Role of inorganic phosphate concentrations in in vitro activity of fosfomycin.

OBJECTIVES: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). METHODS: The wild-type BW25113 strain, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT, and ΔphoB) and six clinical isolates of Escherichia coli with different fosfomycin susceptibilities were used. EUCAST breakpoints were used. Susceptibility was evaluated by agar dilution using standard Mueller-Hinton agar (Pi concentration of 1 mM similar to human plasma concentration) and supplemented with Pi (13 and 42 mM, minimum and maximum urinary Pi concentrations) and/or glucose-6-phosphate (25 mg/L). Fosfomycin transporter promoter activity was assayed using PglpT::gfpmut2 or PuhpT::gfpmut2 promoter fusions in standard Mueller-Hinton Broth (MHB), supplemented with Pi (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC50 under different Pi concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time-kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (Cmax)), 1053 and 4415 mg/L (urine Cmax range), using MHB with 28 mM Pi (mean urine Pi concentration) + 25 mg/L glucose-6-phosphate. RESULTS: All the strains showed decreased susceptibility to fosfomycin linked to increased Pi concentrations: 1-4 log2 dilution differences from 1 to 13 mM, and 1-8 log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing Pi concentrations higher fosfomycin EC50 bacterial viability was observed, except against ΔglpT-uhpT. The increase in Pi reduced the bactericidal effect of fosfomycin. DISCUSSION: Pi variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated Pi concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.

Urinary Tract Conditions Affect Fosfomycin Activity against Escherichia coli Strains Harboring Chromosomal Mutations Involved in Fosfomycin Uptake.

The steps by which Escherichia coli strains harboring mutations related to fosfomycin (FOS) resistance arise and spread during urinary tract infections (UTIs) are far from being understood. The aim of this study was to evaluate the effects of urine, pH, and anaerobiosis on FOS activity against a set of isogenic strains carrying the most prevalent chromosomal mutations conferring FOS resistance (ΔuhpT, ΔglpT, ΔcyaA, and ΔptsI), either singly or in combination. We also studied fosfomycin-resistant E. coli clinical isolates from patients with UTI. Our results demonstrate that urinary tract physiological conditions might have a profound impact on FOS activity against strains with chromosomal FOS resistance mutations. Specifically, acidic pH values and anaerobiosis convert most of the strains categorized as resistant to fosfomycin according to the international guidelines to a susceptible status. Therefore, urinary pH values may have practical interest in the management of UTIs. Finally, our results, together with the high fitness cost associated with FOS resistance mutations, might explain the low prevalence of fosfomycin-resistant E. coli variants in UTIs.