Unveiling a Novel Mechanism of Enhanced Secretion, Cargo Loading, and Accelerated Dynamics of Bacterial Extracellular Vesicles Following Antibiotic Exposure

ABSTRACT Antibiotic exposure substantially alters the production mechanisms of bacterial extracellular vesicles (BEVs), which serve as carriers for intercellular exchange of DNA, proteins, and nutrients, yet the underlying mechanisms remain elusive. Here, using Escherichia coli as a model, we uncover how antibiotic exposure enhances BEV secretion, cargo enrichment, and motility. Our results demonstrate that enrofloxacin (ENR) triggers the SOS response, leading to upregulation of the endolysin genes essd‐1 , rrrd , and rzod , causing peptidoglycan layer damage and promoting modest BEV formation with encapsulated bioactive components such as DNA and proteins. More critically, ENR suppresses ompR , a key regulator in the OmpR/EnvZ two‐component system, downregulating the expression of the outer membrane (OM) protein OmpC and its associated Mla‐OmpC lipopolysaccharide transport complex. This destabilization of the OM further facilitates BEV formation and cargo encapsulation. The Δ ompR mutant in E. coli also exhibits reduced type I fimbriae and enhanced BEV motility, indicating that the OmpR/EnvZ system modulates BEV dynamics via type I fimbriae regulation. These findings reveal a novel mechanism by which E. coli adapts to sub‐inhibitory antibiotic stress by modulating BEV formation and motility, with implications for biomedical nanodelivery applications.