Bacterial extracellular vesicles promote membrane repair and tolerance to polymyxin B

Bacterial extracellular vesicles (EVs) are nanosized lipid structures released under stress, yet their interactions with antibiotics remain poorly understood. We tracked real-time interactions between Escherichia coli , EVs, and fluorescent polymyxin B (Pmb) using single-cell imaging and cytometric approaches. EVs rapidly sequester Pmb, facilitating its removal from bacterial envelopes, and act as plugs by adhering to or fusing with damaged sites. Pmb triggers early Cpx/σE- and Rcs-dependent stress responses, linked to a ∼25% reduction in cell surface area, a ∼50-fold increase in vesiculation, and remodeling of membrane properties. After an adaptive lag phase, sustained EV release supports detoxification and envelope repair, enabling growth recovery and transient tolerance to Pmb. Together, these findings reveal previously unrecognized functions of EVs in membrane repair and tolerance to membrane-active antibiotics.