Manure application primarily drives changes in antibiotic resistome composition rather than abundance in agricultural soil profile.

The prevalence of antibiotic resistance genes (ARGs) in soil has elicited significant concerns about food safety and agricultural sustainability. However, the impact of long-term fertilization on the soil resistome across soil profiles and their associations with both abundant and rare microbial taxa remain unknown. This study employed high-throughput quantitative polymerase chain reaction (HT-qPCR) and 16S rRNA gene sequencing to explore resistome across soil depths under different fertilization regimes (a 12-year field experiment). Compared with the control and chemical-only fertilization, manure amendment increased the ARG richness in the topsoil by 14.1-20 % but had no significant effect on the subsoil. Manure amendment resulted eight unique ARGs into topsoil: sul1, sul2, aadA, aadA2, aadA21, APHA3, ErmY and qacF_H. Compared with the control soil, the manure amendment did not increase the absolute and normalized abundance of ARGs in both top- and subsoil. In addition, abundant microbial taxa exhibited a stronger association with ARGs than rare taxa. Overall, manure amendment had strong and direct impacts on soil ARG composition and indirectly influenced ARG abundance to a limited extent through its effects on soil properties and abundant taxa. These findings strengthen our understanding of the ecological impacts of long-term fertilization and inform sustainable agricultural practices.