Synergistic activity of dual β-lactams against <i>Mycobacterium avium</i> complex

<i>Mycobacterium avium</i> complex (MAC) is a nontuberculous mycobacteria that cause chronic pulmonary infections, particularly in individuals with underlying structural lung disease. Despite prolonged multidrug therapy, treatment outcomes for MAC pulmonary disease remain suboptimal, posing ongoing clinical challenges. Although β-lactams have demonstrated efficacy against <i>Mycobacterium abscessus</i>, β-lactams are not currently recommended for MAC, in part due to limited supporting data. Since <i>M. abscessus</i> and MAC have similar cell wall structures and peptidoglycan synthesis, we hypothesized that β-lactams could be effective against MAC, especially when used in combination to achieve broader inhibition of cell wall-synthesizing enzymes, L,D-transpeptidases, and D,D-transpeptidases. In a systematic screen of clinically relevant β-lactams, we identified combinations-specifically those pairing a penem or carbapenem with a penicillin or cephalosporin-that exhibited potent bactericidal activity and synergy against MAC at clinically achievable concentrations. Of the 91 β-lactam combinations initially screened against <i>Mycobacterium intracellulare</i> (ATCC13950) and <i>M. avium</i> subsp. <i>avium</i> (ATCC25291), 23 pairs demonstrated synergy against <i>M. intracellulare</i> and 16 against <i>M. avium</i> subsp. <i>avium</i>. For <i>M. intracellulare</i>, most synergistic combinations reduced MICs to clinically achievable levels, and the majority of single agents and all combinations achieved ≥40% <i>f</i>T>MIC₅₀. In contrast, although several combinations showed strong synergy against <i>M. avium</i>, their MICs often remained above therapeutic thresholds. Only a few two-drug combinations reached ≥40% <i>f</i>T>MIC₅₀, indicating the need for additional antibiotics. Three β-lactam combinations achieved ≥40% <i>f</i>T>MIC₅₀ against <i>M. avium</i>. These novel β-lactam combinations, leveraging existing antibiotics, may represent promising and immediately deployable treatment strategies for refractory MAC infections.IMPORTANCE<i>Mycobacterium avium</i> complex (MAC) pulmonary disease is increasingly prevalent and difficult to treat, with current multidrug regimens achieving suboptimal and often unsustained responses. Unlike <i>Mycobacterium abscessus</i>, for which β-lactams have shown promise, β-lactams are not recommended for MAC largely due to limited data. Yet, MAC and <i>M. abscessus</i> share similar peptidoglycan structures and reliance on overlapping L,D- and D,D-transpeptidases for cell wall synthesis, suggesting that strategic β-lactam combinations might overcome intrinsic resistance barriers. This study provides the first systematic evaluation of clinically relevant β-lactam pairs against MAC, revealing that specific combinations-particularly those pairing a penem or carbapenem with a penicillin or cephalosporin-exhibit potent bactericidal activity and robust synergy. Several combinations achieved pharmacodynamic targets at clinically achievable exposures, especially against <i>Mycobacterium intracellulare</i>. These findings identify readily accessible β-lactam combinations that could be rapidly translated into new treatment strategies for refractory MAC pulmonary disease, addressing a critical unmet clinical need.