Genetic mutations driving ciprofloxacin resistance in laboratory-evolved Salmonella Typhimurium

Ciprofloxacin resistance is a significant public health concern, and the mechanisms by which this resistance evolves are poorly defined. Here, by serial passaging under antibiotic selection, we isolated ciprofloxacin-resistant S. Typhimurium mutants and subjected them to whole-genome sequencing to identify the major mutations associated with resistance. The Low CipR 35 mutant acquired four chromosomal mutations in ramR, icdA, lipB, and gyrA, and the High CipR 36 mutant gained additional mutations in gyrB, yaiC, and corA. Functional characterization showed that mutations in ramR resulted in efflux pump up-regulation, while disruptions in the TCA cycle caused by mutations in icdA and lipB led to metabolic alterations. These changes indirectly enhanced resistance by increasing the expression of the global regulator MarA and reducing OmpF-dependent membrane permeability. Despite the G105A substitution in GyrA exhibiting a minimal effect on ciprofloxacin resistance in enzymatic assays, GyrB488-489dup 42 was associated with maintained supercoiling in the presence of ciprofloxacin and enhanced fluoroquinolone resistance, suggesting a major role in resistance evolution. Other mutations-yaiC, impairing biofilm formation, and corA, altering intracellular magnesium accumulation-may stabilize the bacterial cell envelope under antibiotic pressure. Together, these findings provide novel insights into the multifaceted mechanisms leading to ciprofloxacin resistance in Salmonella and suggest potential targets for combating antimicrobial resistance.