Experimental evolution of Pseudomonas aeruginosa biofilms exposed to furanone C-30 with and without tobramycin. Experimental evolution of Pseudomonas aeruginosa biofilms exposed to furanone C-30 with and without tobramycin

The aim of this study is to evaluate the evolutionary robustness of the quorum sensing inhibitor (QSI) furanone C-30 for the treatment of P. aeruginosa biofilm infections. We repeatedly exposed P. aeruginosa biofilms to furanone C-30 (with or without tobramycin) in the synthetic cystic fibrosis sputum medium (SCFM2) and characterized the genotype and phenotype of the evolved lineages. P. aeruginosa biofilms were grown in SCFM2 for 24 h after which the treatment in fresh SCFM2 was added to obtain a final concentration of 20 µg/ml tobramycin and 100 µg/ml furanone C-30. The negative control was treated with fresh SCFM2, including the same amount of DMSO (0.25%) as for the biofilms treated with C-30. After 24 h of static incubation at 37°C, biofilms were sonicated and vortexed in order to disintegrate the biofilm aggregates. After each cycle the number of CFU was determined and an aliquot of the culture was stored at -80°C in Microbank vials to allow further tests on the evolved strains. A sample from the treated biofilm was used to prepare a new overnight culture, in order to start a new cycle. For each treatment three independent lineages were established, that were each exposed for 16 cycles. Whole-genome sequencing was performed on the wild type P. aeruginosa PAO1 and on the exposed lineages after 5, 10 and 16 cycles.