Inhibition of Fosfomycin Resistance Protein FosA by Phosphonoformate (Foscarnet) in Multidrug-Resistant Gram-Negative Pathogens

FosA proteins confer fosfomycin resistance to Gram-negative pathogens via glutathione-mediated modification of the antibiotic. In this study, we assessed whether inhibition of FosA by sodium phosphonoformate (PPF) (foscarnet), a clinically approved antiviral agent, would reverse fosfomycin resistance in representative Gram-negative pathogens. The inhibitory activity of PPF against purified recombinant FosA fromEscherichia coli (FosA3),Klebsiella pneumoniae (FosAKP ),Enterobacter cloacae (FosAEC ), andPseudomonas aeruginosa (FosAPA ) was determined by steady-state kinetic measurements. The antibacterial activity of PPF against FosA in clinical strains of these species was evaluated by susceptibility testing and time-kill assays. PPF increased the Michaelis constant (Km ) for fosfomycin in a dose-dependent manner, without affecting the maximum rate (V max ) of the reaction, for all four FosA enzymes tested, indicating a competitive mechanism of inhibition. Inhibitory constant (Ki ) values were 22.6, 35.8, 24.4, and 56.3 μM for FosAKP , FosAEC , FosAPA , and FosA3, respectively. Addition of clinically achievable concentrations of PPF (∼667 μM) reduced the fosfomycin MICs by ≥4-fold among 52% of theK. pneumoniae ,E. cloacae , andP. aeruginosa clinical strains tested and led to a bacteriostatic or bactericidal effect in time-kill assays among representative strains. PPF inhibits FosA activity across Gram-negative species and can potentiate fosfomycin activity against the majority of strains with chromosomally encodedfosA . These data suggest that PPF may be repurposed as an adjuvant for fosfomycin to treat infections caused by some FosA-producing, multidrug-resistant, Gram-negative pathogens.