本文選題：質(zhì)粒介導(dǎo)的耐藥性 + 菌群結(jié)構(gòu)�。� 參考：《山東大學(xué)》2017年碩士論文

【摘要】：抗生素的發(fā)現(xiàn)與應(yīng)用給人們抗感染治療、醫(yī)療健康帶來了前所未有的光明前景,然而耐藥細(xì)菌的出現(xiàn)又給我們的健康蒙上了一層濃重的陰影,科學(xué)家們研究出不同種類、不斷更新的抗菌方略,細(xì)菌卻能一個(gè)一個(gè)地從容應(yīng)對(duì)。自1929年首例青霉素被發(fā)現(xiàn)之后,在這不到100年時(shí)間里,抗生素的發(fā)展經(jīng)歷了顛覆性的變化,而其耐藥性也在我們的生活中顯現(xiàn)地越來越嚴(yán)重。喹諾酮類和β-內(nèi)酰胺類抗生素作為我們平時(shí)抗感染治療的主要用藥,目前的應(yīng)用已經(jīng)不光局限在人體身上,畜牧業(yè)、家禽飼養(yǎng)產(chǎn)業(yè)、漁業(yè)上抗生素的應(yīng)用已經(jīng)遠(yuǎn)遠(yuǎn)超過了人類本身。據(jù)統(tǒng)計(jì),2013年中國(guó)每天抗生素的千人使用量為157噸,這是英國(guó)(27.4噸)的5.7倍,是美國(guó)(28.8噸)的5.5倍,而在動(dòng)物方面,2013年中國(guó)共投入84240噸抗生素,是英國(guó)(420噸)的整整200倍,是美國(guó)(14600噸)的5.8倍。而近80%的抗生素在體內(nèi)并不能完全降解,最后以母體化合物或者代謝產(chǎn)物以糞便或尿液的形式排放到水環(huán)境中,這對(duì)我們耐以生存的環(huán)境帶來了莫大的災(zāi)難,因此對(duì)抗生素及耐藥性的研究在目前的形勢(shì)下顯得極其迫切。質(zhì)粒介導(dǎo)的耐藥性以其自身易傳播、自身耐藥水平較低卻能選擇出高水平耐藥性的特點(diǎn)而在耐藥性的研究中扮演著極其重要的角色,最近幾來,PMQR與β-內(nèi)酰胺類抗生素耐藥性越來越多地被各國(guó)科學(xué)家研究,但是大多研究都局限于對(duì)篩選到的特定菌株進(jìn)行耐藥基因檢測(cè)或者其他耐藥性的研究,而不能從宏觀總體的角度去把握和評(píng)估污水環(huán)境中的耐藥性流行程度,本文以污水中的總細(xì)菌的宏基因組DNA為直接實(shí)驗(yàn)材料,對(duì)濟(jì)南地區(qū)的四個(gè)污水采樣點(diǎn)(齊魯醫(yī)院、山東省中醫(yī)醫(yī)院、光大污水處理一廠、小清河)中的污水進(jìn)行連續(xù)一年的耐藥基因定量檢測(cè),耐藥基因包括質(zhì)粒介導(dǎo)的喹諾酮類耐藥(PMQR)基因qnrA,qnrB,qnrC,qurD,qnrS,qepA,oqxA,oqxB,aac-(6')-Ib-cr以及質(zhì)粒介導(dǎo)的β-內(nèi)憸胺類抗生素耐藥基因blaTEM,blaCMY,blaCTX-M,blaDHA,blaSHV,并對(duì)其中三個(gè)月的樣本DNA進(jìn)行基于16S rDNA高通量測(cè)序的菌群結(jié)構(gòu)分析,研究耐藥基因與各類細(xì)菌在不同時(shí)間里變化的聯(lián)系。結(jié)果表明,省中醫(yī)醫(yī)院的污水中16S rDNA平均拷貝數(shù)為5.49 × 10~8 copies/mL,分別低于齊魯醫(yī)院、光大污水處理廠、小清河采樣點(diǎn)4.70,7.44,15.77倍,而小清河樣本中的生物量是最多的,接下來是其余兩地。齊魯醫(yī)院污水樣本中的耐藥基因含量為最高,尤其是aac-(6')-Ib-cr,oqxA,oqxB,blaCTX-M基因含量遙遙領(lǐng)先于其他采樣點(diǎn)。光大水務(wù)公司經(jīng)過處理的污水中qnrB,qnrS和qnrD,qepA,blaCMY基因的含量都高于其他采樣點(diǎn)。而小清河河水作為里我們?nèi)粘Ｉ钭罱咏乃到y(tǒng),其耐藥基因豐度處于較高的水平。對(duì)比不同的耐藥基因,aac-(6')-Ib-cr基因是所有耐藥基因中含量最高的,至少比其他耐藥基因高出10倍以上,而在β-內(nèi)酰胺酶基因中,blaTEM基因的含量為最高。通過對(duì)各采樣點(diǎn)中各耐藥基因之間的相關(guān)性分析可知,在自然水體中,β-內(nèi)酰胺類耐藥基因與PMQR基因之間的相關(guān)性表現(xiàn)的比較明顯,而當(dāng)污水系統(tǒng)經(jīng)受不同程度的人為處理后,這兩種耐藥基因之間的相關(guān)性變?nèi)?這在其余三個(gè)污水采樣點(diǎn)的結(jié)果中都有體現(xiàn)。在兩個(gè)不同性質(zhì)的醫(yī)院污水系統(tǒng)中,耐藥基因之間的相關(guān)性表現(xiàn)的較為一致,即PMQR基因內(nèi)部的相關(guān)性表現(xiàn)的較強(qiáng),而作為β-內(nèi)酰胺類耐藥基因的主要代表blaTEM與PMQR基因的相關(guān)性變?nèi)?但仍有含量較少的其他β-內(nèi)酰胺類耐藥基因與PMQR基因有著千絲萬縷的聯(lián)系。在受人工處理最為嚴(yán)重的光大污水處理廠采樣點(diǎn),各耐藥基因之間的聯(lián)系表現(xiàn)地雜亂無章,并沒有表現(xiàn)出一致的規(guī)律性。耐藥基因與菌群結(jié)構(gòu)之間的相關(guān)性分析結(jié)果展示了各類細(xì)菌均與耐藥基因之間表現(xiàn)出著顯著的相關(guān)性。變形菌門(Proteobacteria)、擬桿菌門(Bacteroidetes)、放線菌門(Actinobacteria)、撫微菌門(Verrucomicrobia)、藍(lán)菌門(Cyanobacteria)與耐藥基因之間都存在顯著的相關(guān)性,尤其在前兩個(gè)門單元中,耐藥基因與變形菌門中的α,β,γ,δ變形菌綱,與擬桿菌門中的擬桿菌綱(Bacteroidia)、黃桿菌綱(Flavobacteria)、鞘脂桿菌綱(Sphingobacteriia)都有顯著的相關(guān)性,不光是在門水平,在下屬的綱、目、科、屬水平均體現(xiàn)出了這種關(guān)系。與β-內(nèi)酰胺類耐藥基因顯著相關(guān)的細(xì)菌主要有α,β,δ變形菌綱,γ變形菌綱的氣生單胞菌屬和菌萊茵海默氏菌分支,擬桿菌門的黃桿菌目和鞘脂桿菌目分支,以及弗蘭克菌目細(xì)菌。與PMQR基因qnr表現(xiàn)出顯著相關(guān)的菌類有:α,β變形菌綱,弗蘭克菌目以及擬桿菌綱。與外排泵基因表現(xiàn)出顯著相關(guān)性的菌類有:δ變形菌綱,β變形菌綱的伯克霍爾德氏菌目,γ變形菌綱的黃單胞菌科分支,以及弗蘭克菌目細(xì)菌。
