Dousa, K. M.; Shin, E.; Kurz, S. G.; Plummer, M.; Nantongo, M.; Bethel, C. R.; Taracila, M. A.; Nguyen, D. C.; Kreiswith, B. N.; Daley, C. L.; Remy, K. E.; Holland, S.; Bonomo, R. A.

Mycobacterium abscessus (Mab) affects patients with immunosuppression, Cystic Fibrosis (CF), or underlying structural lung diseases. Additionally, Mab poses clinical challenges due to its resistance to multiple antibiotics. Herein, we investigated the synergistic effect of dual {beta}-lactams [sulopenem and cefuroxime (CXM)] or the combination of sulopenem and CXM with a {beta}-lactamase inhibitors [BLI; avibactam (AVI) or durlobactam (DUR)]. The sulopenem-CXM combination yielded low minimum inhibitory concentration MIC values for 54 clinical Mab isolates and ATCC19977 (MIC50 and MIC90 [≤] 0.25 g/mL). Similar synergistic effects were observed in time-kill studies conducted at concentrations achievable in clinical settings. Sulopenem-CXM outperformed monotherapy, yielding ~1.5 Log10 CFU/mL reduction during 10 days. Addition of BLIs enhanced this antibacterial effect, resulting in additional reduction of CFUs (~3 Log10 for sulopenem-CXM and AVI and ~4 Log10 for sulopenem-DUR). Exploration of the potential mechanisms of the synergy focused on their interactions with L,D-transpeptidases (LDTs; LDTMab1-LDTMab4), Penicillin-Binding-Protein B (PBP-B), and D,D-Carboxypeptidase (DDC). Acyl complexes identified via mass spectrometry analysis, demonstrated the binding of sulopenem with LdtMab2-LdtMab4, DDC, and PBP B, and CXM with LdtMab2 and PBP-B. Molecular docking suggested formation of a covalent adduct between sulopenem and LdtMab2 after the nucleophilic attack of the cysteine residue at the {beta}-lactam carbonyl carbon, leading to the cleavage of the {beta}-lactam ring, and the establishment of a thioester bond linking the LdtMab2 with sulopenem. In conclusion, we demonstrated the biochemical basis of the synergy of sulopenem-CXM with or without BLI. These findings potentially broaden selection of oral therapeutic agents to combat Mab.