M. abscessus l,d-transpeptidases 1 to 5 [LdtMab1 to LdtMab5] and d,d-carboxypeptidase) that are targeted by β-lactam antibiotics. A challenge to this approach is the presence of chromosomally encoded β-lactamase (BlaMab). Using a mechanism-based approach, we found that a novel ceftaroline-imipenem combination effectively lowered the MICs of M. abscessus isolates (MIC50 ≤ 0.25 μg/ml; MIC90 ≤ 0.5 μg/ml). Combining ceftaroline and imipenem with a β-lactamase inhibitor, i.e., relebactam or avibactam, demonstrated only a modest effect on susceptibility compared to each of the β-lactams alone. In steady-state kinetic assays, BlaMab exhibited a lower Ki app (0.30 ± 0.03 μM for avibactam and 136 ± 14 μM for relebactam) and a higher acylation rate for avibactam (k2/K = 3.4 × 105 ± 0.4 × 105 M−1 s−1 for avibactam and 6 × 102 ± 0.6 × 102 M−1 s−1 for relebactam). The kcat/Km was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM−1 s−1) than for imipenem (0.056 ± 0.006 μM−1 s−1). Timed mass spectrometry captured complexes of avibactam and BlaMab, LdtMab1, LdtMab2, LdtMab4, and d,d-carboxypeptidase, whereas relebactam bound only BlaMab, LdtMab1, and LdtMab2. Interestingly, LdtMab1, LdtMab2, LdtMab4, LdtMab5, and d,d-carboxypeptidase bound only to imipenem when incubated with imipenem and ceftaroline fosamil. We next determined the binding constants of imipenem and ceftaroline fosamil for LdtMab1, LdtMab2, LdtMab4, and LdtMab5 and showed that imipenem bound >100-fold more avidly than ceftaroline fosamil to LdtMab1 and LdtMab2 (e.g., Ki app or Km of LdtMab1 = 0.01 ± 0.01 μM for imipenem versus 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modeling indicates that LdtMab2 readily accommodates imipenem, but the active site must widen to ≥8 Å for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (l,d-transpeptidases and d,d-carboxypeptidase) and provides a mechanistic rationale for the effectiveness of this dual β-lactam combination in M. abscessus infections.

MYCOBACTERIUM AVIUM complex (MAC) consists of nontuberculous mycobacteria that cause disease in immunocompromised and immunocompetent hosts. The organisms are ubiquitous in the environment, and acquisition occurs through ingestion or inhalation of aerosols from soil, water, or biofilms. Disease may manifest as disseminated infection, soft tissue infection, chronic pneumonia, or hypersensitivity pneumonitis. Nontuberculous mycobacteria are increasingly associated with pulmonary disease, with MAC being the most common nontuberculous mycobacteria to cause pulmonary disease in the United States. Pulmonary symptoms, nodular or cavitary opacities on a chest radiograph or high-resolution computed tomographic scan with multifocal bronchiectasis and multiple small nodules, plus positive culture results from two sputum specimens or one bronchoscopic specimen are consistent with MAC pulmonary disease. Treatment consists of a macrolide, rifamycin, and ethambutol given three times weekly for noncavitary disease and daily with or without an aminoglycoside for cavitary disease.