Antibiotic Susceptibility Assay for E. coli

Stock solution of 10 mg mL⁻¹ of four antibiotics, i.e. trimethoprim, nitrofurantoin, rifampicin and ciprofloxacin (Sigma-Aldrich), are prepared by dissolving in dimethyl sulfoxide, diluting in deionized water and filter sterilising (0.22 µm syringe filter, Fisher Scientific). A microdilution assay was also performed on 96-well plates by exposing E. coli TG1 to a serial dilution of trimethoprim, nitrofurantoin, rifampicin and ciprofloxacin, in order to determine the MIC of each drug. The reporter dye resazurin was added to indicate the viability status of the cells^{46 (link)}. For more details on the protocol, see Supplementary Information. The MIC measurements were performed with OD = 0.2, i.e., 2 × 10⁸ CFU mL⁻¹, and repeated for four times.