Gentamicin susceptibility testing.

The MIC of Gm was determined using both the broth microdilution method and the agar dilution method (45). First, bacteria were inoculated at ca. 5 × 10⁵ CFU/ml in a 96-well microtiter plate containing 200 μl of TSB or LB per well. The Gm concentrations tested ranged from 0 μg/ml to 512 μg/ml, depending on the strain. The MICs were determined to be the lowest concentration of Gm that inhibited visible growth after 16 to 20 h of incubation at 37°C. Similarly, LA or TSA plates were prepared by adding increasing concentrations of Gm (0 μg/ml to 512 μg/ml) in a 20-ml medium volume. In this case, the bacterial concentrations were adjusted in order to obtain ca. 5 × 10⁴ cells per spot. Growth was assessed after incubation at 37°C for 16 to 20 h. The MIC (expressed in micrograms per milliliter) was defined as the lowest concentration of the antimicrobial agent that prevented the visible growth of a microorganism.