Inhibition of fosfomycin resistance protein FosA by phosphonoformate (foscarnet) in multidrug-resistant Gram-negative pathogens [PublishAheadOfPrint]

FosA proteins confer fosfomycin resistance in 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 from Escherichia coli (FosA3), Klebsiella pneumoniae (FosA^KP), Enterobacter cloacae (FosA^EC) and Pseudomonas aeruginosa (FosA^PA) was determined by steady-state kinetics. The antibacterial activity of PPF against FosA was evaluated by susceptibility testing and time-kill assays in clinical strains of these species. PPF increased the Michaelis constant K_m for fosfomycin in a dose dependent manner without impacting the maximum rate of the reaction (V_max) for all four FosA enzymes tested, denoting a competitive mechanism of inhibition. Inhibitory constants (K_i) were 22.6, 35.8, 24.4 and 56.3 μM for FosA^KP, FosA^EC, FosA^PA and FosA3, respectively. Addition of clinically achievable concentrations of PPF (~667 μM) reduced the MICs of fosfomycin by ≥4-fold among 52% of K. pneumoniae, E. cloacae and P. aeruginosa clinical strains tested and lead 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 encoded fosA. These data suggest that PPF may be repurposed as an adjuvant for fosfomycin to treat infections caused by some of FosA-producing, multidrug-resistant Gram-negative pathogens.

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