Mechanistic and evolutionary determinants of cefiderocol resistance in KPC-producing Pseudomonas aeruginosa

Cefiderocol (FDC), a siderophore cephalosporin, shows potent activity against difficult-to-treat Pseudomonas aeruginosa. We analyzed 25 clinical KPC-variant-producing P. aeruginosa from China (2016-2025) and laboratory-induced FDC-resistant strains (KPC-2/ST463, a high-risk P. aeruginosa clone in China), using WGS, RNA-seq, RT-qPCR, metabolomics, and QM/MM simulations. Clinical isolates were dominated by KPC-33/ST463, highly resistant to ceftazidime-avibactam (25/25) but largely FDC-susceptible (22/25). FDC-induced strains acquired blaKPC-33 mutations and siderophore receptor disruptions. QM/MM metadynamics revealed that KPC-33 optimized Ser70 catalytic-site dynamics, reducing the hydrolysis free energy barrier (ΔG‡ from 15.2 to 13.0 kcal/mol) and increasing catalytic efficiency (kcat/KM from 50 to 2000 M⁻¹s⁻¹). The difference in binding pockets between receptor and ligand required the Ser70 hydroxyl oxygen atom of KPC-2 to travel longer distances to complete nucleophilic attack and acylation. We observed a bifurcated response to antibiotic pressure: clinical adaptation involved multi-scale regulatory shifts in iron uptake and efflux, whereas laboratory induction triggered acute genetic transitions in primary resistance determinants. Global epidemiology shows widespread KPC-plasmid dissemination, particularly in Asia. Monitoring plasmid backbones, Ω-loop KPC variants, and siderophore receptor status may guide FDC therapy and anticipate resistance in high-risk strains. Using multi-omics and QM/MM simulations, this work captures a bifurcated evolutionary response to cefiderocol in KPC-producing Pseudomonas aeruginosa, linking KPC-33 catalytic enhancements and regulatory shifts to guide future targeted therapies.