1 Department of Biology, Concordia University, Montréal, Québec, Canada H4B 1R6.
2 Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec, Canada H4B 1R6.

Antibiotic resistance threatens to undo many of the advancements of modern medicine. A slow antibiotic development pipeline makes it impossible to outpace bacterial evolution, making alternative strategies essential to combat resistance. In this study, we introduce cyclic antibiotic regimens composed of three drugs or "tripartite loops" to contain resistance within a closed drug cycle. Through 424 discrete adaptive laboratory evolution experiments we show that as bacteria sequentially evolve resistance to the drugs in a loop, they continually trade their past resistance for fitness gains, reverting back to sensitivity. Through fitness and genomic analyses, we find that tripartite loops guide bacterial strains towards evolutionary paths that mitigate fitness costs and reverse resistance to component drugs in the loops and drive levels of resensitization not achievable through previously suggested pairwise regimens. We then apply this strategy to reproducibly resensitize or eradicate four drug-resistant clinical isolates over the course of 216 evolutionary experiments. Resensitization occurred even when bacteria adapted through plasmid-bound mutations instead of chromosomal changes. Combined, these findings outline a sequential antibiotic regimen with high resensitization frequencies which may improve the clinical longevity of existing antibiotics even in the face of antibiotic resistance.