Role of inorganic phosphate concentrations in in vitro activity of fosfomycin
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Updated: Oct 17, 2025
Assaying for Inorganic Polyphosphate in Bacteria
Published on: January 21, 2019
Miriam Ortiz-Padilla1, Inés Portillo-Calderón1, Natalia Maldonado1
1Unidad de Gestión Clínica de Enfermedades Infecciosas y Microbiología Clínica, Hospital Universitario Virgen Macarena, Seville, Spain; Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen Macarena/CSIC/Universidad de Sevilla, Seville, Spain.
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.
Insights
High inorganic phosphate (Pi) levels reduce fosfomycin effectiveness against Escherichia coli by decreasing its susceptibility and bactericidal effect. This may explain treatment failures, especially in urinary tract infections with non-wild-type strains.
Area of Science:
- Microbiology
- Pharmacology
- Infectious Diseases
Background:
- Fosfomycin is a broad-spectrum antibiotic used to treat various bacterial infections.
- Its efficacy can be influenced by physiological conditions, including inorganic phosphate (Pi) concentrations.
- Understanding these interactions is crucial for optimizing antibiotic therapy.
Purpose of the Study:
- To investigate the in vitro activity of fosfomycin against Escherichia coli under varying physiological concentrations of inorganic phosphate (Pi).
- To determine the impact of Pi on fosfomycin susceptibility and bactericidal effects.
- To explore the role of Pi in fosfomycin treatment outcomes.
Main Methods:
- Evaluated susceptibility of E. coli strains (wild-type, mutants, clinical isolates) using agar dilution with varying Pi concentrations (1 mM, 13 mM, 42 mM) and glucose-6-phosphate.
- Assayed fosfomycin transporter promoter activity using promoter fusions under different Pi and glucose-6-phosphate conditions.
- Quantified fosfomycin EC50 and performed time-kill assays at clinically relevant concentrations in media mimicking plasma and urine Pi levels.
Main Results:
- Increased Pi concentrations significantly decreased fosfomycin susceptibility in all E. coli strains, with greater reductions at higher Pi levels.
- Elevated Pi led to higher fosfomycin EC50 values, indicating reduced bacterial viability, except in the ΔglpT-uhpT mutant.
- The bactericidal effect of fosfomycin was diminished under higher Pi conditions, while transporter expression remained unaffected.
Conclusions:
- Physiological variations in inorganic phosphate concentrations can significantly reduce fosfomycin activity against E. coli.
- Elevated urinary Pi levels may contribute to the failure of oral fosfomycin treatment in susceptible, non-wild-type E. coli strains.
- Further research is warranted to elucidate the clinical implications of Pi concentrations on fosfomycin efficacy.
