Synergy Between the Auranofin Analogue PEt3AuCl and Membrane Disruptors, Efflux-Pump Blockers, and Glutathione-Depletors Uncovers Tolerance Pathways in Pseudomonas aeruginosa

Auranofin (AF), an FDA-approved drug for rheumatoid arthritis, exhibits strong antibacterial activity against Gram-positive bacteria, while Gram-negative species remain largely tolerant. This study assessed the antimicrobial activity of AF and three analogues against clinically relevant Gram-negative pathogens and explored tolerance mechanisms in Pseudomonas aeruginosa. Broth microdilution assays were performed on reference strains and clinical isolates of Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa. Synergy studies with the most active analogue, PEt3AuCl (AF-Cl), were conducted against P. aeruginosa using polymyxin B (PMB), two efflux-pump inhibitors, and two glutathione (GSH) depletors. Gold compounds showed MICs between 4 and >64 µg/mL, with AF-Cl displaying the highest activity. AF-Cl activity was markedly enhanced by PMB and efflux-pump inhibitors, indicating that outer membrane permeability and efflux contribute to tolerance. Additionally, GSH depletion significantly potentiated AF-Cl, implicating redox homeostasis in resistance. Overall, AF-Cl shows potential against Gram-negative bacteria when combined with agents targeting membrane integrity, efflux systems, or redox balance, supporting combinatorial strategies to overcome resistance in P. aeruginosa and related pathogens.