Molecular insights into fosfomycin resistance in Escherichia coli

Abstract

Objectives: Fosfomycin activity in Escherichia coli depends on several genes of unknown importance for fosfomycin resistance. The objective was to characterize the role of uhpT, glpT, cyaA and ptsI genes in fosfomycin resistance in E. coli. Methods: WT E. coli BW25113 and null mutants, ΔuhpT, ΔglpT, ΔcyaA, ΔptsI, ΔglpT-uhpT, ΔglpT-cyaA, ΔglpTptsI, ΔuhpT-cyaA, ΔuhpT-ptsI and ΔptsI-cyaA, were studied. Susceptibility to fosfomycin was tested using CLSI guidelines. Fosfomycin mutant frequencies were determined at concentrations of 64 and 256 mg/L. Fosfomycin in vitro activity was tested using time-kill assays at concentrations of 64 and 307 mg/L (human Cmax). Results: Fosfomycin MICs were: WT E. coli BW25113 (2 mg/L), ΔglpT (2 mg/L), ΔuhpT (64 mg/L), ΔcyaA (8 mg/L), ΔptsI (2 mg/L), ΔglpT-uhpT (256 mg/L), ΔglpT-cyaA (8 mg/L), ΔglpT-ptsI (2 mg/L), ΔuhpT-cyaA (512mg/L), ΔuhpT-ptsI (64 mg/L) and ΔptsI-cyaA (32 mg/L). In the mutant frequency assays, no mutants were recovered from BW25113. Mutants appeared in ΔglpT, ΔuhpT, ΔcyaA and ΔptsI at 64 mg/L and in ΔuhpT and ΔcyaA at 256 mg/L. ΔglpT-ptsI, but not ΔglpT-cyaA, ΔuhpT-cyaA or ΔuhpT-ptsI, increased the mutant frequency compared with the highest frequency found in each single mutant. In time-kill assays, all mutants regrew at 64 mg/L. Initial bacterial reductions of 2-4 log10 cfu/mL were observed for all strains, except for ΔuhpT-ptsI, ΔglpT-uhpT and ΔuhpT-cyaA. Only ΔglpT and ΔptsImutants were cleared using 307 mg/L. Conclusions: Fosfomycin MIC may not be a good efficacy predictor, as highly resistant mutants may appear, depending on other pre-existing mutations with no impact on MIC.