Characterizing the V516M penicillin-binding protein 2 (PBP2) mutation causing zidebactam resistance in Pseudomonas aeruginosa

The triple action of the diazabicyclooctane (DBO) zidebactam (antibacterial activity [penicillin-binding protein 2 {PBP2} inhibitor], β-lactam enhancement [synergy with PBP3 inhibitors], and β-lactamase inhibition [classes A and C]) provides a promising approach to overcome Pseudomonas aeruginosa resistance. However, PBP2 mutations (particularly V516M) are associated with resistance. Thus, we characterized the effect of PBP2 V516M mutation on the activity and PBP-binding affinities (50% inhibitory concentrations [IC 50 s]) of a panel of β-lactams and DBOs using a PBP2 V516M mutant and combinations with dacB , mexR , mexZ , or oprM knockout mutations. V516M dramatically increased zidebactam minimum inhibitory concentration and PBP2 IC 50 and determined specific patterns of β-lactams and DBO susceptibilities and PBP-binding profiles. Nacubactam showed cross-resistance with zidebactam but showed a much lower basal binding affinity against PBP2, explaining its lower intrinsic antipseudomonal activity. Conversely, durlobactam did not show cross-resistance with zidebactam, consistent with its primary target being PBP1b and not PBP2; the low intrinsic activity of durlobactam was related to efflux. Likewise, PBP2 binding of mecillinam was similar to zidebactam, but its lower antipseudomonal activity was related to the higher contribution of efflux (MexAB-OprM). In contrast, imipenem was barely affected by the PBP2 V516M mutation. Zidebactam resistance did not affect its activity as an AmpC inhibitor, restoring cefepime susceptibility in the dacB mutant. The PBP2 V516M mutation was associated with cell morphology changes, but it did not affect growth rate or virulence. Antibiotic resistance and PBP IC 50 profiling of PBP2 V516M mutation help to understand the impact of target modification resistance mechanisms on existing DBOs and provide clues for guiding the development of novel derivatives.