The persistence of antimicrobial resistant (AMR) genes in the soil-environment is a concern, yet practices that mitigate AMR are poorly understood, especially in grasslands. Animal manures are widely deposited on grasslands, which are the largest agricultural land-use in the United States. These nutrient-rich manures may contain AMR genes. The aim of this study was to enumerate AMR genes in grassland soils following 14-years of poultry litter and cattle manure deposition and evaluate if best management practices (rotationally grazed with a riparian (RBR) area and a fenced riparian buffer strip (RBS), which excluded cattle grazing and poultry litter applications) relative to standard pasture management (continuously grazed (CG) and hayed (H)) minimize the presence and amount of AMR genes. Quantitative PCR (Q-PCR) was performed to enumerate four AMR genes (ermB, sulI, intlI, and bla_ctx-m-32 ) in soil, cattle manure, and poultry litter environments. Six soil samples were additionally subjected to metagenomic sequencing and resistance genes were identified from assembled sequences. Following 14-years of continuous management, ermB, sulI, and intlI genes in soil were greatest (P < 0.05) in samples collected under long-term continuous grazing (relative to conservation best management practices), under suggesting overgrazing and continuous cattle manure deposition may increase AMR gene presence. In general, AMR gene prevalence increased downslope, suggesting potential lateral movement and accumulation based on landscape position. Poultry litter had lower abundance of AMR genes (ermB, sulI, and intlI) relative to cattle manure. Long-term applications of poultry litter increased the abundance of sulI and intlI genes in soil (P < 0.05). Similarly, metagenomic shotgun sequencing revealed a greater total number of AMR genes under long-term CG, while fewer AMR genes were found in H (no cattle manure) and RBS (no animal manure or poultry litter). Results indicate long-term conservation pasture management practices (e.g., RBS and RBR) and select animal manure (poultry litter inputs) may minimize the presence and abundance of AMR genes in grassland soils.

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