Abstract Colistin is a last-resort antimicrobial in human and veterinary medicine, yet it remains unclear whether prolonged exposure can drive collateral resistance beyond polymyxins. Here, we used a spatial experimental-evolution platform, the Microbial Evolution and Growth Arena (MEGA)-plate, to examine adaptation of Escherichia coli ATCC 25922 to stepwise colistin selection in three independent runs. Endpoint isolates from all three runs ( n = 3 isolates per exposure zone) were phenotyped, wh