[Abstract]:The discovery and application of antibiotics has brought unprecedented bright prospects for anti infection treatment and medical health. However, the emergence of drug resistant bacteria has cast a heavy shadow on our health. Scientists have studied different types, constantly updated antibacterial strategies, and the fine bacteria can respond one by one. The first case in 1929. After the discovery of penicillin, the development of antibiotics has undergone a subversive change in less than 100 years, and its resistance has become more and more serious in our lives. The quinolones and beta lactam antibiotics are the main use of our usual anti infection treatment, and the current application is not limited to the body. The use of antibiotics in animal husbandry, poultry raising industry, and the application of antibiotics in fisheries has far exceeded the human itself. According to statistics, in 2013, China used 157 tons of antibiotics per day, 5.7 times as much as the UK (27.4 tons) and 5.5 times the United States (28.8 tons). In the animal square, China invested 84240 tons of antibiotics in 2013, and was the whole of Britain (420 tons). 200 times as many times as 5.8 times that of the United States (14600 tons). And nearly 80% of the antibiotics are not completely degraded in the body. Finally, mother compounds or metabolites are discharged into the water environment in the form of feces or urine. This brings a great disaster to our survival environment. The current situation of the study on antibiotics and resistance is in the current situation. The resistance of PMQR and beta lactam has been studied by scientists in various countries, but most recently, more and more researchers have studied the drug resistance of beta lactam antibiotics, but most of them have been studied by various countries. The study is limited to the screening of selected strains for resistance gene detection or other drug resistance studies, but not from a macro perspective to grasp and evaluate the prevalence of drug resistance in the sewage environment. This paper uses the macrogenomic DNA of the total bacteria in the sewage as the direct testing material for four sewage sampling in Ji'nan area. Point (Qilu Hospital, Shandong traditional Chinese medicine hospital, Guangda sewage treatment plant, Xiaoqing River) for a continuous year of drug resistance gene quantitative detection. Resistance genes include plasmid mediated quinolone resistance (PMQR) gene qnrA, qnrB, qnrC, qurD, qnrS, qepA, oqxA, oqxB, aac- (6') -Ib-cr, and plasmid mediated beta amine antibiotics resistance The drug gene blaTEM, blaCMY, blaCTX-M, blaDHA, blaSHV, and the bacterial group structure analysis based on 16S rDNA high throughput sequencing for three months' sample DNA, to study the relationship between the resistance gene and all kinds of bacteria at different time. The results showed that the average copy number of 16S rDNA in the sewage of the provincial hospital of traditional Chinese medicine was 5.49 x 10~8 copies/mL, respectively. Lower than the Qilu Hospital, the Guangda sewage treatment plant, the Xiaoqing River sampling point 4.70,7.44,15.77 times, and the Xiaoqing River