Extracellular vesicle production and membrane uptake promote repair and antibiotic tolerance in E. coli

Bacterial extracellular vesicles (EVs) are nanosized lipid structures released in response to environmental stressors, such as phages and antibiotics. Despite their critical role in bacterial adaptability, the mechanisms by which EVs interact with membranes under stress remain poorly understood, due to challenges in visualizing these dynamic processes in live bacteria. Here, we use high-resolution fluorescence microscopy, flow cytometry, and cryo-electron microscopy to investigate EV production and uptake in Escherichia coli exposed to sub-minimum inhibitory concentration doses of polymyxin B (Pmb), a membrane-active antimicrobial peptide. Using fluorescently labeled Pmb and EVs, we track Pmb insertion and removal from membranes, EV production and uptake, and their effects on cell growth. Our findings demonstrate that EV production rapidly sequesters Pmb in the medium and facilitates its removal from bacterial membranes. For the first time, we demonstrated that EVs act as membrane plugs by adhering to or fusing with Pmb-damaged membranes. These dynamic processes work together to reduce the antibiotic load from the membranes, turn off the RcsA-mediated membrane stress response, and enable cells to resume growth. Although EVs do not provide resistance to Pmb, they enhance the survival and tolerance of bacterial populations. This study uncovers the dual role of EVs in Pmb sequestration and membrane repair, providing new insights into antibiotic tolerance mechanisms and paving the way for innovative approaches to combat antimicrobial resistance.