摘要

     沼液是畜禽粪便发酵的液体最终产物，在农田中广泛用作肥料。沼液应用于土地可将畜牧业生产中的抗生素及相关抗性基因引入农业土壤。然而，反复施用沼液的土壤中微生物耐药性的变化还不完全清楚。本研究对反复施用沼液的土壤中13种兽用抗生素进行了分析，同时采用实时定量PCR和MiSeq测序方法研究了土壤中抗生素耐药基因（ARGs）和细菌群落组成的时间变化。长期重复施用沼液并没有造成土壤中抗生素残留的过量积累，反而增加了土壤中ARG的丰度，促进了潜在宿主间ARG的迁移。虽然定量PCR分析显示随着时间的推移ARGs相对丰度呈下降趋势，但相关网络分析表明长期使用沼液后会出现高度复杂的细菌-ARG共存，这意味着反复施用沼液可以增强土壤中不同宿主间ARGs的水平基因转移。intl1基因相对丰度的增加支持了ARG-细菌共存的这一变化。此外，分类分析表明在沼液处理的土壤环境中，抗生素抗性细菌（ARB）和ARGs的分布与沼液施用持续时间密切相关，而与抗生素残留的影响无关。未经处理土壤中ARG丰度的自然水平间接表明抗生素的存在/缺乏并不是导致微生物耐药性传播的关键决定因素。本研究进一步揭示了长期重复施用沼液对土壤中ARG丰度变化和细菌-ARG共生的影响，强调了土壤环境变化对ARG迁移的影响。

Biogas slurry, a liquid end product of animal manure fermentation, is widely used as fertilizer in crop fields. Land application may introduce antibiotics and related resistance genes from livestock production into agricultural soil. Nevertheless, changes in antimicrobial resistance in soil where biogas slurry has been repeatedly applied are not fully understood. In the present study, 13 veterinary antibiotics were analyzed in soils that were repeatedly sprayed with biogas slurry, and simultaneously, temporal changes in antibiotic resistance genes (ARGs) and bacterial community composition were investigated using a real-time quantitative PCR assay and MiSeq sequencing. Long-term repeated application of biogas slurry did not result in excessive accumulation of antibiotic residuals in the soil but increased the abundance of ARGs and facilitated ARG transfer among potential hosts. Although the quantitative PCR assay showed a decreasing trend for the relative abundance of ARGs over time, a relevance network analysis revealed highly complex bacteria-ARG co-occurrence after long-term application, which implied that repeated application might intensify horizontal gene transfer (HGT) of ARGs among different bacterial hosts in soil. The increased relative abundance of the intl1 gene supported the shift in ARG-bacteria co-occurrence. Furthermore, ordination analysis showed that the distributions of antibiotic resistance bacteria (ARB) and ARGs were closely related to application duration than to the influence of antibiotic residuals in the biogas slurry-treated soil environment. Additionally, natural level of ARG abundance in untreated soils indirectly suggested the presence/absence of antibiotics was not a key determinant causing the spread of antimicrobial resistance. This study provides improved insight into the effects of long-term repeated application of biogas slurry on the shift in ARG abundances and bacteria-ARG co-occurrence in soils, highlighting the need to focus on the influence of changed soil environment on the ARG transfer.

    https://www.sciencedirect.com/science/article/pii/S0269749119321839