Biophysical Features of Outer Membrane Vesicles (OMVs) from Pathogenic Escherichia coli: Methodological Implications for Reproducible OMV Characterization

<b>Background/Objectives</b>: Bacterial outer membrane vesicles (OMVs) play a role in bacterial communication, virulence, antimicrobial resistance, and host-pathogen interaction. OMV isolation is a key step for studying these particles' functions; nevertheless, isolation procedures can greatly influence the yield, purity, and structural integrity of OMVs, thereby affecting downstream biological analyses and functional interpretation. <b>Methods</b>: In this study, we compared the efficacy of two OMV isolation techniques, differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC), in separating and concentrating vesicles produced by two <i>Escherichia coli</i> strains belonging to uropathogenic (UPEC) and Shiga toxin-producing (STEC) pathotypes. The isolated OMVs were characterized using a multi-analytical approach including transmission and scanning electron microscopy (TEM, SEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), ζ-potential measurement, and protein quantification to assess the purity of the preparations. <b>Results</b>: Samples obtained by dUC exhibited higher total protein content, broader particle size distributions, and more pronounced contamination by non-vesicular material. In contrast, SEC yielded morphologically homogeneous and structurally well-preserved vesicles, higher particle-to-protein ratios, and lower total protein content, reflecting reduced co-isolation of protein aggregates. NTA and DLS analyses revealed polydisperse populations in samples obtained with both isolation methods, with DLS measurements highlighting the contribution of larger or transient aggregates. ζ-potential values were close to neutrality for all samples, consistent with limited electrostatic repulsion and with the aggregation tendencies observed in some preparations. <b>Conclusions</b>: This study describes features of OMV produced by two relevant <i>E. coli</i> strains considering two isolation strategies which exert method- and strain-dependent effects on vesicle properties, including size distribution and surface charge, and emphasizes the trade-offs between yield, purity, and vesicle integrity.