Fig. 1: Experimental evolution in biofilms.

a Schematic overview of the general set-up of experimental evolution experiments involving antimicrobial treatment of biofilms. b P. aeruginosa readily forms aggregates in SCFM2, making this a suitable growth medium to study evolution in a relevant microenvironment. c Based on whole-genome sequence analysis (mutations occurring in P. aeruginosa PAO1 after repeated exposure to furanone C-30 shown as example⁸⁹), frequency of mutations can be calculated and effect of mutations on protein function (fusA1 shown as example⁸⁹) can be estimated. d Phenotypic characterization typically starts with determining antimicrobial susceptibility (illustrated here with disk diffusion) and the number of CFU (number of CFU in three replicate B. cenocepacia populations after repeated cycles of exposure to tobramycin are shown as example⁷⁰). Experimental evolution in biofilms frequently leads to the occurrence of small colony variants (SCV) (P. aeruginosa AA2 shown as example, picture courtesy of Dr. A. Sass). Finally, changes in metabolism occur during evolution and can be measured using for example microcalorimetry; metabolic activity after treatment of WT P. aeruginosa PAO1 (left) or the same strain evolved in the presence of tobramycin (right) is shown as example.