水产养殖环境中磺胺类抗生素抗性基因污染的研究

发布时间：2018-08-06 19:29

【摘要】：抗生素不仅可以防治水产生物的细菌性疾病,同时还可以促进养殖动物的生长,因此在水产养殖业中的使用极为广泛。近年来,随着抗生素使用的加剧,养殖生态环境所遭受的抗生素污染日益严重,并由此诱导产生了一种新型的环境污染物——抗生素抗性基因(ARGs)。抗性基因可以通过基因的水平转移进入人体,影响人类的健康,已经引起了国内外的广泛关注。本文试图以磺胺类抗生素为目标化学物,通过研究太湖周边养殖区域(无锡、湖州、常州、苏州、宜兴)不同养殖品种(河蟹、青虾、鲈鱼、青鱼、鲫鱼、鳊鱼、南美白对虾)养殖池塘的水体和底泥沉积物中抗生素的残留、抗性菌株的情况以及抗生素抗性基因的污染现状和变化规律,并与相近的自然水域相比较,了解养殖水环境中抗生素抗性基因的污染残留状况。研究结果如下:1、分析了 2015年不同地区不同品种的养殖池塘在整个养殖周期中水样和沉积物中磺胺类药物的分布情况,发现在整个养殖季节中,池塘水体和沉积物中磺胺类的含量变化均呈现低-高-低的变化趋势;2、整个养殖过程中,不同养殖池塘的水样、沉积物中均可检出磺胺类的耐药微生物。随着磺胺二甲基嘧啶浓度的升高,养殖池塘的水样、沉积物中微生物的耐药率均呈现下降趋势,且浓度越高,耐药菌的耐药率越低。同时,相同药物浓度下,沉积物中细菌的耐药率要高于水中。3、通过抗性平板分离筛选出具有耐药性的抗性菌株4株,结合菌体生理生化特性分析和16SrDNA序列分析,初步确定NHA1401、NHA1402菌株为奇异变形杆菌(Proteus mirabilis),SHA1501、CBA1506 菌株为葡萄球菌属(Chryseobacterium sp.)和金黄杆菌属(Staphylococcus sp.)的细菌。4、采用PCR方法对环太湖不同养殖区域不同养殖品种的养殖池塘,在同一养殖周期的不同时段(2015年的5-6月、7-8月、9-10月)的水样和沉积物进行磺胺类抗性基因sulⅠ和sulⅡ的定性分析,结果显示,在3个不同的养殖时段中,均可检出磺胺类抗性基因sulⅠ和sulⅡ。养殖前期(5-6月)2种磺胺类抗性基因的检出率最低,分别为80%和65%。随着养殖进程的推进,sulⅠ基因呈现出先升高后下降的趋势,sulⅡ抗性基因检出率则在不断升高。与此同时,磺胺类抗性基因sulⅠ、sulⅡ在太湖自然水体和沉积物中也有检出。5、通过荧光定量PCR方法分析了环太湖不同养殖区域不同养殖品种的养殖池塘中的水样和沉积物,在同一养殖周期的不同时段的磺胺类抗性基因sulⅠ和sulⅡ的相对丰度,结果显示,(1)环太湖水产养殖区不同地区相同品种的养殖池塘环境中磺胺类抗性基因的相对丰度差异不大,最大不超过一个数量级;(2)在相同地区不同品种的水产养殖池塘中,南美白对虾、青鱼、鲈鱼养殖池塘中的污染较青虾、鳊鱼为重。除河蟹外,青虾、南美白对虾、鲈鱼、卿鱼、鳊鱼和青鱼6个品种养殖池塘的水体遭受到的污染程度比沉积物要重,而河蟹养殖池塘则沉积物的污染较重。在水产养殖环境中,无论是水中还是沉积物中,抗性基因都是存在的,且差异性最大在1～2个数量级之间;(3)在相同地区、相同养殖品种的同一个养殖周期中,几乎有90%的养殖池塘,其磺胺类抗性基因相对丰度的最高值出现在养殖中期的7-8月,只有极少数的例外。6、在太湖自然水体中,不同地理位置水域的水样和沉积物中磺胺类抗性基因的相对丰度存在一定的差异,沉积物中磺胺类抗性基因的污染较水体为重。与同期附近的养殖区域相比,天然水体水样中的污染程度轻于养殖区,而沉积物中的污染程度与养殖区相当。研究表明,水产养殖环境中的抗生素抗性基因并非长期固定累积存在于池塘中,磺胺类抗性基因的产生与养殖周期、养殖中抗生素的投放和使用量存在一定的联系。因此,严格规范和控制养殖过程中抗生素的使用,对抗性基因的产生和传播可起到一定的防范作用,从而使抗性基因的转移得到有效遏制,为人类健康提供保障。
[Abstract]:Antibiotics can not only prevent and cure the bacterial diseases of aquatic organisms, but also promote the growth of aquaculture animals. Therefore, the use of antibiotics is extremely widespread in aquaculture. In recent years, with the intensification of the use of antibiotics, the antibiotic pollution suffered by the ecological environment of aquaculture is increasingly severe, and a new environmental pollution has been induced. The antibiotic resistance gene (ARGs). Resistance genes can be transferred into the human body through the transfer of genes and affect human health. It has aroused widespread concern at home and abroad. This article attempts to use sulfonamides as the target chemicals, through the study of different aquaculture products in Taihu, Wuxi, Huzhou, Changzhou, Suzhou, Yixing). The residue of antibiotics in water and sediment sediments in aquaculture ponds, the situation of resistant strains and the pollution status and change rules of antibiotic resistance genes in aquaculture ponds, such as river crabs, penicus, perch, carp, bream, bream, and Penaeus prawns, and the pollution residue of antibiotic resistance genes in aquaculture water. The results are as follows: 1, the distribution of sulfonamides in water samples and sediments of different breed aquaculture ponds in different regions in 2015 was analyzed. It was found that the variation of sulfonamides in pond water and sediment showed a trend of low high low in the whole culture season, and 2, the whole culture process. In the water samples of different aquaculture ponds, the sulfonamides could be detected in the sediment. With the increase of the concentration of sulfamethazine, the resistance rate of microbes in the aquaculture pond and the sediment showed a decreasing trend, and the higher the concentration, the lower the resistance rate of the drug resistant bacteria. The resistance rate was higher than that of.3 in water. 