Comparative analysis of ultracentrifugation and size-exclusion chromatography methods for the isolation of Outer Membrane Vesicles (OMVs) produced by two unrelated pathogenic Escherichia coli strains

Introduction Outer Membrane Vesicles (OMVs) are proteolipid structures blebbing from bacterial outer membrane, contributing to the release of toxins, antimicrobial resistance factors and molecules involved in the infection process, as demonstrated for Uropathogenic (UPEC) and Shiga toxin-producing Escherichia coli (STEC), the primary cause of urinary and gastrointestinal infections in humans, respectively. Conventional techniques, such as ultracentrifugation (UC) and size-exclusion chromatography (SEC), are widely used to isolate OMVs. However, each method may alter the characteristics of the isolated OMVs, impacting their morphology and causing OMV aggregation and unwanted cell debris. Thus, achieving suitable yields and purity of OMVs for research and clinical applications remains challenging. We compared preparations of OMVs produced by an UPEC LC2 strain, from a pyelonephritis case, and a STEC O80:H2 strain, from a milk sample, in terms of morphology, purity, size and concentration, obtained using differential UC (dUC) and SEC, to optimize OMV recovery for downstream studies. Methods For both strains, OMVs were purified via dUC using two sequential steps at 100k and 200k x g of 2h each, obtaining two OMV-enriched fractions (F1 and F2), and via SEC, from the same overnight cultures, in triplicate. The OMVs collected were analyzed by Electron Microscopy (EM) and Nanoparticle Tracking Analysis (NTA) to define their morphology, size and concentration. Results EM observation of dUC fractions revealed that, in both strains, F1 contained a high abundance of cellular debris and contaminants, including pili and flagella alongside OMVs. In contrast, F2 of both strains showed decreased levels of debris and contaminants. Thus, F2 was selected for further analyses. As for STEC strain, NTA showed an average concentration of 2,2e+11 particles/ml for F2 preparation and of 9,8e+10 particles/ml for SEC-isolated OMVs, with an average diameter-distribution of 142 nm and 100 nm, respectively. As for UPEC strain, NTA showed an average concentration of 4,6e+10 particles/ml for F2 preparation and of 6,1e+10 particles/ml for SEC-isolated OMVs, with an average diameter-distribution of 149 nm and 159 nm, respectively. In general, F2 from dUC resulted in quite homogeneous particles preparations comparable to SEC-isolated preparations. Discussion and conclusions Our data validated the efficacy of sequential centrifugation in obtaining fractions with an acceptable depletion of unwanted non-OMV materials (F2), which could interfere with downstream investigations. Similarly, SEC method allows the isolation of homogenous and measurable OMV-enriched preparations, comparable to dUC fractions obtained at 200k x g. These results may help in selecting the most suitable method for OMV recovery for cellular and molecular applications