Investigating the divergence of β-lactamase PDC variants conferring various antibiotic resistance profiles in Pseudomonas aeruginosa, coupled with the molecular profiling of PDC-266

Pseudomonas aeruginosa is a critical pathogen in hospital-acquired infections, with resistance primarily mediated by chromosomally encoded blaPDC alleles. While wild-type PDCs typically hydrolyze penicillins and cephalosporins, specific amino acid substitutions can broaden their hydrolytic profiles. This study investigates the molecular epidemiology and biochemical characteristics of clinical PDC variants, with a focus on comprehensive functional profiling of the previously documented PDC-266 enzyme. We sequenced the genomic DNA of 14 P. aeruginosa clinical isolates. Antimicrobial susceptibility testing was performed via agar dilution. The plasmids expressing blaPDC−3, blaPDC−5, blaPDC−10, and blaPDC−266 were transformed into E. coli DH5α. Site-directed mutagenesis (targeting residues Q79R, N131S and L205V) was conducted to evaluate the impact of individual amino acid residues. Steady-state kinetic parameters and inhibition assays were determined using purified enzymes. Molecular docking was utilized to analyze the structural interactions between PDC variants and meropenem. Among the 14 isolates, four PDC variants were identified: PDC-3 (n = 6), PDC-5 (n = 5), PDC-10 (n = 2), and PDC-266 (n = 1). The PDC-266-producing isolate (PA1CSR) exhibited a significantly elevated MIC for meropenem (64 µg/mL). Transformation assays confirmed that PDC-266 conferred an 8-fold increase in meropenem MIC relative to strains expressing PDC-3, PDC-5, or PDC-10. Kinetic analysis revealed that PDC-266 and the engineered variant PDC-3-N131S possessed moderate catalytic efficiency against meropenem (kcat/Km: 15–16.5 mM⁻¹·s⁻¹). Molecular docking simulations generated a structural model hypothesizing that the N131S substitution may reshape the active site cavity into a narrower, deeper pocket, which may facilitate enhanced hydrogen-bonding interactions between meropenem and the catalytic Ser64 residue. This work provides the first full biochemical and structural characterization of the documented PDC-266 enzyme, which suggests the enzyme possesses functional capacity to hydrolyze meropenem. The N131S substitution appears to act as the primary residue likely driving expanded substrate breadth, via a modeled remodeling of the active-site pocket architecture derived from in silico simulations. Despite this expansion, PDC-266 remains susceptible to avibactam and sulbactam, highlighting the potential of these inhibitors in treating PDC-266-mediated resistance. Functional Profiling of PDC-266: First comprehensive biochemical and structural characterization of the PDC-266 cephalosporinase recovered from clinical P. aeruginosa isolates. Unique Carbapenem-Hydrolyzing Trait: PDC-266 exhibits atypical carbapenemase-like activity against meropenem, a rarely observed substrate profile for native PDC enzymes, validated via enzyme kinetic assays. Key Mutation: The N131S substitution likely functions as the major residue associated with elevated meropenem catalytic efficiency and higher MIC values. Structural Hypothesis from Docking: N131S may trigger conformational rearrangement of the active site to form a narrower, deeper pocket, which potentially improves meropenem binding affinity.