Field-based evidence for the prevalence of soil antibiotic resistomes under long-term antibiotic-free fertilization

Growing evidence suggests that the use of manure containing residual antibiotics universally leads to an increase in soil antibiotic resistance genes (ARGs). However, there is limited understanding of the influence of long-term antibiotic-free fertilization and the differences between antibiotic-free manure and chemical fertilizer on soil ARGs. This study aimed to quantify the assembly patterns of the antibiotic resistome by in situ probing bacterial community and environmental variations in field soils that have been subjected to long-term exposure to chemical fertilizer and/or manure from animals without antibiotic amendments. Long-term fertilization slightly impacts the diversity of antibiotic resistomes, with 85.5 % of total ARGs and mobile genetic elements (MGEs) being common across all treatment types, while significantly increasing their abundances from 0.68 to a maximum of 0.90 copies/16S rRNA. The rise in ARG abundances was less pronounced when using antibiotic-free manure compared to chemical fertilizer, particularly for Rank Ⅱ ARGs. However, when antibiotic-free manure and chemical fertilizer were combined, a significant increase in nutrients (such as available nitrogen and organic matter) and MGEs occurred, leading to the enrichment of soil microbial populations, especially in certain resistant species, and Rank Ⅰ and Ⅱ ARGs. Despite the influence of various factors like bacterial communities, soil properties, heavy metals, and MGEs, the MGEs had the most significant standardized effects on shaping ARGs through both direct and indirect pathways. Our findings indicates that while of antibiotic-free manure can lower the risk of antibiotic residues and promote sustainable farming practices, it may not fully eliminate the prevalence of ARGs, highlighting the need for more comprehensive strategies to address antibiotic resistance in agriculture rather than simply prohibiting the use of antibiotics.