Comparative analysis of isolation and characterization methods of outer membrane vesicles (OMVs) produced by Uropathogenic Escherichia coli

Introduction: Uropathogenic Escherichia coli (UPEC) represent the primary cause of urinary tract infections (UTIs), affecting community and hospital settings. Among virulence factors, Outer Membrane Vesicles (OMVs) could actively contribute to the progression of the infection in humans. However, achieving suitable yields and purity of UPEC-OMVs remains challenging. Methods: We investigated different preparations of OMVs produced by a UPEC strain comparing two isolation methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC). All preparations were analyzed by Scanning and Transmission electron microscopy, Nanoparticle Tracking Analyses (NTA) and Dynamic Light Scattering (DLS) to estimate OMVs morphology, size, concentration and polidispersion index (PDI). Results: Electron microscopy analyses of OMVs obtained from both methods showed preparations rich in vesicles with diameters distribution between 40-200 nm. NTA showed a concentration of about 3e+10 particles/ml for UC-isolated OMVs and 1.50e+11 particles/ml for SEC-isolated OMVs, with an average diameter-distribution of 143 nm and 160 nm, respectively. For UCisolated OMVs, the average size detected by DLS was too high to be attributed to separated vesicles, while the average size of SEC-isolated OMVs was 156 nm, in line with NTA measurement. The PDI index of SEC- isolated OMVs (0.29), respect to that measured with UC (0.59), being lower of 0.3 indicated a mono- disperse sample. Conclusion: Both isolation methods could be used to obtain good quality OMVs preparations from UPEC cultures. This evidence will allow to standardize and improve the OMV recovery from UPEC cultures and select the most suitable combination of techniques for different research application.