Leached Nitrate Reduces the Abundance of Antibiotic Resistance Genes in Deep Soil Layers up to 10 m

ABSTRACT Antibiotic resistance genes (ARGs) represent a threat to human health. Factors impacting ARGs have been well studied in surface soils, but how such factors affect ARGs in deep soils (> 2 m) remains unknown. This in situ study investigated how long‐term excessive nitrogen (N) fertilisation, specifically leached nitrate (NO 3 − ), influenced ARGs up to 10 m depth. Under replicated 26‐year fertilisation (600 kg N ha −1 ) plots, significant reductions (10%–65%) were observed across multiple ARG classes, including beta‐lactam, fluoroquinolone, glycopeptide, macrolide‐lincosamide‐streptogramin (MLS), multidrug, and rifamycin resistance genes, correlating with elevated NO 3 − concentrations. KEGG pathways related to antibiotic biosynthesis and the relative abundance of S‐strategy ARG hosts, such as Achromobacter , were both reduced. Additionally, network analysis revealed more positive and fewer negative microbial associations under N treatment. These findings suggest that NO 3 − leaching alleviates microbial N limitation, modulates ecological strategies and microbial interaction networks, and ultimately contributes to lower ARG abundance in deep soil layers.