施用粪肥的设施菜地土壤中抗生素及抗性基因多样性及丰度的研究

发布时间：2018-04-23 19:17

本文选题：菜地土壤 + 抗生素　；参考：《山东农业大学》2017年硕士论文

【摘要】：抗生素作为20世纪最重要的发现之一,在保障人类及动物健康以及促进畜禽养殖业发展方面起着不可替代的作用。然而,由于抗生素的误用和滥用,使得大量的抗生素以原药或代谢物的形式随人或动物体的排泄而进入环境,导致环境中,特别是土壤环境和水环境中存在大量抗生素残留,进而增加了土壤或水环境中微生物的抗性压力,加速了大量抗性微生物的产生。抗性微生物对抗生素产生耐药性的主要原因是其携带有各种抗生素抗性基因,因此,抗生素的大量使用直接或间接增加了环境中抗性基因的含量。设施菜地是使用畜禽粪便较为频繁的场所,因此,研究设施菜地土壤中抗生素含量、耐药菌数量以及抗性基因的含量是十分必要的。本研究采集了山东潍坊、泰安、滨州、东营四个地区长期使用粪肥的9个典型设施菜地土壤,研究了设施菜地土壤中抗生素及抗性基因含量等。本实验选取了四类抗生素进行实验,利用固相萃取(Solid Phase Extraction,SPE)以及高效液相色谱(High Performance Liquid Chromatography,HPLC)检测的方法,检测了土壤样品中三种抗生素的含量;另外,对土样中含有的可培养细菌进行了数量的统计及菌种的鉴定;选取了五类共12种抗性基因和可移动遗传元件(MGE)利用real-time qPCR定量检测的方法对其丰度进行了测定。最后,将抗性基因的含量与土样理化性质、抗生素含量、耐药菌的数量以及MGE的含量进行综合分析。该论文的主要研究结果如下:(1)在土壤样品抗生素含量检测实验中,强力霉素在所有土壤样品中均有检出,浓度范围在52.37μg/kg~428.16μg/kg之间;磺胺二甲嘧啶除了在土样S4中没有检出外,其他土样中均有检出,浓度范围在14.12μg/kg~81.31μg/kg之间;泰乐菌素只在S0、S2、S3、S4和S8这5个土壤样品中有检出,浓度范围为70.01μg/kg~482.36μg/kg。(2)在可培养抗生素耐药菌实验中,土样中的耐药菌对抗生素的耐药性因抗生素种类和浓度的不同而在数量上呈现出较大差异。在未使用过粪肥的土样S0中,细菌对抗生素仍有一定的抗性存在,长期使用粪肥的菜地土样S1~S9耐药率要高于土样S0。随着抗生素浓度的增加,细菌耐药率逐渐降低。土壤耐药菌对四种抗生素的耐药率大小依次为:磺胺二甲嘧啶泰乐菌素恩诺沙星强力霉素。(3)在抗生素耐药菌分离鉴定实验中,共筛选出强力霉素耐药菌12个属,17个种,优势菌株为链霉菌属;筛选出磺胺二甲嘧啶耐药菌9个属,16个种,优势耐药菌为芽孢杆菌属;筛选出恩诺沙星耐药菌9个属,16个种,优势菌株为微杆菌属;筛选出泰乐菌素耐药菌9个属,12个种,优势菌株为寡养单胞菌属和假单胞菌属。芽孢杆菌属、金黄杆菌属和假单胞菌属属于多重耐药菌。(4)在选取的五类共16种抗性基因中,tet W、tetM、tetO、sul1、sul2、qepA、qnrB、qnrS、ermB、ermF的检出频率均为100%。在定量实验中,各种抗性基因在不同土样中的含量存在较大差异。四环素类抗性基因中,tetM的含量最高,磺胺类抗性基因中sul2的含量较高,喹诺酮类抗性基因中,qepA的含量相对较高,大环内酯类抗性基因中,ermF的含量相对较高。可移动元件intI1的含量相对较高。各抗性基因总量关系为:磺胺类大环内酯类四环素类喹诺酮类。(5)从土样理化性质方面分析,土样pH值与恩诺沙星耐药菌数量之间呈现极显著相关,重金属含量与喹诺酮类抗性基因qnr呈现极显著相关;从抗生素含量角度分析,土样中强力霉素含量与泰乐菌素的含量及磺胺类抗性基因的含量sul呈现极显著相关,与强力霉素耐药菌及磺胺二甲嘧啶耐药菌呈现显著相关,泰乐菌素的含量与磺胺类抗性基因sul之间也呈现出极显著相关;从可移动元件角度分析,土样中可移动元件总量intI与磺胺类抗性基因sul及大环内酯类抗性基因erm呈现极显著相关。
[Abstract]:As one of the most important discoveries in twentieth Century, antibiotics play an irreplaceable role in protecting human and animal health and promoting the development of livestock and poultry industry. However, due to the misuse and abuse of antibiotics, a large number of antibiotics enter the environment with the excretion of human or animal bodies in the form of original drugs or metabolites, leading to the environment, In particular, there are a large number of antibiotic residues in the soil environment and water environment, thus increasing the resistance pressure of microorganisms in the soil or water environment and accelerating the production of a large number of resistant microorganisms. The main reason for the resistance of resistant microorganisms to antibiotics is that they carry a variety of antibiotics resistance genes. Therefore, a large number of antibiotics are used directly. Therefore, it is necessary to study the content of antibiotics, the number of drug-resistant bacteria and the content of resistance genes in the soil of vegetable plots. This study collected four areas in Weifang, Tai'an, Binzhou and Dongying, Shandong, for the long use of manure. The contents of antibiotics and resistance genes in vegetable soil soil were studied in 9 typical vegetable plots soil, and four kinds of antibiotics were selected in the experiment. The soil samples were detected by solid phase extraction (Solid Phase Extraction, SPE) and high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). The content of the three kinds of antibiotics; in addition, the quantity of the cultivated bacteria contained in the soil samples and the identification of the strains were carried out. The abundance of the five classes of resistance genes and the removable genetic elements (MGE) were determined by the method of real-time qPCR quantitative detection. Finally, the content of the resistant genes and