水环境中质粒介导的喹诺酮和β-内酰胺类耐药性研究

发布时间：2018-05-05 07:58

本文选题：质粒介导的耐药性 + 菌群结构　；参考：《山东大学》2017年硕士论文

【摘要】：抗生素的发现与应用给人们抗感染治疗、医疗健康带来了前所未有的光明前景,然而耐药细菌的出现又给我们的健康蒙上了一层浓重的阴影,科学家们研究出不同种类、不断更新的抗菌方略,细菌却能一个一个地从容应对。自1929年首例青霉素被发现之后,在这不到100年时间里,抗生素的发展经历了颠覆性的变化,而其耐药性也在我们的生活中显现地越来越严重。喹诺酮类和β-内酰胺类抗生素作为我们平时抗感染治疗的主要用药,目前的应用已经不光局限在人体身上,畜牧业、家禽饲养产业、渔业上抗生素的应用已经远远超过了人类本身。据统计,2013年中国每天抗生素的千人使用量为157吨,这是英国(27.4吨)的5.7倍,是美国(28.8吨)的5.5倍,而在动物方面,2013年中国共投入84240吨抗生素,是英国(420吨)的整整200倍,是美国(14600吨)的5.8倍。而近80%的抗生素在体内并不能完全降解,最后以母体化合物或者代谢产物以粪便或尿液的形式排放到水环境中,这对我们耐以生存的环境带来了莫大的灾难,因此对抗生素及耐药性的研究在目前的形势下显得极其迫切。质粒介导的耐药性以其自身易传播、自身耐药水平较低却能选择出高水平耐药性的特点而在耐药性的研究中扮演着极其重要的角色,最近几来,PMQR与β-内酰胺类抗生素耐药性越来越多地被各国科学家研究,但是大多研究都局限于对筛选到的特定菌株进行耐药基因检测或者其他耐药性的研究,而不能从宏观总体的角度去把握和评估污水环境中的耐药性流行程度,本文以污水中的总细菌的宏基因组DNA为直接实验材料,对济南地区的四个污水采样点(齐鲁医院、山东省中医医院、光大污水处理一厂、小清河)中的污水进行连续一年的耐药基因定量检测,耐药基因包括质粒介导的喹诺酮类耐药(PMQR)基因qnrA,qnrB,qnrC,qurD,qnrS,qepA,oqxA,oqxB,aac-(6')-Ib-cr以及质粒介导的β-内憸胺类抗生素耐药基因blaTEM,blaCMY,blaCTX-M,blaDHA,blaSHV,并对其中三个月的样本DNA进行基于16S rDNA高通量测序的菌群结构分析,研究耐药基因与各类细菌在不同时间里变化的联系。结果表明,省中医医院的污水中16S rDNA平均拷贝数为5.49 × 10~8 copies/mL,分别低于齐鲁医院、光大污水处理厂、小清河采样点4.70,7.44,15.77倍,而小清河样本中的生物量是最多的,接下来是其余两地。齐鲁医院污水样本中的耐药基因含量为最高,尤其是aac-(6')-Ib-cr,oqxA,oqxB,blaCTX-M基因含量遥遥领先于其他采样点。光大水务公司经过处理的污水中qnrB,qnrS和qnrD,qepA,blaCMY基因的含量都高于其他采样点。而小清河河水作为里我们日常生活最接近的水系统,其耐药基因丰度处于较高的水平。对比不同的耐药基因,aac-(6')-Ib-cr基因是所有耐药基因中含量最高的,至少比其他耐药基因高出10倍以上,而在β-内酰胺酶基因中,blaTEM基因的含量为最高。通过对各采样点中各耐药基因之间的相关性分析可知,在自然水体中,β-内酰胺类耐药基因与PMQR基因之间的相关性表现的比较明显,而当污水系统经受不同程度的人为处理后,这两种耐药基因之间的相关性变弱,这在其余三个污水采样点的结果中都有体现。在两个不同性质的医院污水系统中,耐药基因之间的相关性表现的较为一致,即PMQR基因内部的相关性表现的较强,而作为β-内酰胺类耐药基因的主要代表blaTEM与PMQR基因的相关性变弱,但仍有含量较少的其他β-内酰胺类耐药基因与PMQR基因有着千丝万缕的联系。在受人工处理最为严重的光大污水处理厂采样点,各耐药基因之间的联系表现地杂乱无章,并没有表现出一致的规律性。耐药基因与菌群结构之间的相关性分析结果展示了各类细菌均与耐药基因之间表现出着显著的相关性。变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)、抚微菌门(Verrucomicrobia)、蓝菌门(Cyanobacteria)与耐药基因之间都存在显著的相关性,尤其在前两个门单元中,耐药基因与变形菌门中的α,β,γ,δ变形菌纲,与拟杆菌门中的拟杆菌纲(Bacteroidia)、黄杆菌纲(Flavobacteria)、鞘脂杆菌纲(Sphingobacteriia)都有显著的相关性,不光是在门水平,在下属的纲、目、科、属水平均体现出了这种关系。与β-内酰胺类耐药基因显著相关的细菌主要有α,β,δ变形菌纲,γ变形菌纲的气生单胞菌属和菌莱茵海默氏菌分支,拟杆菌门的黄杆菌目和鞘脂杆菌目分支,以及弗兰克菌目细菌。与PMQR基因qnr表现出显著相关的菌类有:α,β变形菌纲,弗兰克菌目以及拟杆菌纲。与外排泵基因表现出显著相关性的菌类有:δ变形菌纲,β变形菌纲的伯克霍尔德氏菌目,γ变形菌纲的黄单胞菌科分支,以及弗兰克菌目细菌。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X131.2;X703

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