Addressing Which Pathogens Are More Suitable to Ensure Water Reuse Safety—A Proof-of-Concept

Current regulations and legislation promote water reuse as a response to the global water crisis, while simultaneously highlighting the need for an urgent revision to ensure the protection of public health. Changes to these regulatory frameworks are generally supported by scientific evidence, particularly when defining the most appropriate water quality parameters to be monitored. In a previous study, our group proposed that Norovirus genogroups I and II should be further investigated as potential indicators for water quality monitoring, and, consequently, for assessing wastewater treatment efficiency. In the present study, we evaluated the presence of these enteric viruses, together with hepatitis A and hepatitis E viruses, using real-time PCR. Additionally, we assessed the occurrence of <i>Clostridioides difficile</i>-an undervalued pathogen that represents a major cause of healthcare-associated diarrhea, increasingly raising concern in community settings-alongside currently established microbiological parameters. Samples were collected from two stages of wastewater treatment and from water intended for reuse. Water quality was further evaluated using a bioluminescence-based bioassay. Overall, this proof-of-concept study supports the inclusion of Norovirus genogroup II and <i>Clostridioides difficile</i> as complementary microbial indicators for assessing the safety of water for reuse, particularly within the context of European regulatory standards.