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

Treated-wastewater (TWW) irrigation and manure amendment are critical for sustainable agriculture in water- and nutrient-stressed regions. Yet, these practices can potentially disseminate pathogens and antimicrobial resistance (AMR) determinants to crops, which can be transmitted through the food chain to humans. Previous studies indicate that pathogen and AMR indicators from wastewater and manure survive poorly in the environment, suggesting that ecological barriers prevent their dissemination. However, a recent study in our lab demonstrated 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-like 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 (ARGs) in the anthropogenically treated 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 with multiple resistance genes to a susceptible strain. Overall, results highlight the capacity of enrichment to reveal presence of gold-standard bacterial pathogen and ARG indicators in anthropogenically affected agro-environment that are below detection limits in direct culture-based and culture-independent analyses s, providing a platform that can be adopted to improve quantitative microbial risk assessment models in the future.