Reused water in leafy vegetables production: analytical needs to support microbiological hazard and risk assessment

Climate changes, certification of sustainability in the agrisector prompt farmers to refrain from groundwater use for irrigation. Effluents from urban Wastewater Treatment Plants (uWWTPs) have been proposed for direct irrigation on crops and vegetables, after a secondary phase treatment. uWWTPs could receive inputs both from anthropogenic and animal effluents, thus determining a potential reassortments of pathogens and of associated virulence genes, Antimicrobial Resistance Genetic Elements (ARGE), included. Pathogens and virulence factors other than those linked to Salmonella may resist to secondary phase treatments. According to basic microbiological requirements from EU Regulations Salmonella spp. is considered as the unique pathogen indicator. Recorded increase in food consumption of ready-to-eat vegetables may expose communities to food-born intoxication outbreaks from emerging pathogens. Target PCR for the detection of virulence genes associated with the different E. coli pathogroups and Salmonella spp. was performed from DNA extracted directly from irrigation water samples (pre-enrichment) and after enrichment in non-selective medium (postenrichment). V3-V4 hypervariable regions using Illumina MiSeq platform sequencing of the 16SrRNA encoding gene were studied to detect the Pathobiome from genomic libraries of a comprehensive list of 1,516 human bacterial pathogens (Bartlett A. et al., 2022. doi: 10.1099/mic.0.001269. Metagenomic sequencing (Shotgun) was performed on Ion Torrent S5 sequencing platform. Bioinformatic analysis of the platform ARIES (https://aries.iss.it/platform/), accounting for the NCBI-NR database (non-redundant) (ftp://ftp.ncbi.nlm.nih.gov/ ) and the MEGAN V6 Community Edition software (http://wwwab.informatik.uni-tuebingen.de/software/megan6/ ). Resistome has been currently proposed as an emerging hazard to be assessed & managed within the EU Commission proposal on urban wastewaters. Response: Broadening hazard assessment to pathogens other than Salmonella spp., via microbiology, genomic and metagenomic tools. Develop a knowledge hub, under the FAIR scheme, with the support of metadata. Prompt the knowledge hub feeding by stakeholders, also supporting the traceability. Support to risk management; testing WWTPs treatments effectiveness; conditional risk-based use of re-used waters; support to source attribution in food outbreaks investigations.