Combinations comprising dual β-lactams and a β-lactamase inhibitor achieve optimal synergistic inhibition of <i>Mycobacterium abscessus</i> growth

The historical model, which posits that β-lactams inhibit bacterial growth while β-lactamase inhibitors (BLIs) merely protect β-lactams from enzymatic degradation, fails to fully explain their activity against <i>Mycobacterium abscessus</i> (<i>Mab</i>). This study demonstrates that synergistic effects extend beyond the traditional one β-lactam + one BLI paradigm, refuting the oversimplified mechanistic framework. First, β-lactam-based BLIs such as clavulanic acid, sulbactam, and tazobactam exhibit intrinsic antibacterial activity against <i>Mab</i>. These agents synergized not only with β-lactams but also with one another, undermining their historical classification as mere β-lactamase inhibitors. The data indicate that their activity is not limited to inhibiting β-lactamases but extends to directly targeting critical bacterial processes. Second, dual β-lactam combinations exhibit synergism against <i>Mab</i> even in the absence of BLIs. For example, despite being rapidly hydrolyzed by the native β-lactamase Bla_Mab, amoxicillin demonstrates strong synergism with β-lactams such as imipenem or ceftaroline. This suggests that the second β-lactam either acts as a functional BLI surrogate or targets complementary pathways. Supporting this, experiments using penicillin- and carbapenem-based probes revealed that β-lactams bind to multiple <i>Mab</i> proteins simultaneously, reinforcing the idea that their synergy arises from targeting complementary essential proteins. Finally, triple combinations comprising dual β-lactam and one BLI, such as amoxicillin + ceftaroline + avibactam, achieved very high synergy, underscoring the complementary roles of dual β-lactams and BLIs. The evidence in this study necessitates a revised model that can more accurately explain the activities of β-lactams and BLIs and underscores the potential for optimizing β-lactam/BLI regimens against <i>Mab</i>.