磺胺类抗生素抗性基因在畜禽粪便堆肥系统中的消长及影响机制研究

Antibiotic resistance genes (ARGs), which would impart the ability of disease-causing microorganisms to resist medical treatment, are recognized to represent a global threat to human health. Owing to the growing serious pollution of ARGs in livestock manure and the wide application of composting as a practical, economical, environmentally safe way for manure management, this project is proposed and aims at monitoring the selected ARGs responsible for sulfonamides during composting and understanding how composting affects the disturbance and the spread of ARGs. This project can be divided into three sections: (1) The first section aims to clarify responding dynamic behaviors of sulfonamide ARGs to physicochemical parameters and sulfonamide antibiotics concentrations and to isolate key composting influencing factors. Large-scale composting and laboratory incubation experiments would be performed with the application of qPCR and HPLC/MS/MS. (2) In the second section, relationships between microorganism community profiles and dynamic behaviors of ARGs would be analyzed using molecular biology tests such as MiSeq sequencing. The number of resistant bacteria and their community composition would be tracked as well. (3) In the last section, effects of composting on the spread of ARGs are investigated by conducting conjugation test to determine the efficiency of horizontal gene transfer and by characterizing the antibiotic plasmids. According to the above-mentioned studies, mechanisms of effects of composting on the dynamic behaviors of sulfonamides ARGs could be elaborated. The project can provide a reference for improving composting process to eliminate ARGs in manure. Besides, it could be essential for the elucidation of the source and the distribution of ARGs in agricultural soils.

抗生素抗性基因作为细菌耐药产生的根源正在世界范围威胁人类健康。针对抗生素滥用引发的畜禽养殖粪便抗性基因污染日益加剧这一现状，也基于堆肥作为一项粪便资源化处理方式广为应用的前提，本项目对磺胺抗性基因在堆肥系统中的消长行为及影响机制开展系统研究：首先结合实地堆肥和室内培养试验，采取qPCR、HPLC/MS/MS等技术，在监测主要堆肥过程因子和磺胺抗生素浓度变化的基础上，明确抗性基因消长对堆肥进程的响应，筛选影响抗性基因扩散的主控因素；接着利用PLFA、MiSeq等技术考察堆肥群落演替与抗性基因消长行为的作用关系，追踪抗性菌数量及群落组成，探索主要宿主菌分布；开展细菌接合实验分析堆肥前后磺胺抗性转移水平差异及抗性质粒遗传特征，明确堆肥对磺胺抗性传播的影响；最终阐明堆肥进程中磺胺抗性基因消长的影响机制。研究可为抗性基因污染控制研究提供理论依据，对解析粪肥施用土壤中抗性基因来源也具有重要参考价值。

针对抗生素滥用引发的畜禽粪便抗性基因污染日益加剧这一现状，为明确堆肥中抗生素抗性基因（ARGs）的消长机制，研究以磺胺ARGs为主要对象，开展如下内容：. 1、探索好氧堆肥中ARGs的消长规律，发现磺胺ARGs可随堆肥进程呈波动变化和平稳下降，其中同一类ARGs的消长在不同堆肥场地、不同处理之间存在差异，其中不同场地之间的差异明显大于同一场地的不同处理。. 2、分析了堆肥中典型作用因子对ARGs消长的影响，表明：（1）磺胺抗生素与堆肥中sul1和IntI1消长无明显相关性，但与sul2显著相关，其浓度增加可促进sul2扩散。抗生素和抗性基因对环境改变的应答不完全一致，特别是不同来源粪便中ARGs丰度与抗生素浓度之间没有直接联系。（2）高温能有效降低猪粪中ARGs相对丰度和耐药菌数量，但受温度范围和抗性种类的影响。相比磺胺ARGs，四环素ARGs对温度更为敏感。原料pH调节可以在一定程度上控制ARGs和磺胺耐药菌扩散，但不同类型ARGs对pH的响应不同。木屑添加增加sul1和IntI1相对丰度，降低sul2相对丰度和磺胺耐药菌数量，促进抗生素降解。光照增加sul1相对丰度，降低sul2相对丰度，对IntI1基因没有显著影响。多因素方差分析指出，磺胺耐药菌、磺胺ARGs和磺胺抗生素对堆肥参数改变的响应不完全一致；不同磺胺ARGs的主控因素也不相同，例如温度和pH变化是sul2消长的主控因素，而辅料添加是sul1消长的主控因素。. 3、通过细菌群落结构和磺胺耐药转移水平分析探索了堆肥中耐药消长的微生物学基础，指出：（1）猪粪中ARGs消长与细菌总量变化没有明显联系，但与细菌群落组成密切相关。（2）堆肥改变了猪粪中磺胺耐药菌的组成分布，堆肥早期猪粪中的优势磺胺耐药菌主要是Idiomarina、Pseudomonas、Ignatzschineria，而后期则为Bacillus和Staphylococcus。（3）堆肥处理降低了猪粪中微生物的磺胺耐药转移水平，其中sul2和IntI1转移频率的降低幅度大大高于sul1。sul1是接合子获取磺胺耐药的必要元件，而sul2不是必备的，接合子无法通过单独获取sul2获得磺胺抗性。