Fig. 1.

Experimental biofilm evolution of P. aeruginosa under antibiotic stress. A The setup of experimental biofilm evolution of P. aeruginosa. The biofilm of six independent lineages of the P. aeruginosa PAO1 were grown on 5 mm glass bead. After 24 h cultivation, one bead was vortex and sonicated for CFU counts, another bead was transferred to a 24 well microplate and treated with 160 μg/mL imipenem. After 24 h treatment, the surviving cells were grown overnight in fresh medium and start another cycle. B Evolution of biofilm bacteria exposed to imipenem resulted in a rapid increase in biofilm bacteria CFU on bead. C Crystal violet (CV) staining of biofilms formed by ancestral and hyperbiofilm variant strains on PVC plate. Data are presented as the mean ± s.d. of five biological replicates. Significance was determined using a Student’s t test: *P < 0.05, **P < 0.01 and ***P < 0.001. D and E The time frame of emergence of hyperbiofilm variants in linage W1 (D) and W6 (E). The biofilm formation by the different colonies was displayed with CFU of biofilm cells on 5 mm glass bead