Multistep resistance generation evaluation

1. For the first passage, the broth microdilution method for MIC determination of compounds was performed against the bacteria. An overnight starter culture of bacteria in CAMHB was diluted 1000-fold in fresh media, and grown at 37 °C to reach an OD600 of approximately 0.3.
2. This log-phase culture was diluted in fresh media to generate the working solution containing 5×10⁵ CFU/mL bacteria.
3. 100 µL of this working solution was transferred into every well of a flat-bottom 96-well plate, except for the first column (which was reserved for CAMHB as negative control) and the first row (reserved for initial compound solutions).
4. Compounds were added at specific starting concentrations with a total volume of 200 µL working solution into the first row.
5. The compounds were then sequentially diluted twofold across 8 wells with working solution. Plates were incubated at 37 °C, shaking at 220 rpm, for 24 h.
6. The OD₆₀₀ values were measured to determine MIC values of compounds.
7. For each subsequent passage, the inoculum for MIC determination was adjusted to a final density of approximately 5 × 10⁵ CFU/mL using the contents of a well containing compounds at a sub-inhibitory concentration (at which bacterial growth was observed from the previous passage). 
8. To measure the MIC of each passage, bacteria were transferred to a new 96-well microtiter plate. Then repeat the step 4-7.
9. Resistance was classified as a greater than a 4-fold increase in the initial MIC.

Figure 1. Flow chart for multistep resistance generation experiment.

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