Durlobactam in combination with β-lactams to combat .

() presents significant clinical challenges. This study evaluated the synergistic effects of a β-lactam and β-lactamase inhibitor combination against 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 (; ≤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 and LDT/PBPs more potently than avibactam. The s of durlobactam for PBP B, PBP-lipo, and LDT2 were below clinically achievable unbound concentrations, while avibactam's for LDT/PBPs exceeded the clinical concentrations. Single β-lactam treatments resulted in minimal killing (1 log reduction). Although avibactam yielded no effect, combinations with avibactam showed a significant reduction (4 log CFU/mL). Durlobactam alone showed ~2 log reduction, and when combined with imipenem or two β-lactams, durlobactam achieved near-eradication of , 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 .