河口及近岸海洋环境中抗生素抗性基因的传播方式及环境因子影响机制研究

Antibiotics are widely used in human medicine and stockbreeding operations, large amounts of antibiotics and their metabolites are potentially released into different compartments of the environment, which are regarded as the most important factor for the evolution and selection of antibiotic resistance genes. The increase in the number of antibiotic resistance genes is a major concern of health officials world-wide. The transfer of antibiotic resistance genes (ARGs) could have more adverse effects on environment than the ARGs themselves. However, many studies have been carried out in waste water treatment plants and rivers but few research were implemented in estuaries or coastal environmental. Meanwhile, the major transfer pathway of ARGs and its influencing factors of the transfer are not clear. Therefore, the present study will select Liao River estuary and the coastal areas of Dalian, investigate the distribution characteristic of antibiotics resistance genes in the area and determine the most important propagation pathway of the ARGs. The seawater and sediments will be sampled, twenty-one antibiotics (6 fluoroquinolones, 15 sulfonamides) will be simultaneously quantified with solid-phase extraction and liquid chromatography-tandem mass spectrometry. DNA will be extracted and quantified by real-time (SYBR Green) PCR methods after the samples be filtered through 0.22 μm membranes. Furthermore, multiple environmental factors will be analyzed to discover the main factors which affect the transfer of ARGs. The experiment of transfer simulation will be designed according to the influence factors to reveal the effect mechanism of different factors on ARGs propagation process. Based on all the studies, we will summarize the horizontal distribution of ARGs in Liao River estuary and the coastal areas of Dalian. The project will clarify the major propagation pathway to better understand the environmental behavior of ARGs. This project also provide effective strategies to reduce the transfer and proliferation of ARGs in the environment.

抗生素抗性基因作为一类新兴环境污染物，其在不同环境介质中的传播扩散造成了比抗生素本身更大的危害。然而关于抗性基因在环境中的传播方式、环境因子及其他主要污染物对其传播扩散的影响等研究尚少，尤其在近岸海洋环境中的研究尚处于空白。本项目针对这一问题，选取辽河口与大连近岸海域为代表，应用HPLC-MS/MS与RT-PCR对磺胺、喹诺酮两类抗生素及其抗性基因、整合子基因进行定量分析，评估抗生素抗性污染水平的同时分析确定抗性基因在近岸海洋环境中传播的规律；通过多功能检测仪、ICP-MS、GC等对各环境因素进行检测，分析可能存在的影响因子，从而确定实验室控制条件；并利用构建的抗性质粒，在实验室内模拟各环境因素对抗性基因水平转移过程的影响，揭示其影响机制。项目的实施将有助于理解抗生素抗性基因在近岸海洋环境中的动态变化过程，为制定相应的防治措施提供理论依据与技术支持。

抗生素抗性在环境中广泛的存在和传播扩散目前已经成为全球公共安全问题，尽管近年来关于环境中抗生素抗性污染状况的研究日益增多，但研究区域主要集中于河流、城市污水与土壤等内陆环境，关于其在河口和近岸海洋环境中分布状况和动态变化特征尚不清楚，环境因子及其他污染物是否能影响抗生素抗性的环境行为也有待研究。为了解决以上问题，本研究选取辽河口和大辽河口、莱州湾和大连近岸为河口和近岸海洋环境代表，首先，建立了适用于河口及近岸海洋环境中痕量抗生素分析、大肠杆菌(E. coli)与金黄色葡萄球菌(S. aureus)抗性水平评估和抗生素抗性基因(ARGs)定量的研究方法。其次，应用该方法分析了河口和近岸海洋环境中的磺胺类抗生素(SAs)和喹诺酮类抗生素(QNs)的含量水平及其抗性基因的污染特征，评估了E. coli 与S. aureus 抗生素抗性水平与分布特征，同时对各环境因素进行检测，分析可能存在的影响因子并开展了环境因子影响下的ARGs水平转移实验。实验结果表明，(1)河口及近岸海洋环境普遍存在SAs和QNs抗生素污染问题，但污染水平较轻，抗生素残留浓度远低于河流、城市污水等内陆环境。(2)虽然河口及近岸海洋环境的抗生素残留量远低于内陆环境，但仍然普遍存在着抗生素抗性污染问题：河口及近岸海洋环境中sul1、sul2的检出率为100%，sul3、aac(6’)-Ib、qnrS也均有不同程度检出，整体分布呈自河端向海端递减的趋势，说明冲淡水对抗性基因的分布产生一定的影响；辽河口和大辽河口、莱州湾和大连近岸中E. coli 与S. aureus 的平均抗性率分别为35.5%、26.0%和14.5%，整体分布呈现由河端向海端递增的特征。(3)抗性基因丰度和抗性率与抗生素残留量之间不具有显著相关性，反而与部分环境因子(盐度、温度、pH及COD)、重金属（Fe、Al、Zn、Pb等）以及有机污染物(多环芳烃、多氯联苯、有机氯农药、酚类等)之间具有显著相关性。该研究共发表学术论文10余篇，有助于理解抗生素抗性污染在河口和近岸海洋环境中的分布特征与传播机制，为及时采取针对性措施解决日趋严重的环境抗生素抗性污染问题提供技术支持。