the physicochemical properties of the soil samples were determined. The content of antibiotics, the number of resistant bacteria and the content of MGE were analyzed synthetically. The main results of this paper were as follows: (1) in the test of soil sample antibiotic content, the concentration of doxycycline was detected in all soil samples, the concentration range was between 52.37 Mu and g/ kg, and the sulfonamydrine was not detected in the soil sample S4. Out of the other soil samples, the concentration range was between 14.12 g/kg~81.31 and g/kg, and tylosin was detected only in the 5 soil samples of S0, S2, S3, S4 and S8. The concentration range was 70.01 mu g/kg~482.36 mu g/kg. (2) in the experiment of antibiotic resistant bacteria. The resistance of the bacteria to antibiotics in the soil samples was due to the type and concentration of antibiotics. In the soil sample S0 of unused manure, the bacteria still have some resistance to antibiotics, and the resistance rate of the soil sample S1~S9 of the vegetable soil sample with manure for a long time is higher than that of the soil sample S0., with the increase of the antibiotic concentration, the resistance rate of the bacteria is gradually reduced. The resistance of the soil resistant bacteria to the four antibiotics The rate was as follows: (3) in the isolation and identification experiments of antibiotic resistant bacteria, 12 genera of doxycycline resistant bacteria were screened, 17 species were selected and the dominant strain was Streptomyces. 9 genera, 16 species of sulfonamillacine resistant bacteria were screened, and the dominant bacteria were bacillus. There are 9 genera and 16 species of Nofloxacin resistant bacteria and the dominant strain is microbacilli; 9 genera and 12 species of tylosin resistant bacteria are screened. The dominant strains are oligomeric and Pseudomonas, bacillus, Pseudomonas and Pseudomonas. (4) among the selected 16 resistance genes, Tet W, tetM, tetO, sul1, The detection frequencies of sul2, qepA, qnrB, qnrS, ermB, ermF were 100%. in quantitative experiments, and there was a large difference in the content of various resistance genes in different soil samples. Among the tetracycline resistant genes, the content of tetM was the highest, the content of sul2 in the sulfonamides resistance genes was higher, and the content of qepA was relatively high in the quinolone resistance genes, and the macrolides were found. Among the resistant genes, the content of ermF is relatively high. The content of the mobile element intI1 is relatively high. The total resistance genes are related to the quinolones of the sulfonamide macrolide tetracycline class. (5) the analysis of the physicochemical properties of the soil samples shows that the pH values of the soil samples are highly correlated with the number of enrofloxacin resistant bacteria, and the heavy metal content and quinolone content The resistance gene qnr showed very significant correlation. From the point of view of antibiotic content, the content of doxycycline in soil samples was significantly correlated with the content of tylosin and the content of sulfonamides resistance gene Sul, and was significantly related to the drug resistant and sulfadiazine resistant strains of doxycycline, and the content of tylosin and the sulfonamides resistance gene s UL also showed significant correlation. From the angle of removable components, the total intI of the mobile element in the soil samples was highly correlated with the sulfonamide resistance gene Sul and the macrolide resistance gene ERM.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S626;X53

【参考文献】