Antibiotic resistance gene prevalence in soils due to animal manure deposition and long-term pasture management

The persistence of antibiotic resistant (AMR) genes in the soil-environment is a concern, yet practices that mitigate AMR are poorly understood, especially in grasslands (the largest agricultural land use in the US). Animal manures, which are valuable sources of nutrients, may also contain AMR genes and residues. The aim of this study was to enumerate AMR bacteria and genes in grassland soils following 14-years of poultry litter and cattle manure deposition and evaluate if pasture management [hayed (H), continuously grazed (CG), rotationally grazed with a fenced riparian buffer (RBR), and a fenced riparian buffer strip (RBS), which excluded cattle grazing and poultry litter applications] impacted the presence and amount of AMR genes. Quantitative PCR (Q-PCR) were performed to enumerate four AMR genes (ermB, sulI, intlI, and blactx-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 had the highest abundances in soil (P<0.05) following long-term continuous grazing (relative to conservation best management practices), 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. Additionally, soil sulI and intlI gene abundance increased following poultry litter applications, however, ermB, sulI, and intlI had greater gene copy abundances per gram dry weight in cattle manure than poultry litter (P<0.05). Similarly, metagenomic shotgun sequencing revealed a greater total number of AMR genes under long-term CG systems, while fewer AMR genes were found in H (no cattle manure) and RBS (no cattle manure or poultry litter). These results illustrate that manure and conservation pasture management practices (e.g. RBS and RBR) may minimize the presence and number of AMR genes in grassland soils.