Antibiotic Susceptibility in Shigella

In order to determine MIC of erythromycin, tetracycline and streptomycin, M90T wild-type strain and its derivatives M90T ΔacrAB, M90T ΔacrA, M90T ΔacrB pSfacrB_D408A, and M90T ΔacrD, were inoculated into LB and grown at 37 °C by shaking for 16 h. Cultures were then diluted to OD₆₀₀ 0.02 in LB and 100 µL aliquots were transferred to 96-well plate, each well containing 100 µL of 2-fold serial dilutions of the compounds to be tested (erythromycin 0.001 mg/mL to 2 mg/mL, tetracycline 0.0078 μg/mL to 16 μg/mL, and streptomycin 0.0625 μg/mL to 128 μg/mL). After 16 h incubation at 37 °C, the lowest concentration of antibiotic inhibiting bacterial growth was estimated, as previously described [40 (link)]. At least three biological replicates were performed. Bile salts (B8756, Sigma-Aldrich, St. Louis, MO, USA), consisting of an approximate 1:1 mixture of cholate and deoxycholate, were used at 0.25% (w/v).