(T1130-10-59) Simultaneously Inactivating Three or all Four Penicillin-Binding Proteins (PBPs) among PBP1a, 1b, 2 and 3 Synergistically Kills Acinetobacter baumannii (AB)

Purpose: AB is among the most clinically challenging Gram-negative pathogens. The PBPs are the high-affinity target sites of all β-lactams and each β-lactam preferentially binds to one or multiple different PBPs. However, no systematic study identified the PBPs that need to be inactivated to kill AB. We aimed to identify the PBP occupancy patterns that elicit rapid and substantial killing of AB.

Methods: Static in vitro time-kill experiments over 24 h were performed in duplicate at an initial inoculum of ~5 log₁₀ CFU/mL in AB quadruple knockout strain AL3681 (a derivative of AB307-0294 that lacked two efflux pumps and both β-lactamases, Δbla_ADC Δbla_OXA-51, ΔadeABC, ΔadeIJK). Based on our PBP IC₅₀ data on 50 compounds in lysed AL3681 using the Bocillin^TM FL binding assay, we studied all possible PBP occupancy patterns for 33 β-lactams, and 22 double or triple β-lactam combinations (498 time-kill profiles in total). Confocal microscopy and flow cytometry with live-dead staining were used to characterize bacterial damage. Data were analyzed by non-compartmental methods and linear mixed-effects modeling.

Results: Inhibiting a single PBP yielded ≤0.5 log₁₀ killing at 1h and ≤1 log₁₀ killing at 5h, and required β-lactam concentrations ≥4 × IC₅₀ of the targeted PBP. Inhibiting two PBPs yielded ≤0.5 log₁₀ killing at 1h and ≤1.5 log₁₀ killing at 5h, and required β-lactam concentrations ≥2 × IC₅₀s. Simultaneous inhibition of three or four PBPs among PBPs 1a, 1b, 2 and 3 led to 0.5 to 1.5 log₁₀ killing at 1h and 1.0 to 3.4 log₁₀ killing at 5h, and required lower β-lactam concentrations (0.25-0.5 × IC₅₀s for the PBP with the highest IC₅₀). Synergistic killing and morphology changes were confirmed by microscopy and flow cytometry. PBP1a/1b or PBP3 binding led to filaments, and PBP2 binding to spheres.

Conclusion: Inhibiting 3 or all 4 PBPs among PBP1a, 1b, 2 or 3 killed AB near-maximally. This study is the first to identify and systematically evaluate the PBP occupancy patterns that elicit rapid and substantial killing of AB. These novel insights were uniquely enabled by creating a resistance-deprived quadruple knockout strain and the PBP IC₅₀ database in this strain.

Acknowledgements: JBB performed research consulting for MicuRx Pharmaceuticals. This work was supported by NIH/NIAID R01 AI130185 and R01 AI136803.