Biophysical Features of Outer Membrane Vesicles (OMVs) from Pathogenic Escherichia coli

Outer membrane vesicles (OMVs) blebbing from bacterial outer membrane represent a key weapon in interspecies and interkingdom communication, virulence and spread of antimicrobial resistance. OMV isolation is a fundamental step to the study of their functions; however, the yield, purity, and structural integrity of OMVs can be influenced by the purification procedure. In this work, we compared the efficacy of two commonly used isolation techniques, size-exclusion chromatography (SEC) and differential ultracentrifugation (dUC), in separating and concentrating vesicles produced by Shiga toxin-producing (STEC) and uropathogenic (UPEC) Escherichia coli strains. The obtained OMVs were characterized using a comprehensive multi-analytical approach including scanning and transmission electron microscopy (SEM, TEM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), polydispersity index (PDI) and ζ-potential measurements, and protein quantification for purity assessment. dUC-derived samples were characterized by broader particle size distributions, higher protein concentration, and more noticeable contamination by non-vesicular material. In contrast, SEC-derived samples yielded structurally well-preserved and morphologically homogeneous vesicles, higher particle-to-protein ratios, and lower total protein content, showing reduced co-isolation of protein-aggregates. NTA and DLS analyses unveiled polydisperse populations in samples obtained with both isolation methods, with DLS measurements emphasizing the presence of larger or transient aggregates. ζ-potential values were close to neutrality for all samples, coherent with limited electrostatic repulsion and with the aggregation tendencies observed in some preparations. Of interest, a dense proteinaceous matrix was observed in all samples, more pronounced in UPEC samples, suggesting strain-specific biophysical interference. Overall, the two methods provided complementary information, emphasizing the trade-offs between yield, purity and vesicle integrity, exerting also strain-dependent effects. These findings may support informed selection of OMV isolation strategies and reproducible characterization of bacterial vesicles for downstream application in host-pathogen interaction and vesicle-based studies.