Antibacterial activity of enmetazobactam against <i>Acinetobacter</i> spp.: a molecular dissection of mechanism of action and resistance determinants

The persistence of multidrug-resistant <i>Acinetobacter baumannii</i> remains a clinical challenge. Cefepime/enmetazobactam is a novel combination with demonstrated activity against extended-spectrum β-lactamase-producing Enterobacterales, but its activity against <i>Acinetobacter</i> has not yet been thoroughly explored. We aimed to assess its activity against <i>Acinetobacter</i> spp., including multidrug-resistant strains producing carbapenem-hydrolyzing class D β-lactamases (CHDLs). We analyzed 208 clinical isolates of <i>Acinetobacter</i> spp., including 67 carbapenem-resistant <i>Acinetobacter baumannii</i> (CRAB). Antibiotic susceptibility testing was conducted with cefepime, sulbactam, and imipenem, alone and in combination with enmetazobactam; the latter was also tested individually. Additionally, MICs of enmetazobactam/durlobactam and sulbactam/durlobactam were determined for CRAB and CHDL-producing <i>A. baumannii</i> ATCC 17978 transformants. PBP binding assays (IC₅₀), molecular docking, simulation studies with the enmetazobactam/OXA-23 adduct, hydrolysis kinetics (<i>k</i>_cat, <i>K</i>_m), and OXA-23 inhibition assays (IC₅₀, <i>k</i>_off, <i>t</i>_₁/₂) were performed to elucidate the mechanism of enmetazobactam and detect reduced susceptibility. Enmetazobactam showed high intrinsic activity against <i>Acinetobacter</i> spp., displaying reduced MICs against carbapenem-susceptible isolates. MIC_50/90 of the enmetazobactam/durlobactam combination was 2/2 mg/L for CHDL-producing <i>A. baumannii</i>. Enmetazobactam exhibited bactericidal activity comparable to sulbactam. Binding assays revealed that the antimicrobial activity is driven by selective affinity for PBP2 (IC₅₀ 3.6 mg/L) and PBP3 (IC₅₀ 4.2 mg/L). OXA-23 readily inactivated enmetazobactam, confirming the major role of CHDLs in resistance to enmetazobactam, via substrate-assisted de-acylation. This study evidences the potent antimicrobial activity of enmetazobactam against <i>A. baumannii</i> via inhibition of PBP2 and PBP3. Its combination with new OXA-type inhibitors (e.g., durlobactam) represents a potential therapeutic alternative for multidrug-resistant <i>A. baumannii</i>.