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

Background Pseudomonas aeruginosa is a critical pathogen in hospital-acquired infections, with resistance primarily mediated by chromosomally encoded PDC genes. While wild-type PDCs typically hydrolyze penicillins and cephalosporins, mutations can expand their substrate spectrum. This study investigates the molecular epidemiology and biochemical characteristics of PDC variants, particularly PDC-266. Methods 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 (Q79R, N131S, L205V) was conducted to evaluate the impact of individual residues. Steady-state kinetic parameters and inhibition assays were determined for purified enzymes. Molecular docking was utilized to analyze the structural interaction between PDC variants and meropenem. Results 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 compared to PDC-3, PDC-5, and 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 demonstrated that the N131S substitution alters the active site cavity, making it narrower and deeper, facilitating stronger hydrogen bonding between meropenem and the catalytic residue SER-64. Conclusion This study characterizes PDC-266, a PDC variant capable of hydrolyzing Meropenem. The N131S substitution is identified as the critical determinant for this expanded substrate specificity by remodeling the active site architecture. Despite this expansion, PDC-266 remains susceptible to Avibactam and Sulbactam.