Differential impact of swine, bovine and poultry manure on the microbiome and resistome of agricultural grassland

Land spreading of animal manure is an essential process in agriculture. Despite the importance of grassland in global food security the potential of the grass phyllosphere as a reservoir of antimicrobial resistance (AMR) is unknown. Additionally, the comparative risk associated with different manure sources is unclear. Due to the One Health nature of AMR there is an urgent need to fully understand the risk associated with AMR at the agriculture - environmental nexus. We performed a grassland field study to assess and compare the relative and temporal impact of bovine, swine and poultry manure application on the grass phyllosphere and soil microbiome and resistome over a period of four months, using 16S rRNA amplicon sequencing and high-throughput quantitative PCR (HT-qPCR). The soil and grass phyllosphere contained a diverse range of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). Manure treatment was found to introduce ARGs belonging to clinically important antimicrobial classes, such as aminoglycoside and sulphonamide into grass and soil. Temporal analysis of ARGs and MGEs associated with manure treatment indicated ARGs patterns were similar across the different manure types in the manure treated soil and grass phyllosphere. Manure treatment resulted in the enrichment in members of the indigenous microbiota and the introduction of manure associated bacteria, with this impact extending past the recommended six-week exclusion period. However, these bacteria were in low relative abundance and manure treatment was not found to significantly impact the overall composition of the microbiome or resistome. This provides evidence that the current guidelines facilitate reduction of biological risk to livestock. Additionally, in soil and grass samples MGEs correlated with ARGs from clinically important antimicrobial classes, indicating the key role MGEs play in horizontal gene transfer in agricultural grassland. These results demonstrate the role of the grass phyllosphere as an under-studied sink of AMR.