Recurrent Extinction of Resistance Mutations Leads to Convergent Multidrug Resistance in Sequential Antibiotic Treatment

To overcome the antibiotic resistance problem, exploiting reproducible evolutionary tradeoffs is considered key for designing evolution-proof antibiotic therapies. Yet the predictability of resistance evolution has been viewed as limited, given the often idiosyncratic genetic trajectories observed in laboratory evolution experiments. To address this, we partially mimicked clinical antibiotic pharmacodynamics by imposing strong selection and evolved Escherichia coli under single or sequential antibiotic treatment. Under single-antibiotic selection, endpoint resistance, persistence, and tolerance phenotypes were reproducible, but populations evolving in parallel frequently followed divergent genetic trajectories. Remarkably, sequential antibiotic use redirected these divergent paths toward genotypic and phenotypic convergence, driven by extinction of resistance-conferring mutations when switching to the next effective antibiotic. Single-cell RNA sequencing demonstrated that evolved cultures contain cells occupying distinct metabolic niches and include more cells in states with lower translational activity and higher expression of toxin-antitoxin genes. These findings provide evolutionary insights to inform clinically effective antibiotic treatment strategies that employ sequential treatment.