Abstract:

The enrichment of antibiotic-resistant pathogens and the spread of antibiotic resistance genes (ARGs) caused by the long-term accumulation of antibiotics in soil has become an environmental issue of great concern. To investigate the effect of reductive soil disinfestation (RSD) on antibiotics and associated ARGs, four antibiotics (tetracycline, oxytetracycline, sulfadiazine, and sulfamethoxazole) were selected, which are commonly used and are difficult to degrade, and a mixed antibiotic-contaminated soil was constructed. Each antibiotic was administered at a dose of 20 mg∙kg⁻¹. Three treatments, CK (control without soil treatment), RCS (RSD incorporated with 2% corn straw), and RSR (RSD incorporated with 2% sugarcane bagasse), were performed and destructive sampling was conducted on days 7, 14, and 28. High-performance liquid chromatography and fluorescence quantitative PCR were used to analyze the changes in antibiotic concentrations as well as the abundance of ARGs and mobile genetic elements (MGEs) during RSD treatment. The results showed that RSD treatment effectively degraded tetracyclines and sulfonamides in the soil. After 28 days of treatment, the degradation rate of sulfonamides was 45.2%-100%, which was better than that of tetracyclines, ranging from 33.5% to 57.2%. The RSR treatment had a better degradation effect on tetracycline and sulfadiazine than the RCS treatment, with degradation rates of up to 57.2%-64.8%, while the opposite was true for oxytetracycline, which was degraded up to 44.7% by the RCS treatment. The degradation of sulfamethoxazole by both RSD treatments was up to 100% after 14 days of treatment. The antibiotic concentration gradually decreased with the treatment time, and the two RSD treatments had different degradation effects on antibiotics. The absolute abundance of ARGs in the soil increased significantly after RSD treatment, and the effect of RCS treatment was stronger than that of the RSR treatment. With the extension of the treatment time, the relative abundance of ARGs such as tetM and qacH decreased in the RSD treatments, suggesting that the negative ecological effects of the increased ARGs were mitigated. The RSD treatment significantly reduced the relative abundance of IS26 in soil, suggesting that it can impair the horizontal transfer capacity of ARGs by ablating specific MGEs. Overall, RSD treatment can effectively degrade tetracyclines and sulfonamides in soil, and has the potential to reduce the abundance of ARGs and the risk of their spread. This study provides some theoretical and technical support for solving the problem of soil antibiotic contamination and reducing the risk of ARGs spread.

Key words: reductive soil disinfestation, antibiotics, antibiotic resistance genes, mobile genetic elements, degradation effects

摘要：

土壤中抗生素长期累积所引发的抗性致病菌富集以及抗性基因（Antibiotic resistance genes，ARGs）的传播扩散，已成为当前备受关注的环境问题。为研究强还原处理（Reductive soil disinfestation，RSD）对土壤中抗生素及其ARGs的影响，以常用且难降解的抗生素四环素、土霉素、磺胺嘧啶和磺胺甲恶唑所构建的质量分数为20 mg∙kg⁻¹的抗生素混合污染土壤为研究对象，分别设置以玉米秸秆（2%，RCS）、甘蔗渣（2%，RSR）为有机物料的RSD处理，同时设置不处理对照，于处理第7、14、28天进行破坏性采样，利用高效液相色谱和荧光定量PCR技术分析RSD处理过程中抗生素质量分数及ARGs和可移动遗传元件（Mobile genetic elements，MGEs）丰度的变化。 结果表明，RSD处理可有效降解土壤中的四环素类和磺胺类抗生素，处理28 d后磺胺类抗生素的降解率为45.2%－100%，优于四环素类的33.5%－57.2%；RSR处理对四环素和磺胺嘧啶的降解效果优于RCS处理，其降解率可达57.2%－64.8%，而RCS处理对土霉素的降解率可达44.7%；两种RSD处理对磺胺甲恶唑的降解率在14 d时即可达100%。RSD处理后土壤中ARGs绝对丰度显著增加，RCS处理的增加作用强于RSR处理；随处理时间延长，tetM、qacH等基因相对丰度在RSD处理中呈下降趋势，一定程度上表明ARGs增加带来的生态负面效应得到缓解。RSD处理可显著降低IS26基因的相对丰度，表明其能通过消减特定MGEs削弱ARGs水平转移能力。综上，RSD处理可有效降解土壤中四环素类和磺胺类抗生素，具有消减土壤中ARGs并降低其传播风险的潜力。研究结果可为解决土壤抗生素污染问题、降低ARGs扩散风险提供一定的理论与技术支持。

关键词: 强还原土壤处理, 抗生素, 抗生素抗性基因, 可移动遗传元件, 降解效应