First Penicillin-Binding Protein Occupancy Patterns of β-Lactams and β-Lactamase Inhibitors in Klebsiella pneumoniae

Abstract

Penicillin-binding proteins (PBPs) are the high-affinity target sites of all β-lactam antibiotics in bacteria. It is well known that each β-lactam covalently binds to and thereby inactivates different PBPs with various affinities. Despite β-lactams serving as the cornerstone of our therapeutic armamentarium against Klebsiella pneumoniae, PBP binding data are missing for this pathogen. We aimed to generate the first PBP binding data on 13 chemically diverse and clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae PBP binding was determined using isolated membrane fractions from K. pneumoniae strains ATCC 43816 and ATCC 13883. Binding reactions were conducted using β-lactam concentrations from 0.0075 to 256 mg/liter (or 128 mg/liter). After β-lactam exposure, unbound PBPs were labeled by Bocillin FL. Binding affinities (50% inhibitory concentrations [IC₅₀]) were reported as the β-lactam concentrations that half-maximally inhibited Bocillin FL binding. PBP occupancy patterns by β-lactams were consistent across both strains. Carbapenems bound to all PBPs, with PBP2 and PBP4 as the highest-affinity targets (IC₅₀, <0.0075 mg/liter). Preferential PBP2 binding was observed by mecillinam (amdinocillin; IC₅₀, <0.0075 mg/liter) and avibactam (IC₅₀, 2 mg/liter). Aztreonam showed high affinity for PBP3 (IC₅₀, 0.06 to 0.12 mg/liter). Ceftazidime bound PBP3 at low concentrations (IC₅₀, 0.06 to 0.25 mg/liter) and PBP1a/b at higher concentrations (4 mg/liter), whereas cefepime bound PBPs 1 to 4 at more even concentrations (IC₅₀, 0.015 to 2 mg/liter). These PBP binding data on a comprehensive set of 13 clinically relevant β-lactams and β-lactamase inhibitors in K. pneumoniae enable, for the first time, the rational design and optimization of double β-lactam and β-lactam-β-lactamase inhibitor combinations.

Keywords: BLIs; Enterobacteriaceae; Klebsiella pneumoniae; PBPs; beta-lactamase inhibitors; beta-lactams; drug-resistant bacteria; occupancy patterns; penicillin-binding proteins; pharmacodynamics; principal component analysis; receptor binding.