Long-term manure applications promote persistent antibiotic resistance in soil

Manure application has the potential to influence soil microbial composition and the antibiotic resistome; however, its long-term effects remain largely unknown. This study investigated the prolonged impacts of manure applications (pig and chicken manure/compost) on soil antibiotic resistance over a two-year period. Compared with the control (51.9-85.1 ng/g), manure-amended soils contained markedly higher antibiotic concentrations (356-26100 ng/g), remaining 4-300 times higher after 730 days, especially in pig compost treatments. The abundances of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in amended soils (4.48 × 10⁸-2.74 × 10 ¹² copies/g) consistently exceeded those in controls (1.24 × 10⁷-6.10 × 10⁷ copies/g). Notably, 62.5 % of ARGs were located on plasmid-associated contigs, and high-risk genes such as floR and aph(3')-III persisted throughout incubation. Elevated MGE levels after 730 days indicated sustained horizontal gene transfer (HGT) potential under antibiotic selection pressure. Overall, these results reveal the long-lasting enrichment of ARGs and highlight the need for improved manure management and long-term.