Durlobactam in combination with β-lactams to combat <i>Mycobacterium abscessus</i>

<i>Mycobacterium abscessus</i> (<i>Mab</i>) presents significant clinical challenges. This study evaluated the synergistic effects of a β-lactam and β-lactamase inhibitor combination against <i>Mab</i> and explored the underlying mechanisms. Synergy was assessed through MIC tests and time-kill studies, and binding affinities of nine β-lactams and BLIs to eight target receptors (L,D-transpeptidases [LDT] 1-5, D,D-carboxypeptidase, penicillin-binding protein [PBP] B, and PBP-lipo) were assessed using mass spectrometry and kinetic studies. Thermal stability and morphological changes were determined. Imipenem demonstrated high binding affinity to LDTs and PBPs, with extremely low inhibition constants (<i>K_i</i>_,<i>app</i>; ≤0.002 mg/L for LDT1-2, ≤0.6 mg/L for PBPs), while cephalosporins, sulopenem, tebipenem, and amoxicillin exhibited moderate to low binding affinity. Durlobactam inactivated Bla_Mab and LDT/PBPs more potently than avibactam. The <i>K_i,app</i>s of durlobactam for PBP B, PBP-lipo, and LDT2 were below clinically achievable unbound concentrations, while avibactam's <i>K_i,app</i> for LDT/PBPs exceeded the clinical concentrations. Single β-lactam treatments resulted in minimal killing (~1 log₁₀ reduction). Although avibactam yielded no effect, combinations with avibactam showed a significant reduction (~4 log₁₀ CFU/mL). Durlobactam alone showed ~2 log₁₀ reduction, and when combined with imipenem or two β-lactams, durlobactam achieved near-eradication of <i>Mab</i>, surpassing the current therapy (amikacin + clarithromycin + imipenem/cefoxitin). Inactivation of PBP-lipo by sulopenem, imipenem, durlobactam, and amoxicillin (with avibactam) led to morphological changes, showing filaments. This study demonstrates the mechanistic basis of combinations therapy, particularly imipenem + durlobactam, in overcoming β-lactam resistance in <i>Mab</i>.