Role of inorganic phosphate concentrations in in vitro activity of fosfomycin

Abstract

Objectives: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (P_i).

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 (P_i concentration of 1 mM similar to human plasma concentration) and supplemented with P_i (13 and 42 mM, minimum and maximum urinary P_i 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 P_i (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC₅₀ under different P_i concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time-kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (C_max)), 1053 and 4415 mg/L (urine C_max range), using MHB with 28 mM P_i (mean urine P_i concentration) + 25 mg/L glucose-6-phosphate.

Results: All the strains showed decreased susceptibility to fosfomycin linked to increased P_i concentrations: 1-4 log₂ dilution differences from 1 to 13 mM, and 1-8 log₂ dilution differences at 42 mM P_i. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing P_i concentrations higher fosfomycin EC₅₀ bacterial viability was observed, except against ΔglpT-uhpT. The increase in P_i reduced the bactericidal effect of fosfomycin.

Discussion: P_i variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated P_i concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.

Keywords: Escherichia coli; Fosfomycin; Fosfomycin activity; Fosfomycin transporters; Inorganic phosphate; Resistance.