Experimental evolution of enrofloxacin resistance and cross-resistance in Escherichia coli using a MEGA-plate system

Fluoroquinolones are widely used in veterinary medicine, and their extensive application has raised concerns regarding the selection of antimicrobial resistance and cross-resistance. This study investigated the experimental evolution of enrofloxacin resistance and associated cross-resistance in Escherichia coli using a Microbial Evolution and Growth Arena (MEGA)-plate system. A reference E. coli strain (ATCC 25922) was exposed to stepwise increasing concentrations of enrofloxacin, enabling the spatial separation and isolation of bacterial populations adapted to different levels of selective pressure. Isolates recovered from distinct exposure zones were subjected to phenotypic antimicrobial susceptibility testing, including minimum inhibitory concentration (MIC) determination, and whole-genome sequencing to identify genetic changes associated with resistance development. Progressive exposure to enrofloxacin increased the enrofloxacin MIC from 0.003 μg/mL (parental) to 4 μg/mL in 1000 × -zone isolates (1333-fold). Cross-resistance was observed for selected non-fluoroquinolone agents, with MIC increases ranging from 8 to 33-fold for β-lactams, 4-fold for tetracyclines, and 16-fold for florfenicol. Genomic analysis identified mutations in genes encoding fluoroquinolone target enzymes (gyrA, parC) as well as regulatory genes linked to multidrug efflux systems (acrR, marR, robA, emrR). Together, these findings indicate that enrofloxacin-driven selection can promote cross-resistance through a combination of target-site alterations and changes in the regulation of efflux-associated resistance mechanisms. The results show that the MEGA-plate system is a suitable experimental model for studying stepwise resistance evolution and underscore the need for prudent use of enrofloxacin in veterinary settings.