Closed-loop steering locates an evolutionary barrier to multidrug resistance in <i>Escherichia coli</i>

Abstract Resistance to one drug often raises susceptibility to another; such collateral sensitivity is expected to constrain multidrug resistance. This is hard to test: failure to reach a state is indistinguishable from never having been driven toward it. We therefore recorded the change we imposed: a closed-loop system updated each lineage’s drug environment daily based on its measured distance to a target. Steered toward triple chloramphenicol–norfloxacin–kanamycin resistance for 70 days, 52 Escherichia coli lineages gained substantial resistance without converging on the target. The mismatch between imposed and observed movement mapped accessibility, locating a region immediately before the target where progress was reproducibly slowed. Crossing it was associated not with broad resistance mutations shared by most lineages, but with recurrent mutations upstream of the efflux pump gene cmr , which raised chloramphenicol resistance threefold without detectable cost. Progression toward multidrug resistance was therefore constrained without being prevented, at a location consistent with a known physiological trade-off; recording the imposed change makes accessibility measurable during evolution.