Occurrence of "under-the-radar" antibiotic resistance in anthropogenically affected produce

With global climate change, treated-wastewater (TWW) irrigation and manure amendment are becoming increasingly important in sustainable agriculture in water- and nutrient-stressed regions. Yet, these practices can potentially disseminate pathogens and antimicrobial resistance (AMR) determinants to crops, resulting in serious health risks to humans through the food chain. Previous studies demonstrated that pathogen and AMR indicators from wastewater and manure survive poorly in the environment, suggesting that ecological barriers prevent their dissemination. However, we recently found that these elements can persist below detection levels in low quality TWW-irrigated soil, and potentially proliferate under favorable conditions. This "under the radar" phenomenon was further investigated here, in TWW irrigated- and poultry litter-amended lettuce plants, using an enrichment platform that resembles gut conditions, and an analytical approach that combined molecular and cultivation-based techniques. Enrichment uncovered clinically-relevant multidrug resistant pathogen indicators and a myriad of antibiotic resistance genes in the litter amended and TWW-irrigated lettuce that were not detected by direct analyses, or in the enriched freshwater irrigated samples. Selected resistant E. coli isolates were capable of horizontally transferring plasmids carrying multiple resistance genes to a susceptible strain. Overall, our study underlines the hidden risks of under-the-radar pathogen and AMR determinants in anthropogenically affected agroenvironments, providing a novel platform to improve quantitative microbial risk assessment models in the future.