The Quorum-Sensing Inhibitor Furanone C-30 Rapidly Loses Its Tobramycin-Potentiating Activity against Pseudomonas aeruginosa Biofilms during Experimental Evolution.

The use of quorum-sensing inhibitors (QSI) has been proposed as an alternative strategy to combat antibiotic resistance. QSI reduce the virulence of a pathogen without killing it and it is claimed that resistance to such compounds is less likely to develop, although there is a lack of experimental data supporting this hypothesis. Additionally, such studies are often carried out in conditions that do not mimic the situation. In the present study, we evaluated whether a combination of the QSI furanone C-30 and the aminoglycoside antibiotic tobramycin would be "evolution-proof" when used to eradicate Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. We found that the biofilm-eradicating activity of the tobramycin/furanone C-30 combination already decreased after 5 treatment cycles. The antimicrobial susceptibility of P. aeruginosa to tobramycin decreased 8-fold after 16 cycles of treatment with the tobramycin/furanone C-30 combination. Furthermore, microcalorimetry revealed changes in the metabolic activity of P. aeruginosa exposed to furanone C-30, tobramycin, and the combination. Whole-genome sequencing analysis of the evolved strains exposed to the combination identified mutations in , , and , genes known to be involved in antibiotic resistance. In P. aeruginosa treated with furanone C-30 alone, a deletion in was also observed. Our data indicate that furanone C-30 is not "evolution-proof" and quickly becomes ineffective as a tobramycin potentiator.