Tobramycin Adaptation Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa Quorum Sensing‐Null Mutants

Abstract

The bacterium Pseudomonas aeruginosa causes serious disease in immunocompromised patients and is a model for studying quorum sensing, a cell‐cell signaling system that becomes activated at a certain population or “quorum.” The P. aeruginosa master quorum sensing regulator is LasR, which drives transcription of dozens of genes including those coding for virulence and antibiotic resistance. Paradoxically, lasR‐null mutants are common in infections of tobramycin‐treated patients, presenting a challenge to ongoing efforts to develop anti‐LasR therapeutics. To understand how these mutants might arise, we performed a laboratory evolution experiment. We grew P. aeruginosa populations with sublethal tobramycin. Every day, we transferred the population to fresh medium and every three days we increased the tobramycin concentration. Variants from these populations became highly tobramycin resistant. Surprisingly, inactivating LasR in some of the variants further increased resistance, which was the opposite effect as that observed in the ancestral strain where LasR inactivation decreased tobramycin resistance. This phenomenon is known as sign epistasis. The evolved variants with the altered LasR phenotype all encoded a point mutation in the translation elongation factor fusA1. fusA1frequently incurs mutations as a mechanism of adaptation to tobramycin in antibiotic‐treated patient infections. We showed that the fusA1 mutation reversed the effect of LasR on antibiotic resistance, and thus is responsible for sign epistatic effects on LasR. Our results suggest that antibiotic selection might drive the accumulation of mutations that reverse the effect of LasR mutants on tobramycin resistance. These results could possibly explain how lasR mutants emerge in infected patients and have implications for developing novel therapeutics to treat antibiotic‐resistant infections.