Pathogenic escherichia coli virulome in top soil improvers and irrigation waters devoted to leafy vegetable production. Hazard assessment from a case study in Italy

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.