Manure-driven antibiotic resistance in agroecosystems: Decadal evidence of accumulation dynamics and food chain transmission

The global dependence on organic fertilizers raises concerns about the proliferation of antibiotic resistance in agricultural ecosystems. However, the long-term (decade-scale) dynamics of antibiotic resistance genes (ARGs) and their potential transmission into the food chain via ready-to-eat vegetables remain poorly understood. This study investigates the accumulation of ARGs in soils subjected to 10 years of pig manure application at two intensities, and assesses the subsequent transfer of soil ARGs and human pathogenic bacteria (HPB) to lettuce. Our results demonstrate that long-term manure fertilization drives the linear or exponential accumulation of specific "accumulative ARGs" in soil, with the total abundance increasing by 196% (under 9.0 t/ha) and 145% (4.5 t/ha) over the decade. This accumulation was dose-dependent and linked to the co-enrichment of specific bacterial hosts and mobile genetic elements, establishing persistent soil resistomes. While ARGs in lettuce roots were mainly derived from freshly applied manure rather than from the historically enriched soil ARGs pool, their presence in stems and leaves was negligible. Notably, lettuce cultivated in long-term manure-amended soil did not show an increased load of pathogenic bacteria. Overall, this study establishes a mechanistic framework for understanding the evolution of antibiotic resistance in manured paddy soil, highlighting that fresh manure inputs, rather than historically accumulated soil ARGs, are the primary driver of immediate resistance risk in lettuce.