4 strains resistant to resistance were screened by the resistance plate separation. Combined with the analysis of physiological and biochemical characteristics and 16SrDNA sequence analysis, NHA1401 was preliminarily identified, the strain NHA1402 was Proteus mirabilis, SHA1501, and CBA1506 strain was Staphylococcus (Chryseobacterium sp.) and golden yellow. Bacteriobacterium (Staphylococcus sp.) bacterial.4, the qualitative analysis of sulfonamide resistance genes Sul I and sul II in water samples and sediments at different periods of the same period (5-6 months, 7-8 months, 9-10 months, 2015) by PCR method was used to analyze the qualitative analysis of Sul I and sul II in the water samples and sediments at the same period of the same breeding cycle (5-6 months, 7-8 months, 9-10 months). Sulfonamides resistance genes Sul I and sul II were detected during the breeding period. The detection rates of 2 sulfonamides resistance genes were the lowest in the early stage of culture (5-6 months), respectively, 80% and 65%., respectively, with the advance of the breeding process, the Sul I gene showed a tendency to rise first and then decline, while the detection rate of the Sul II resistance gene was increasing. Gene Sul I, Sul II also detected.5 in Taihu natural water and sediment, and analyzed the water samples and sediments in different aquaculture ponds of different aquaculture regions around Taihu by fluorescence quantitative PCR method. The relative abundance of the sulfonamides resistance based on Sul I and sul II at the same period of the same breeding period showed that (1) The relative abundance of sulfonamides resistance genes in the environment of the same breed aquaculture ponds in different regions of the Taihu aquaculture area is not much different, and the maximum is not more than one order of magnitude. (2) in the aquaculture ponds of different varieties in the same area, the pollution of Penaeus prawns, green fish and perch ponds is more serious than the penaeid shrimp and bream. The water body of the 6 aquaculture ponds of shrimp, white white prawns, perch, Qing fish, bream and green fish in aquaculture ponds are heavier than the sediments, while the river crab aquaculture ponds have heavy pollution. In aquaculture environment, both in water and in sediment, the resistance base is present, and the difference is at the maximum of 1~2 orders of magnitude. (3) in the same area, almost 90% of the aquaculture ponds were found in the same breeding period of the same breed. The highest relative abundance of the sulfonamides resistance gene appeared at 7-8 months in the middle period of the culture, and only a few exceptions.6, in the natural waters of Taihu, the sulfonamides resistant base in the water samples from different geographical locations and the sediments. There is a certain difference in relative abundance, the pollution of the sulfonamides resistance gene in the sediments is heavier than that in the water body. Compared with the aquaculture area near the same period, the pollution degree of the natural water samples is lighter than that in the aquaculture area, and the pollution in the sediments is equivalent to that in the aquaculture area. The non long-term fixed accumulation exists in the pond. The production of the sulfonamides resistance gene is related to the breeding cycle and the dosage of antibiotics in the culture. Therefore, it is necessary to strictly regulate and control the use of antibiotics in the culture process and to prevent the production and transmission of the antagonistic genes, thus making the resistant gene. Transfer is effectively curbed, providing protection for human health.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S948

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