Agnieszka Razim Agnieszka Razim Astrid Laimer-Digruber (Centre of Pathobiology, Department of Biological Sciences and Pathobiology, Departments, University of Veterinary Medicine Vienna) Astrid Laimer-Digruber Anna M. Schmid Anna M. Schmid Tanja V. Edelbacher (Centre of Pathobiology, Department of Biological Sciences and Pathobiology, Departments, University of Veterinary Medicine Vienna) Tanja V. Edelbacher Magdalena E. Paschall Magdalena E. Paschall Tamara Weinmayer Tamara Weinmayer Michael Thaler Michael Thaler Rim Kebreab Rim Kebreab Magdalena E. Skalska Magdalena E. Skalska Paweł Migdał Paweł Migdał Mattia Morandi Mattia Morandi Catherine Daniel Catherine Daniel Dagmar Srutkova Dagmar Srutkova Martin Schwarzer Martin Schwarzer Jiri Hrdy Jiri Hrdy Monika Ehling-Schulz (Centre of Pathobiology, Department of Biological Sciences and Pathobiology, Departments, University of Veterinary Medicine Vienna) Monika Ehling-Schulz Sabina Górska Sabina Górska Aleksandra Inic-Kanada Aleksandra Inic-Kanada Ursula Wiedermann Ursula Wiedermann Irma Schabussova Irma Schabussova OAE Publishing 2026 Aim: To determine how exposure to bile influences the production, physicochemical properties, and molecular composition of extracellular vesicles (EVs) released by the probiotic Lactiplantibacillus plantarum (L. plantarum) NCIMB 8826. Methods: L. plantarum cultures were incubated with and without a physiologically relevant cocnentartion of bile. EVs were isolated by ultracentrifugation followed by size-exclusion concentration (SEC) and characterized per Minimal Information for Studies of Extracellular Vesicles guidelines. Assessments included purity [protein and peptidoglycan (PGN) content], particle size distribution, zeta potential (ZP), lipid profiling, stability assays under varying pH, ionic strength (NaCl) and detergent challenge, Fourier-transform infrared spectroscopy (FTIR), and proteomic analysis were performed. Comparisons were made between purified EVs and parent bacterial cells, and between EVs from bile-exposed and control conditions. Results: SEC markedly reduced copurifying proteins and PGNs, improving EV compositional purity. Purified L. plantarum EVs (LpEVs) displayed surface lipid profiles and ZP that were distinct from parent cells and maintained stability across a range of pH values, elevated NaCl, and low detergent concentrations. Bile exposure induced release of larger LpEVs enriched in proteins involved in bile metabolism. FTIR detected bile-induced molecular changes in LpEVs that differed from alterations observed in whole bacterial cells. Conclusion: L. plantarum modulates EV size and cargo in response to bile stress, producing stable, compositionally distinct vesicles enriched in bile salt hydrolase. These findings indicate that bacterial EVs act as dynamic responders to environmental stress and may mediate early host–microbe communication prior to detectable cellular changes. PDFDocument eng 2026-10-21T07:44:53.414573975Z 2026 EVs probiotics stress gut environment bacterial communication Fourier-transform infrared spectroscopy ultracentrifugation 11623799 b application/pdf http://creativecommons.org/licenses/by/4.0/