Adaptation of biofilms to tobramycin and the quorum sensing inhibitor C-30 during experimental evolution requires multiple genotypic and phenotypic changes.

In the present study we evaluated the fitness, antimicrobial susceptibility, metabolic activity, gene expression, production of virulence factors and virulence of experimentally evolved PAO1. These strains were previously evolved in the presence of tobramycin and the quorum sensing inhibitor furanone C-30 (C-30) and carried mutations in and . Compared to the wild-type (WT), the evolved strains show a different growth rate and different metabolic activity, suggesting they have an altered fitness. mutants were less susceptible to C-30 than WT strains; they also show reduced susceptibility to chloramphenicol and ciprofloxacin, two substrates of the MexEF-OprN efflux pump. mutants had a decreased susceptibility to aminoglycoside antibiotics, and an increased susceptibility to chloramphenicol. The decreased antimicrobial susceptibility and decreased susceptibility to C-30 was accompanied by a changed metabolic activity profile during treatment. The expression of was significantly increased in mutants and induced by C-30, suggesting that MexEF-OprN exports C-30 out of the bacterial cell. The production of virulence factors as well as virulence in two models of the strains evolved in the presence of C-30 was unchanged compared to the virulence of the WT. Finally, the evolved strains were less susceptible towards tobramycin (alone and combined with C-30) in an mouse model. In conclusion, this study shows that mutations acquired during experimental evolution of biofilms in the presence of tobramycin and C-30, are accompanied by an altered fitness, metabolism, expression and and antimicrobial susceptibility.