Multipassage resistance selection.

<p>A, plot of MICs against <i>E</i>. <i>coli</i> lineages adapted to increasing concentrations of temporins or ampicillin. B, control: MICs against lineages grown in the same conditions without antimicrobial agents (MilliQ water). The following temporins were tested: SHa, D-SHa (SHa with all residues in D-configuration), and [K³]SHa. The conventional antibiotic ampicillin was also used for comparison. <i>E</i>. <i>coli</i> ATCC 25922 was continuously re-cultured in the presence of doubling concentrations of antimicrobial agents from 1/16 of the MIC until adaptation to the MIC (50 passages, 10 bacterial lineages with 1/16 MIC, 1/8 MIC, 1/4 MIC, 1/2 MIC, and MIC) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174024#sec011" target="_blank">Materials and Methods</a>). The MIC of the antimicrobial agent was determined against the adapted <i>E</i>. <i>coli</i> lineages originating from different last passages: passage 5 (<i>E</i>. <i>coli</i> with no antimicrobial agent), 15 (<i>E</i>. <i>coli</i> with a concentration of antimicrobial agent equal to 1/16 MIC), 25 (1/8 MIC), 35 (1/4 MIC), 45(1/2 MIC), and 55 (MIC). MIC values were obtained in triplicate and represent the average of three independent experiments. Curves representing the MIC as a function of the passage number were obtained from the means ± SEM of MIC values of at least three independent experiments.