Dousa KM, et al. Antimicrob Agents Chemother 2020.
Mycobacterium abscessus (Mab) is a highly drug-resistant nontuberculous mycobacteria (NTM). Efforts to discover new treatments for M. abscessus infections are accelerating with a focus on cell wall synthesis proteins (L, D-transpeptidases, LdtMab1-5, 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 show that a novel ceftaroline-imipenem combination effectively lowered the minimal inhibitory concentrations (MICs) of Mab isolates (MIC50 ≤ 0.25, MIC90 ≤ 0.5). Ceftaroline and imipenem combined with a β-lactamase inhibitor, relebactam or avibactam, demonstrated only a modest effect on susceptibility, compared to each of the beta-lactams alone. In steady state kinetic assays, BlaMab exhibited a lower K i app (K i app = 0.30 ± 0.03 μM, avibactam; 136 ± 14 μM, relebactam) and a faster acylation rate for avibactam (k 2/K = 3.4 ± 0.4 x 105 M-1s-1, avibactam; 6 ± 0.6 x 102 M-1s-1, relebactam). The k cat/K m was nearly 10-fold lower for ceftaroline fosamil (0.007 ± 0.001 μM-1s-1) compared to imipenem (0.056 ± 0.006 μM-1s-1). Timed mass spectrometry captured complexes of avibactam and BlaMab, LdtMab1, 2, and 4, and D,D-carboxypeptidase, whereas relebactam bound only BlaMab and LdtMab1 and 2 Interestingly, LdtMab1, 2, 4 and 5 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 to LdtMab1, 2, 4 and 5 and showed that imipenem bound > 100 fold more avidly than ceftaroline fosamil for LdtMab1 and LdtMab2 (e.g. K i app or K m LdtMab1 = 0.01 ± 0.01 μM for imipenem vs 0.73 ± 0.08 μM for ceftaroline fosamil). Molecular modelling indicates that LdtMab2 readily accommodates imipenem, but the active site must widen to ≥ 8Å for ceftaroline to enter. Our analysis demonstrates that ceftaroline and imipenem binding to multiple targets (L, D-transpeptidases and D, D-carboxypeptidase) provides mechanistic rationale for the effectiveness of this dual β-lactam combination in Mab infections.