水产品中抗生素抗性基因的污染特征研究

发布时间：2018-09-12 19:43

【摘要】：抗生素在水产养殖中有着不可取代的作用,但抗生素的长期滥用导致的细菌耐药性问题愈发引起人们的关注。抗生素抗性基因(antibiotic resistance genes,ARGs)是细菌耐药性传播扩散的关键因子。它可以通过可移动遗传元件在细菌间传播,进而会对食品安全和人类健康产生严重威胁。然而,目前关于ARGs的污染特征研究多集中于水产养殖的养殖用水和底泥中,水产品作为进入人体的直接载体,水产品中的致病菌的耐药性会对人体产生直接影响。然而,目前水产品批发市场中水产品中ARGs的污染特征却并不清楚。因此,为了更清楚的了解水产品中ARGs的污染特征,更好的解决食源性致病菌的耐药问题和临床用药问题,本研究做了以下3部分工作:(1)上海市水产品批发市场排污沟淤泥中抗生素抗性基因的污染分析;(2)中国东南沿海地区水产品中抗生素抗性基因的特征及与抗生素残留的关联分析;(3)副溶血性弧菌链霉素耐药表型和基因型的不对应性原因分析。(1)上海市水产品批发市场排污沟淤泥中抗生素抗性基因的污染分析为了探究待售水产品中抗生素抗性基因(Antibiotic resistance genes,ARGs)在水产品批发市场中的扩散污染情况,选取上海市3个大型水产品批发市场作为研究对象,采用PCR的方法,对市场内部和市场附近的排污沟表层淤泥中5大类(4种磺胺类、8种四环素类、6种β-内酰胺类、5种氯霉素类、4种链霉素类)共27种ARGs进行了检测,并利用PCR-DGGE技术分析了细菌种群多样性。结果显示共有17种ARGs在批发市场淤泥中被检出,其中磺胺类、四环素类和链霉素类ARGs的检出率较高且检出种类较多,表明水产品批发市场含有种类丰富的ARGs,水产品以磺胺类、四环素类和链霉素类ARGs污染为主。对市场外围的检测显示市场外围区域ARGs的种类分布略低于市场内部,表明水产品批发市场中ARGs的存在会通过污水排放途径扩散到周边环境。DGGE图谱的分析结果显示,3个批发市场污泥样品细菌种类差异性较大,表明环境中ARGs的种类分布与细菌的种类并无直接关联。本研究首次通过调查水产品批发市场中饭排污沟淤泥中ARGs的污染证实水产品批发市场已经成为ARGs的重要储存库,以此造成的污染会进一步传播到周边环境,此外,这些种类丰富的ARGs重新扩散到待售水产品中的风险很大,需引起人们足够的重视。(2)中国东南沿海地区水产品中抗生素抗性基因的特征及与抗生素残留的关联分析抗生素抗性基因(Antibiotic resistance genes,ARGs)作为一种新型环境污染物,对人体健康构成了潜在的威胁。本研究通过PCR和q PCR定性定量检测94个水产品(其中18个为抗生素检测阳性)鱼皮上37种抗性基因的污染特征。此外,本研究分离出10株副溶血性弧菌,以验证水产品中副溶血性弧菌ARGs的存在性。结果显示,相比与海水鱼,淡水鱼中含有的ARGs的种类更多,丰度更高。tet S、str A、str B、aad A、flo R、sul I和sul II被认为是中国东南沿海地区水产品中的主导基因,它们的检出率均大于30%并且在五个城市中均有检出。分离的副溶血性弧菌抗性基因检出了水产品中这7个主导基因,表明这7大ARGs在副溶血性弧菌中也已普遍存在。抗生素检测阳性的水产品中含有的ARGs明显高于其他水产品,表明抗生素的使用不仅仅可能会诱导该种类下的抗性基因,还可能对诱导其他类ARGs的产生。本次研究并无磺胺类抗生素检出,表明在没有磺胺类抗生素的选择性压力下,sul I和sul II在水产品中已普遍存在。本研究表明淡水鱼已成为ARGs的重要储存库,抗生素的使用可能不仅仅诱导该种类下的抗性基因,还可能诱导其他类ARGs的产生,需引起人们足够的重视。本研究首次监控了东南沿海地区水产品中ARGs的污染特征,为东南沿海地区水产品及水产品致病菌中抗性基因提供数据支持,为临床医疗诊断提供基础数据。(3)副溶血性弧菌链霉素耐药表型和基因型的不对应性分析近些年来,由于抗生素的滥用,细菌耐药性带来的临床治疗难题引起人们愈发的关注。本课题组之前的调查发现,副溶血性弧菌耐药表型和对应的基因型并不一致,具体表现为有些副溶血性弧菌携带有抗性基因,而药敏实验表现为对抗生素敏感。结合水产品中ARGs污染的主导基因和本课题组副溶血性弧菌的耐药情况,选取抗链霉素类的aad A、sul I和sul II作为目标基因。实验材料选取课题组10株携带有aad A却对链霉素敏感的副溶血性弧菌菌株以及46株含有sul I或sul II却对复方新诺明表现为敏感的菌株。结果发现,副溶血性弧菌aad A基因有包括3大类8种多种突变形式。而sulⅠ突变主要发生在231位点缺失A,sulⅡ主要发生的是106位点缺失A。这些突变都有可能导致基因不能正常编码。本实验表明在抗性基因(ARGs)扩散传播的过程中,由于各种原因造成的抗性基因突变如缺失、重复、移码、插入等可能是造成细菌耐药菌株表型缺失的一个重要原因。本实验首次通过分析抗性基因序列中碱基突变的方法阐述副溶血性弧菌耐药表型和基因型不一致现象背后的原因。
[Abstract]:Antibiotics play an irreplaceable role in aquaculture, but the long-term abuse of antibiotics leads to the problem of bacterial resistance. Antibiotic resistance genes (ARGs) are a key factor in the spread of bacterial resistance. However, the current study on the pollution characteristics of ARGs is mostly concentrated in aquaculture water and sediment. Aquatic products as a direct carrier into the human body, the drug resistance of pathogenic bacteria in aquatic products will have a direct impact on the human body. However, the current wholesale market of aquatic products. The contamination characteristics of ARGs in aquatic products are not clear. Therefore, in order to better understand the contamination characteristics of ARGs in aquatic products, and to better solve the problem of drug resistance of foodborne pathogens and clinical drug use, the following three parts of work were done in this study: (1) The contamination fraction of antibiotic resistance genes in sewage silt of Shanghai aquatic products wholesale market. Analysis; (2) Characteristics of antibiotic resistance genes in aquatic products from southeastern coastal areas of China and their association with antibiotic residues; (3) Analysis of phenotype and genotype of streptomycin resistance of Vibrio parahaemolyticus. (1) Pollution analysis of antibiotic resistance genes in sewage sludge of Shanghai aquatic products wholesale market The spread of antibiotic resistance genes (ARGs) in aquatic products wholesale markets was investigated. Three large-scale aquatic products wholesale markets in Shanghai were selected as the research objects. PCR was used to detect 5 major types (4 sulfonamides, 8 tetracyclines, 6 tetracyclines) in the surface mud of sewage drains in and near the market. 