Fosfomycin disodium salt

Antimicrobial susceptibility profiles were obtained via VITEK 2 using N272 cards. fosfomycin susceptibility was determined in duplicate using Disk Diffusion (DD) and Agar Dilution (AD) methods. DD was performed on Mueller–Hinton agar [23 (link)] (Difco^TM) using 200 μg fosfomycin/50 μg G-6-P disks (Oxoid). AD was done on Mueller–Hinton agar with fosfomycin disodium salt (Sigma Chemical Co., St Louis, MO, USA) supplemented with 25 mg/L of glucose-6-phosphate (G6P) (Sigma Chemical Co., St Louis, MO, USA). Both techniques were executed according to the CLSI guidelines M02-A12 and M07-A10, respectively. A two-fold dilution method from 16 to 256 μg/mL was used to evaluate MICs results. Susceptibility results were interpreted according to the CLSI M100 2018 breakpoints for E. coli extrapolated to Enterobacteriaceae (DD: ≥16 susceptible, 13-15 intermediate, and ≤12 resistant; AD: ≤64 susceptible, 128 intermediate, and ≥256 resistant). Escherichia coli ATCC^® 25922 was used as a quality control strain.

The susceptibility of all E. coli isolates to fosfomycin was initially screened by the disk diffusion method according to CLSI guidelines, using 200 μg fosfomycin disks containing 50 μg of glucose-6-phosphate on Mueller Hinton agar [17 ]. The Minimum Inhibitory Concentration (MIC) of strains showing zone diameters above CLSI breakpoints (≥16 mm) were further determined in duplicate by the agar dilution method [17 ]. Fosfomycin disodium salt (Sigma Aldrich) was added to Mueller–Hinton agar containing 25 mg/L glucose-6-phosphate to obtain two-fold dilutions, spanning concentrations from 16 to 2048 mg/L.