MexS mediated heteroresistance of Pseudomonas aeruginosa to ciprofloxacin

Bacterial heteroresistance has been increasingly identified as an important phenomenon for many antibiotic/bacterium combinations. However the mechanisms underlying ciprofloxacin heteroresistance in <i>P. aeruginosa</i>, a key drug for treating this pathogen, remain poorly understood. In this study, 11 ciprofloxacin-heteroresistant <i>P. aeruginosa</i> isolates were identified by screening 226 clinical strains. Whole-genome sequencing (WGS) identified recurrent mutations within the <i>mexS</i> gene among these isolates. Crucially, CRISPR-Cas9-mediated deletion of <i>mexS</i> in the representative heteroresistant isolate (7318HR) abolished the heteroresistance phenotype, thereby confirming its functional necessity. Transcriptomic profiling following <i>mexS</i> deletion identified 532 differentially expressed genes involved in multiple biological processes, including phenazine biosynthesis, propanoate metabolism, bacterial secretion systems, and quorum sensing. Notably, genes associated with type IVb-tad pili were significantly downregulated. Consistent with this, the <i>mexS</i> mutant exhibited concurrent impairments in virulence-associated traits, including markedly reduced twitching motility (by approximately 38%) and polystyrene adhesion capacity (by approximately 50%) compared to the wild-type strain. These findings demonstrate that <i>mexS</i> is a key genetic determinant of ciprofloxacin heteroresistance in <i>P. aeruginosa</i> and concurrently modulates pilus-mediated virulence-associated functions.