Metagenomic Assessment of Critical Management Points for Antibiotic Resistance Genes through an Integrated Lab-, Microcosm-, and Greenhouse-Scale Study. Lettuce_Lot_1

Prior studies have demonstrated the potential for agricultural production systems to contribute to the spread of antibiotic resistance genes (ARGs). However, integrated study is needed to effectively identify critical control points that can be targeted to optimize mitigation efforts. Here we applied shotgun metagenomic sequencing to comprehensively compare resistomes (i.e., total ARG profiles) characteristic of amendment (manure or compost), soil (loamy sand or silty clay loam), and vegetable (lettuce or radish) samples collected across studies carried out at laboratory-, microcosm-, and greenhouse-scale, respectively. Five hundred and twenty-five ARGs were found to be common to all three sample types, with vegetables carrying the greatest diversity of ARGs (n=838). Additionally, vegetables carried the most ARG- mobile genetic element co-occurrences when assessed via metagenomic assembly. Notably, radishes grown in soils fertilized with manure or compost harbored a higher relative abundance of total ARGs and clinically-relevant ARGs than samples from other experimental conditions, although lettuce was found to contain the highest relative abundance of pathogen gene markers quantified in the metagenomes. The findings emphasize the need to harmonize manure management with efforts to combat the spread of antibiotic resistance and indicate that post-harvest processes should also be considered in a multi-barrier approach to achieving this goal.