EnvZ/OmpR-driven cooperative behavior promotes cefiderocol resistance in a hanging-droplet evolution system

Understanding how bacteria rapidly adapt to recently introduced antibiotics increasingly demands experimental models that move beyond classical evolution systems. We developed a microbial evolution hanging-droplet system (MEHS) that uses gravity-driven flow to sustain continuous exponential growth, doubling daily reproduction rate. Using this MEHS, <i>Klebsiella pneumoniae</i> rapidly adapted to fluctuating cefiderocol (CFDC) exposure. However, resistant clones comprised only a minor fraction of the evolved populations. Many mutations overlapped with variants previously observed in clinical practice, including alterations in EnvZ/OmpR two-component system that reprogrammed siderophore biosynthesis. These changes promoted cross-protection of susceptible subpopulations, alleviating the fitness costs typically associated with resistance and facilitating population-level adaptation. Moreover, the detection of similar variants in clinical isolates collected before CFDC use highlights their latent potential to evolve under selective pressure. Our findings establish MEHS as a powerful platform for resolving clinically relevant resistance trajectories and point to regulatory nodes as potential targets for disrupting cooperative behaviors that undermine antibiotic efficacy.