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 P. aeruginosa , a key drug for treating this pathogen, remain poorly understood. In this study, 11 ciprofloxacin-heteroresistant P. aeruginosa isolates were identified by screening 226 clinical strains. Whole-genome sequencing (WGS) identified recurrent mutations within the mexS gene among these isolates. Crucially, CRISPR-Cas9-mediated deletion of mexS in the representative heteroresistant isolate (7318HR) abolished the heteroresistance phenotype, thereby confirming its functional necessity. Transcriptomic profiling following mexS 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 mexS 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 mexS is a key genetic determinant of ciprofloxacin heteroresistance in P. aeruginosa and concurrently modulates pilus-mediated virulence-associated functions.