家畜粪便堆肥过程中生物气溶胶负载抗生素耐药菌和抗性基因逸散特征及机制研究

Antibiotic resistance genes (ARGs) are emergent contaminants that pose a threat to worldwide human health. Concentrated animal husbandry and feeding operations are believed to be a major contributor to the increased environmental burden of ARGs. For the benefit of recycling waste, composting has been widely used to treat livestock manure. However, Antibiotic resistance genes and Antibiotic resistance bacteria (ARB) tend to escape and can become aerosolized during the composting process. Bioaerosol may represent an entry point in the transimission of ARB to humans and spread of ARG into the environment. To date, most research on ARGs and ARB found in bioaerosols were mainly on livestock and poultry farms. No published papers are available on these emerging contaminants during the composting process. In this project, the distribution characteristics of ARGs and ARB found in bioaerosols were studied by site investigation and laboratory studies. The research places emphasis on the influencing factors of ARGs emission, the distribution characteristics of ARGs among different ARBs and particle sizes (PM2.5/PM10). The dynamic relationship between the distribution characteristics and the spread distance would be uncovered. The aerosolized mechanism, escaping pathway and spread process of bioaerosols containing ARGs and ARB would be clarified gradually. The model would be established to describe the escaping process of ARGs and ARB from composting to air. The results in this project would provide a theoretical basis to emerging contaminant control, reduction of health risks from bioaerosols containing ARGs and ARB, and prediction of potential hazards.

抗生素抗性基因作为一种新型污染物正在全世界范围内威胁到人类健康。集约化畜禽养殖粪便是环境中日益严重的抗性基因负担的主要贡献者。堆肥作为一种资源化处理畜禽粪便的方式广为应用，但粪便微生物在堆肥过程中很容易生成生物气溶胶，耐药菌和抗性基因随之逸散至环境中，对人类健康产生危害。目前，关于生物气溶胶负载抗生素耐药菌和抗性基因的研究多集中在畜禽养殖场，堆肥过程中相关研究未见报道。因此，本项目通过现场调查和实验室研究，对典型堆肥厂生物气溶胶负载耐药菌和抗性基因的分布规律进行研究；重点考察抗性基因的逸散影响因素、研究其在不同粒径生物胶体（PM2.5/PM10）和耐药菌的分布特性、揭示该特征与传播距离的动态关系，阐明发生机制、逸散规律和传播过程；建立逸散过程模型，阐述抗性基因由堆肥向空中及周边逸散轨迹。为控制新型空气污染物产生，降低耐药菌和抗性基因经生物气溶胶传播风险、减少人类暴露、预测危害提供理论依据。

堆肥过程中逸散的生物气溶胶负载抗生素耐药菌和抗性基因，对人类和环境健康产生危害。本项目对典型堆肥厂生物气溶胶负载耐药菌和抗性基因的分布规律进行研究。考察了抗性基因的逸散影响因素、研究其在不同粒径生物胶体和耐药菌的分布特征、揭示该特征与传播距离的动态关系，阐明发生机制、逸散规律和传播过程。.本研究对北京市平谷、怀柔和门头沟的四家堆肥厂进行调查。对上风向（1次）、堆肥区（9次）、包装区（7次）、办公区（8次）和下风向（4次）5个地点空气中，总基因拷贝数（16SrDNA）、2种条件致病菌特异性基因（Escherichia coli, Staphylococcus spp）、一类整合子（int 1）、7种β-内酰胺类抗性基因（blaCARB-4, blaOXA-18, blaOXA1, blaOXAII, blaOXAIII, blaPSE, blaTEM*）、10种四环素抗性基因（tetQ, tetM, tetS, tetT, tetW, tetA/P, tetG, tetL, tetZ, tetX）、4种磺胺类抗性基因（sul1, sul2, sul3, dfrA1,）和1种红霉素抗性基因（ermB）的浓度。目标基因最高浓度主要在装料区和堆肥区检出。值得注意的是，本研究在5个采样点均检测到了一类整合子（intl1）。其中包装区和堆肥区浓度分别达到104和103gene number•m-3。..多数包装区和堆肥区与所考查的基因具有相关性，而办公区和下风向与所考查基因均无明显相关。其中，Staphylococcus spp、一类整合子（int 1）和总基因拷贝数（16SrDNA）主要和四环素抗性基因相关，而Escherichia coli主要和β-内酰胺类抗性基因相关。Escherichia coli和Staphylococcus spp分别都于intl1和16SrDNA具有相关性。堆肥区和包装区空气中的抗性基因变化规律相似，下风向和办公区的抗性基因可能来源于堆肥区和包装区。. 本研究堆肥厂不同地点生物气溶胶的生物多样性进行分析。结果显示，堆肥区和包装区的生物多样性高于办公区和场外。非度量多维尺度分析(non-metric multi-dimensional scaling, NMDS)分析和Heatmap图结果均显示，堆肥区和包装区的细菌群落结构相似，办公区的菌群和

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