Antimicrobial Susceptibility and Synergy Testing

Minimum inhibitory concentrations (MICs) for cefazolin and fosfomycin were determined by broth microdilution method in cation-adjusted Mueller-Hinton broth (CA-MHB) supplemented with glucose-6-phosphate (G6P) at a final concentration of 25 mg/L which was also used for synergy testing.

Synergy-testing was performed using a chequerboard assay as previously described (Li et al., 2018). Briefly, serial dilutions of cefazolin and fosfomycin were made in u-bottomed 96-well microtiter plates with a final inoculum of approximately 5 × 10⁵ CFU/ml and a final volume of 200 µL per well. Plates were read after an incubation of 18–24 h at 36°C (±1°C). After calculation of the fractional inhibitory concentration indices (FICI) results were interpreted as synergism ≤0.5, >0.5–4 = no interaction and >4 antagonism. The susceptible breakpoint index (SBPI) was calculated according to the following formula: SBPI = (susceptible breakpoint of antimicrobial A/combined MIC of antimicrobial A) + (susceptible breakpoint of antimicrobial B/combined MIC of antimicrobial B), using the clinical breakpoint of 32 mg/L for fosfomycin and the pharmacokinetic/pharmacodynamic breakpoint of 2 mg/L for cefazolin (Milne and Gould, 2010; The European Committee on Antimicrobial Susceptibility Testing, 2021. Breakpoint tables for interpretation of MICs and zone diameters. Version 11.0, 2021. http://www.eucast.org). An SBPI ≥2 indicates that the combined MICs of the tested antimicrobials are equally or lower than their respective breakpoints. It follows that the greater the SBPI value, the more effective the antimicrobial combination is. All experiments were performed in duplicates.