FIG 1.

Parallel evolution of tobramycin resistance level across species and environments. Populations of A. baumannii and P. aeruginosa were propagated in minimal medium with either increasing concentrations of tobramycin or no drug and in either a planktonic or a biofilm lifestyle. Five replicate populations were propagated per treatment. (A) Populations were either propagated for 12 days in no antibiotic or inoculated into half the MIC of tobramycin, with the concentrations being doubled every 72 h. Samples of each population were archived for later phenotypic analysis and sequencing periodically throughout the experiment (red arrows). (B) Populations were propagated with selection for either planktonic growth through a daily 1:100 dilution or biofilm growth through a daily bead transfer, which forces cells to undergo the entire biofilm life cycle of attachment, growth, dispersion, and reattachment every 24 h, as described in previous work (79). (C) Tobramycin resistance level relative to that for the ancestral clone for three randomly chosen populations per treatment after 12 days of evolution. MICs were determined by microdilution in Mueller-Hinton broth according to CLSI guidelines. The fold change in the MIC for three replicates per population is shown, with the median fold change and range indicated. The A. baumannii ancestral clone MIC was 1.0 mg/liter and the P. aeruginosa ancestral clone MIC was 0.5 mg/liter in Mueller-Hinton broth. Populations had to acquire resistance to TOB at a concentration 4× the MIC for the ancestral strain in order to survive the experiment (gray dashed line).