Effects of long-term application of different livestock manures on the spread characteristics of antibiotic resistance genes and exposure risks in the soil-lettuce system

Long-term application of livestock manures can lead to the accumulation of antibiotic residues and the enrichment of antibiotic resistance genes (ARGs), thereby posing potential risks to human health via the food chain. However, the effects of prolonged, repeated applications of different manure types on ARG distribution and exposure risk remain insufficiently understood. In this study, we examined lettuce cultivation systems that had received more than a decade of continuous application of broiler manure, layer manure, or pig manure. Using HT-qPCR, 16S rRNA sequencing, host association analysis, and variance partitioning analysis (VPA), together with assessments of the average daily dose of soil-derived ARGs (ADD_T) and the estimated daily intake of leaf-associated ARGs (EI_ARGs), we elucidated the relationships and mechanistic drivers among ARGs, mobile genetic elements (MGEs), and microbial communities within the manure-soil-lettuce systems. Our results revealed that tolC and sul1 were the predominant ARGs in the systems, accounting for 57.7-94.0 % of the total ARG abundance. Firmicutes and Proteobacteria were identified as the major microbial reservoirs of ARGs, and both MGEs and microbial community composition played key roles in shaping the overall ARG profile. Risk assessment demonstrated that the risk of ARG exposure was significantly greater in the broiler manure-amended system than in the layer and pig manure systems. Children faced higher soil exposure risks (7.7 × 10⁴ copies·g⁻¹·d⁻¹) due to their lower body weight, whereas adults faced greater risks from dietary lettuce consumption (9.5 × 10¹⁰ copies·d⁻¹) due to their higher intake levels. Overall, this study provides essential evidence for the risk management of ARGs and supports the sustainable use of organic fertilizers.