本文選題：菜地土壤 + 抗生素�。� 參考：《山東農(nóng)業(yè)大學》2017年碩士論文

【摘要】：抗生素作為20世紀最重要的發(fā)現(xiàn)之一,在保障人類及動物健康以及促進畜禽養(yǎng)殖業(yè)發(fā)展方面起著不可替代的作用。然而,由于抗生素的誤用和濫用,使得大量的抗生素以原藥或代謝物的形式隨人或動物體的排泄而進入環(huán)境,導致環(huán)境中,特別是土壤環(huán)境和水環(huán)境中存在大量抗生素殘留,進而增加了土壤或水環(huán)境中微生物的抗性壓力,加速了大量抗性微生物的產(chǎn)生。抗性微生物對抗生素產(chǎn)生耐藥性的主要原因是其攜帶有各種抗生素抗性基因,因此,抗生素的大量使用直接或間接增加了環(huán)境中抗性基因的含量。設施菜地是使用畜禽糞便較為頻繁的場所,因此,研究設施菜地土壤中抗生素含量、耐藥菌數(shù)量以及抗性基因的含量是十分必要的。本研究采集了山東濰坊、泰安、濱州、東營四個地區(qū)長期使用糞肥的9個典型設施菜地土壤,研究了設施菜地土壤中抗生素及抗性基因含量等。本實驗選取了四類抗生素進行實驗,利用固相萃取(Solid Phase Extraction,SPE)以及高效液相色譜(High Performance Liquid Chromatography,HPLC)檢測的方法,檢測了土壤樣品中三種抗生素的含量;另外,對土樣中含有的可培養(yǎng)細菌進行了數(shù)量的統(tǒng)計及菌種的鑒定;選取了五類共12種抗性基因和可移動遺傳元件(MGE)利用real-time qPCR定量檢測的方法對其豐度進行了測定。最后,將抗性基因的含量與土樣理化性質(zhì)、抗生素含量、耐藥菌的數(shù)量以及MGE的含量進行綜合分析。該論文的主要研究結果如下:(1)在土壤樣品抗生素含量檢測實驗中,強力霉素在所有土壤樣品中均有檢出,濃度范圍在52.37μg/kg~428.16μg/kg之間;磺胺二甲嘧啶除了在土樣S4中沒有檢出外,其他土樣中均有檢出,濃度范圍在14.12μg/kg~81.31μg/kg之間;泰樂菌素只在S0、S2、S3、S4和S8這5個土壤樣品中有檢出,濃度范圍為70.01μg/kg~482.36μg/kg。(2)在可培養(yǎng)抗生素耐藥菌實驗中,土樣中的耐藥菌對抗生素的耐藥性因抗生素種類和濃度的不同而在數(shù)量上呈現(xiàn)出較大差異。在未使用過糞肥的土樣S0中,細菌對抗生素仍有一定的抗性存在,長期使用糞肥的菜地土樣S1~S9耐藥率要高于土樣S0。隨著抗生素濃度的增加,細菌耐藥率逐漸降低。土壤耐藥菌對四種抗生素的耐藥率大小依次為:磺胺二甲嘧啶泰樂菌素恩諾沙星強力霉素。(3)在抗生素耐藥菌分離鑒定實驗中,共篩選出強力霉素耐藥菌12個屬,17個種,優(yōu)勢菌株為鏈霉菌屬;篩選出磺胺二甲嘧啶耐藥菌9個屬,16個種,優(yōu)勢耐藥菌為芽孢桿菌屬;篩選出恩諾沙星耐藥菌9個屬,16個種,優(yōu)勢菌株為微桿菌屬;篩選出泰樂菌素耐藥菌9個屬,12個種,優(yōu)勢菌株為寡養(yǎng)單胞菌屬和假單胞菌屬。芽孢桿菌屬、金黃桿菌屬和假單胞菌屬屬于多重耐藥菌。(4)在選取的五類共16種抗性基因中,tet W、tetM、tetO、sul1、sul2、qepA、qnrB、qnrS、ermB、ermF的檢出頻率均為100%。在定量實驗中,各種抗性基因在不同土樣中的含量存在較大差異。四環(huán)素類抗性基因中,tetM的含量最高,磺胺類抗性基因中sul2的含量較高,喹諾酮類抗性基因中,qepA的含量相對較高,大環(huán)內(nèi)酯類抗性基因中,ermF的含量相對較高。可移動元件intI1的含量相對較高。各抗性基因總量關系為:磺胺類大環(huán)內(nèi)酯類四環(huán)素類喹諾酮類。(5)從土樣理化性質(zhì)方面分析,土樣pH值與恩諾沙星耐藥菌數(shù)量之間呈現(xiàn)極顯著相關,重金屬含量與喹諾酮類抗性基因qnr呈現(xiàn)極顯著相關;從抗生素含量角度分析,土樣中強力霉素含量與泰樂菌素的含量及磺胺類抗性基因的含量sul呈現(xiàn)極顯著相關,與強力霉素耐藥菌及磺胺二甲嘧啶耐藥菌呈現(xiàn)顯著相關,泰樂菌素的含量與磺胺類抗性基因sul之間也呈現(xiàn)出極顯著相關;從可移動元件角度分析,土樣中可移動元件總量intI與磺胺類抗性基因sul及大環(huán)內(nèi)酯類抗性基因erm呈現(xiàn)極顯著相關。
[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.

【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S626;X53

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