Circular economy and pathogenic Escherichia coli: unfolding the paradigm of human exposure to E. coli infections, including STEC, through crops

Irrigation waters and soil improvers deriving from recycled sludges are increasingly used to save water in agriculture and to enhance the productivity of soils intended for growing crops for human nutrition. Their use can lead to the efficient recycle of carbon and nitrogen sources and mitigate the impact of climate changes on the water consumption in agriculture but can introduce potential food safety risks. As a matter of fact, the characterization of the hazards and the risks associated with the use of municipal and livestock waste, including the possible spread of pathogens and AMR genes, has not yet been framed in a comprehensive and combined assessment. Vegetables are increasingly reported as sources of STEC infections and as cause of STEC outbreaks, sometimes involving large numbers of cases. We assessed the presence and viability of pathogenic E. coli including STEC in irrigation waters and organic materials used to amend agricultural soils, to evaluate the potential for their transfer from these end-of-waste products to crops. A convenient sampling was performed in 2022, in Italy. Biosolids of different origin (municipal sludge-derived compost; manure and compost from intensive animal farming systems; compost from green and household wastes) and irrigation waters (reclaimed and surface waters) were assayed for the presence of pathogenic E. coli (STEC, EPEC, EAEC, EIEC, ETEC) virulence genes by Real Time PCR. Samples were analysed as such and after overnight enrichment at 37 °C in Buffered Peptone Water. Virulence genes associated to all E. coli pathogroups were detected in most of the samples. As regards biosolids, most samples showed positive signals for the presence of stx1, stx2 and eae genes when analysed without enrichment, while only few samples showed positive signals for aggR, aaiC, ipaH, lt, stp, sth genes. Interestingly, after enrichment more signals for the presence of E. coli pathogroups were detected across all the samples. As for irrigation waters, few samples were positive for stx2, aggR, aaiC, lt, stp and ipaH genes and many samples showed positive signals for eae gene when the DNA directly extracted from the samples was assayed. In analogy to what observed with biosolids, the same specimens displayed positivity for a wider spectrum of pathogenic E. coli virulence genes when tested after enrichment. These results suggested the presence of viable bacteria in biosolids and irrigation waters, which raised above the limit of detection by Real Time PCR after the enrichment step. This hypothesis was confirmed by cultivation of the enrichment cultures on MacConkey and TBX media. In some cases, the bacteria responsible for the positive signal observed in Real Time PCR could also be isolated in purity, as in the case of one EPEC (eae+) and one ETEC (stp+), from pig manure and one STEC strain (stx1+) from one sample of surface water. The detection of viable pathogenic E. coli, including STEC, in irrigation waters and biosolids is of concern, as the analysed samples were collected in the last step of their production process, immediately before their application on cultivated soils. Moreover, the biosolids are routinely subjected to stabilization treatments and our result suggest that these may not be completely effective in abating the microbiological hazards and highlight the risk that these may be introduced into the agro-industry and expose consumers to the infections.