samples are the most, followed by the rest of the other two. The drug resistance gene content in the sewage samples is the highest, especially the aac- (6') -Ib-cr, oqxA, oqxB, blaCTX-M is far ahead of the other sampling points. The contents of qnrB, qnrS, qnrD, qepA, blaCMY genes in the treated sewage of the Water Service Company were all higher than those of the other sampling points. While the Xiaoqing River river was the closest water system in our daily life, its resistance gene abundances were at a higher level. The aac- (6') -Ib-cr gene was the highest in all drug resistance genes compared to the different resistance genes. In the beta lactamase gene, the blaTEM gene is the highest in the beta lactamase gene, and the correlation between the beta lactam resistance gene and the PMQR gene in the natural water body is more obvious, while in the sewage system, the blaTEM gene is the highest in the beta lactamase gene, and the correlation between the beta lactam resistance gene and the gene is more obvious in the natural water body. The correlation between the two resistant genes was weakened after different levels of human treatment, which was reflected in the results of the other three sewage sampling points. In the two different nature hospital sewage systems, the correlation between the resistance genes was more consistent, that is, the correlation between the PMQR genes was stronger and was beta - as beta. The correlation between the main representative of the drug resistance gene of the lactam blaTEM and the PMQR gene is weak, but the other beta lactam resistance genes, which are still less content, are closely related to the PMQR gene. The correlation analysis showed the correlation between the resistance genes and the structure of the bacteria group. The results showed that all kinds of bacteria showed significant correlation with the drug resistant genes. Deformable bacteria gate (Proteobacteria), Bacteroidetes, Actinobacteria, Verrucomicrobia, Cyanobact Eria) has a significant correlation with the resistance genes, especially in the first two gate units. The resistance genes and the alpha, beta, gamma, and delta strains in the Proteus doors have significant correlation with the Bacteroidia, Flavobacteria and Sphingobacteriia, not only at the gate level. This relationship is reflected in the class, order, family and level of the subordinates. The bacteria that are significantly related to the beta lactam resistance genes are alpha, beta, Delta, Aeromonas, and the branch of the bacteria Rhine, the Xanthomonas and the branch of the Pseudomonas aeruginosa, and the Frank bacteria. And the PMQR base. The bacteria that were significantly related to qnr were alpha, beta Proteus, Frank, and Pseudomonas. The bacteria that showed significant correlation with the efflux pump genes were: Delta strain, Burke Holder of beta Proteus, the branch of Xanthomonas in the gamma deforminosa, and the bacteria of the order Frank.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X131.2;X703

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