FIG 1.

Schematic overview of the in vitro experimental evolution assay. (A) A single colony is cultured in RPMI-MOPS medium (2% glucose) for 24 h at 37°C after which a standardized inoculum (10⁶ cells) is resuspended in medium containing no drug (control), the drug at a concentration of 2× MIC₅₀, 1× MIC₅₀, and 0.5× MIC₅₀ (shown here) of the particular starting strain. Daily, the culture is rediluted (1/10) in fresh RPMI-MOPS medium (2% glucose) with a concentration of drug based on the OD₆₀₀ of the control culture (see Materials and Methods). All strains were evolved in triplicate. Daily aliquots of evolving populations were stored in RPMI-MOPS medium containing 25% glycerol at −80°C for later analysis. (B) Ancestry of the five evolved strains that were sequenced. WGS was performed on a single colony. The name of each strain represents the experimental treatment (letter) and day of isolation (number), respectively.