Background/Objectives: 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. Methods: 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 Escherichia coli 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. Results: 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. Conclusions: This study describes features of OMV produced by two relevant E. coli 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.

Shiga toxin (Stx)–producing Escherichia coli (STEC) harboring virulence determinants of Extraintestinal pathogenic E. coli (ExPEC) are currently emerging in Europe as a cause of severe disease. Among the ExPEC features identified in STEC, the gene hlyF is associated with an augmented production of Outer Membrane Vesicles (OMV). OMVs produced by E. coli strains have been shown to deliver toxins and small RNAs, but information on the latter genetic component is still scanty. We investigated the small RNAs contained in the OMVs produced by two hlyF-positive STEC strains producing Stx2 belonging to O26:H11 and O80:H2 serotypes, isolated from a human case of Hemolytic Uraemic Syndrome (HUS) and from a milk sample, respectively. The OMVs were purified from overnight cultures using two sequential ultracentrifugation steps at 100.000 and 200.000 x g of 2h each. The OMVs collected were analyzed by Nanoparticle Tracking Analysis (NTA) and Electron Microscopy (EM) to define their morphology, size and concentration. The whole genome sequences of the two test strains and three additional hlyF-positive STEC strains were analyzed to identify sequences of small RNAs. Specific Real Time PCR assays were deployed to detect the small RNAs within OMVs. The EM and NTA characterization confirmed the presence of spherical particles, and showed that a cleaner preparation was obtained after the second step of ultracentrifugation (200.000 x g). We identified the sequences of 27 putative small RNAs present in the genome of hlyF-positive STEC strains and absent in that of a non-pathogenic E. coli. Nine of them were identified and quantified in the OMVs produced by the two test strains. A regulatory role in bacterial DNA replication, integration and in stress-response was hypothesized for the identified small RNAs, some of which encompassing all the domains of life. STnc_100 was the only small RNA identified in the OMVs produced by both strains. Interestingly, a copy of STnc_100 sequence is harbored downstream the stx2 operon carried by Stx2-encoding phages in both the strains and has a complementarity region for stxB gene, suggesting a modulation in Stx production or release. Our results indicate that OMVs release may exert regulatory functions, putatively influencing the crosstalk with the host and the gut microbiota during the infection process.

Top soil improvers (TSIs) and reused water from municipal wastewater treatment plants (WWTPs) represent useful and sustainable sources of organic carbon and nutrients for agriculture. However, from their regular use, emerging Escherichia coli enterophatotypes, particularly Shiga toxin‐producing E. coli (STEC), were responsible for severe foodborne outbreaks from contaminated ready‐to‐eat vegetables. We report the hazard characterization of 31 top soil improvers (TSIs) and 15 irrigation water samples of different origins, sampled at the farm level. The increased PCR signals of the marker virulence genes stx1, stx2, aggR, aaiC, ipaH, lt, stp, and sth for enteropathogenic, enteroaggregative, enteroinvasive, enterotoxigenic, and Shiga toxin‐producing E. coli from post‐enrichment samples were considered proof of the presence of viable bacteria. Despite positive signals from TSI‐enriched samples, we isolated one enteropathogenic and one enterotoxigenic strain from the same pig slurry sample and one STEC strain carrying the enterotoxigenic heat‐stable enterotoxin from bovine slurry. From irrigation water samples, the most detected genes were stx1, stx2, and eae, followed by aggR, aaiC, ipaH, lt, stp, and sth, leading to one STEC (stx1+) isolation. E. coli pathotype virulotyping could be proposed to assess if combined TSI and water inputs would potentially lead to hybrid strains with shuffled virulence features to support safe and sustainable ready‐to‐eat vegetable production.

The use of reclaimed water from wastewater treatment plants (WWTPs) in agriculture represents a sustainable way to reduce water footprint. However, outbreaks linked to the consumption of contaminated ready-to-eat food (RTE) have frequently been associated with the presence of foodborne pathogens in irrigation water. To deepen the hazard characterization of water used for crop irrigation, reclaimed and surface water samples (n=15), collected in Northern Italy in spring and summer, were analysed via Real Time PCR, before and after an enrichment treatment, to detect virulence genes of STEC (Shiga toxin producing - E. coli); EPEC (Enteropathogenic E. coli); EAEC (Enteroaggregative E. coli); EIEC (Enteroinvasive E. coli); ETEC (Enterotoxigenic E. coli) and Salmonella spp. Virulence genes of Salmonella spp. and all E. coli pathogroups were detected in 14/15 samples. 11/14 samples showed the presence of pathogenic E. coli both pre- and post-enrichment. The most detected genes were stx1, stx2, eae, followed by aggR, aaiC, ipaH, lt, stp, sth genes. Four enriched samples were positive for invA gene. Four strains were isolated from enriched samples, including one STEC (stx1+) strain and two Salmonella Veneziana strains from surface water samples, and one strain of S. Taksony from a WWTP sample. The detection of viable pathogens in irrigation water suggests that the depurative treatments may not be fully effective in eliminating pathogenic E. coli and Salmonella spp. in reclaimed water. A conditioned use of surface water is needed to reduce the pathogen input on crops. Furthermore, the higher positive signals recorded after rainfalls suggest that pathogen mobilization is linked to precipitation patterns. Since the detection of pathogenic E. coli in agricultural water is not regulated by the Italian and European legislation, except for STEC O157 in spent irrigation water, their likely presence should be adequately considered and risk-oriented, depending on climatic conditions, processing technologies and type of agricultural use.