27 kinds of ARGs including beta-lactams, 5 kinds of Chloramphenicols and 4 kinds of streptomycins were detected, and the diversity of bacterial population was analyzed by PCR-DGGE. The results showed that 17 kinds of ARGs were detected in the sludge of wholesale market, among which sulfonamides, tetracyclines and streptomycins were detected more frequently, indicating that there were many kinds of ARGs in aquatic products. The wholesale market contains abundant ARGs, and the aquatic products are mainly polluted by sulfonamides, tetracyclines and streptomycins. The detection of the periphery of the market shows that the distribution of ARGs in the periphery of the market is slightly lower than that in the interior of the market. The results showed that the bacterial species of sludge samples from the three wholesale markets were different, indicating that the distribution of ARGs in the environment was not directly related to the bacterial species. In addition, the risk of these ARGs reappearing in aquatic products for sale is very high, which needs to be paid enough attention. (2) Characteristics of antibiotic resistance genes in aquatic products in southeastern coastal areas of China and their association with antibiotic residues Resistance genes, ARGs, as a new type of environmental pollutants, pose a potential threat to human health. In this study, 37 resistance genes in 94 aquatic products (18 of which were positive for antibiotics) were detected qualitatively and quantitatively by PCR and q-PCR. In addition, 10 strains of Vibrio parahaemolyticus were isolated to verify the contamination characteristics of aquatic products. The results showed that freshwater fish contained more ARGs than seawater fish. Tet S, str A, str B, aad A, flo R, Sul I and sul II were considered to be the dominant genes in aquatic products in southeastern coastal areas of China. The detection rates of ARGs in freshwater fish were more than 30% and were detected in five cities. Seven major ARGs were found in aquatic products, indicating that the seven major ARGs were also widespread in Vibrio parahaemolyticus. Antibiotic-positive aquatic products contained significantly higher ARGs than other aquatic products, suggesting that the use of antibiotics may not only induce resistance genes in this species. No sulfonamide antibiotics were detected in this study, suggesting that Sul I and sul II were common in aquatic products without the selective pressure of sulfonamide antibiotics. This study monitored the pollution characteristics of ARGs in aquatic products in southeastern coastal areas for the first time, providing data support for the resistance genes in pathogenic bacteria of aquatic products and aquatic products in southeastern coastal areas, and providing basic data for clinical diagnosis. (3) Vibrio parahaemolyticus In recent years, due to the abuse of antibiotics, the problem of clinical treatment caused by bacterial drug resistance has attracted more and more attention. Previous investigations by our team found that the phenotype and genotype of V. parahaemolyticus were not consistent, and some V. parahaemolyticus were specific. Combining the dominant gene of ARGs contamination in aquatic products and the drug resistance of Vibrio parahaemolyticus in our research group, we selected aad-A, sul-I and sul-II as the target genes. Vibrio parahaemolyticus strains and 46 strains containing Sul I or Sul II but susceptible to compound trimethoprim were found. The results showed that the AAD A gene of Vibrio parahaemolyticus had three major classes and eight kinds of mutations. The Sul I mutation mainly occurred at 231 locus deletion A, and the Sul II mutation mainly occurred at 106 locus deletion A. This experiment shows that the phenotypic deletion of resistant strains may be caused by mutations such as deletion, repetition, code shifting and insertion in the diffusion of resistant genes (ARGs). The reasons behind the drug resistance phenotypes and genotype incompatibility of Vibrio parahaemolyticus are discussed.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS254.1;TS201.6

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