Land application of manure is common agricultural practice potentially leading to dispersal and propagation of antibiotic resistance genes (ARGs) in environmental settings. In this study, high-throughput quantitative PCR and high-throughput sequencing were employed to assess the impact of long-term manure application on the patterns of ARGs and bacterial community. A total of 154 unique ARGs encoding for resistance to a broad-spectrum of antibiotics were detected, and long-term manure application highly increased the abundance and diversity of ARGs in the present soil. Resistance genes for aminoglycoside, tetracycline, and sulfonamide were the three most dominant types of ARGs, and antibiotic deactivation was the most dominant resistance mechanism. The total enrichment of ARGs was higher in manure fertilized soils, up to 3640.3-fold compared to control samples. Eight mobile genetic elements (MGEs) were detected with the enrichment varied from 8.7-fold to 256.0-fold compared with controls. Significant correlations were found between MGEs and most types of ARGs, indicating that MGEs played an important role in the dissemination of ARGs. Besides, ARGs ermF, aacC and blaOCH were identified as potential indicators for estimating the abundance of co-occurring ARGs subtypes based on network analysis. In addition, microbial communities significantly differed among treatments with manure fertilizer, only chemical fertilizer and controls, and bacterial community shifts, rather than MGEs was the major factor shaping the antibiotic resistome, suggested by variation partitioning analysis (VPA). Furthermore, based on the network analysis, 7 bacterial families might be possible hosts of the detected ARGs. These results provide comprehensive and systematic insights into evaluating the effects of manure application on the propagation of ARGs in agricultural soil.
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