蚯蚓堆肥降解畜禽污泥中四环素抗性菌和磺胺嘧啶抗性菌的效果及机理研究

Antibiotic-resistant bacteria pollution is severe in animal breeding industry, and it is not conducive to the sustainable development of agriculture. Besides, the earthworm as disintegrator can play an important role in the ecological environment, vermicomposting may be a effective way to reduce the resistant bacteria in animal sludge. Therefore, we plan to take tetracycline-resistant bacteria and sulfadiazine-resistant bacteria as models in this project, and the relationship between the earthworms and the microorganisms of substrate will be investigated to reveal the degradation mechanism of earthworms to antibiotic-resistant bacteria in sludge. The main jobs are as following: To determine the influence of earthworm on bacterial community structure and composition in the sludge by using 16S sequencing; To confirm that the drug-resistant bacteria can be degraded by earthworm after the antibiotic-resistant bacteria were separated, purified and identified, also, the degradation effect and the preference of worm to bacteria were maesured; To obtain the migration patterns of drug-resistant bacteria between earthworm gut and substrate by bioinformatics data analysis; To detect the contents of antibiotics, measure its potential on drug-resistant inducement, and reveal its connection with overall bacteria communities and/or part of them; To explore the transformation and decomposition of antibiotic-resistant genes, and clarify its regulation of resistant bacteria. All the researches can help to reveal the degradation mechanism of earthworm on antibiotic-resistant bacteria. And this study could provide theoretical basis for introducing earthworm into antibiotic-resistant bacteria treatment, and it will be benefit to environmental-friendly development of animal breeding industry.

畜禽养殖业耐药菌污染严重，不利于农业的可持续发展。蚯蚓作为分解者能在生态环境中起重要作用，蚯蚓堆肥或许可以有效降解粪污中的耐药菌。为此，本项目计划以四环素抗性菌和磺胺嘧啶抗性菌为模型，从蚯蚓与基质中微生物之间的关系入手，展开蚯蚓对畜禽污泥中耐药菌降解效果和机理的研究。主要内容包括：采用16S高通量测序技术，确定蚯蚓对污泥中细菌群落结构和组成的影响；在分离、纯化和鉴定好目标耐药菌的前提下，证实耐药菌可被蚯蚓降解这一推理，并确定降解效果，探索蚯蚓对某些菌种的偏好或针对性；通过生物信息学数据分析，得出耐药菌在基质和蚯蚓肠道内的迁移规律；检测抗生素含量，衡量其耐药菌诱导潜力，揭示其与耐药菌群整体结构和某些个体的关系；探索耐药基因的转移和分解，阐明其对耐药菌的调控。通过上述研究，可揭示蚯蚓有效降解耐药菌的机理，为蚯蚓引入耐药菌处理提供理论依据，有助于养殖业的绿色健康发展。

耐药菌和耐药基因在我国畜禽粪污中广泛存在，其在环境中扩散又引起了严重的问题，是粪污无害化处理的难点之一。本研究以金霉素和磺胺甲恶唑为模板，外源添加抗生素到猪粪中并进行高温-蚯蚓联合堆肥，探究该堆肥处理方式对基质中抗生素、抗性基因及耐药菌的去除效果，同时采用高通量测序手段测定蚯蚓肠道中抗性基因的种类及丰度，进一步探讨抗性基因在基质和蚯蚓肠道中的迁移变化。结果发现，高温-蚯蚓联合堆肥可有效降解基质中92%的磺胺甲恶唑和36%的金霉素，可有效去除基质中99.98%金霉素耐药菌和75.82%磺胺甲恶唑耐药菌，对31种四环素类、4种磺胺类、9种MEGs、24种Multidrug类抗性基因去除率达43%-99%。蚯蚓肠道中的抗性基因丰度也随着堆肥显著降低，大多数ARGs不会在蚯蚓肠道中富集，堆肥过程中的高温和蚯蚓肠道消化是抗性基因去除的主要原因，其丰度降低途径分为直接途径和间接途径。该结果可为畜禽粪便无害化、降害化处理提供理论指导,有助于养殖业绿色健康发展。