鸡粪中多重耐药细菌的分离鉴定及介导抗性基因水平转移元件的检测

发布时间：2018-07-03 21:53

本文选题：质粒 + 抗生素　；参考：《河南师范大学》2015年硕士论文

【摘要】：由于抗生素在禽畜养殖、临床医药等方面的大量使用,近年来出现了大量的多重耐药细菌,但是,多重耐药菌产生的机制并不完全清楚。研究表明,质粒经常携带一些可移动的元件(如转座子),捕捉不同的抗性基因介导抗生素抗性基因的扩散,这被认为是多重耐药细菌产生的主要机制之一。本论文围绕质粒在环境抗生素抗性基因水平传播中所起的作用进行以下几方面的研究:一、鸡粪中多重耐药细菌的分离鉴定及抗性检测。用含有庆大霉素、氯霉素、阿莫西林、头孢氨苄、环丙沙星、四环素、红霉素、卡那霉素这八种抗生素的培养基(LB培养基、SS培养基),我们从一规模化养鸡场的鸡粪中筛选出35株多重耐药细菌。这些多重耐药细菌分布在13个不同的属,以革兰氏阴性细菌为主导。在LB培养基中G-细菌与G+细菌分别占63%和37%,在SS培养基中G-细菌与G+细菌分别占87%和13%。这些细菌中对红霉素具有耐药性的最多,对阿莫西林具有耐药性的最少,它们分布于变形菌门、放线菌门以及厚壁菌门。抗生素抗性基因常与重金属抗性基因定位在同一个遗传元件上,我们将没有抗生素抗性的大肠杆菌与2株耐8种抗生素细菌进行对比实验。结果表明,这些多重耐药菌对Zn、Pb、Cu、Mn的抗性比没有抗生素抗性的细菌强。二、多重耐药细菌质粒的检测及遗传特性研究。我们提取了各多抗菌的抗性质粒并转化入模式菌株DH5a中。进一步检测发现,这些多抗菌中均有一大小在15kb左右的质粒被成功转入DH5a中。携带该质粒的DH5a可以在加有四环素、卡那霉素、头孢氨苄的LB固体培养基上生长,这表明该质粒上可能携带这三种抗生素抗性基因。测序结果显示,该质粒大小为14591bp,将其与NCBI中现有质粒进行同源性对比分析,发现其与一植物双元穿梭表达载体p ZGA22的相似度达到99%。通过生物信息学分析,我们发现其携带编码四环素类(tet R_C、tet R_N)、磷酸转移酶类(NTPII、NTPIII)抗性基因以及一个能同时控制多种抗生素抗性和重金属抗性的超家族域Glo_EDI_BRP。这些结果表明,该质粒很可能具有介导环境中多种耐药基因(包括多种重金属抗性基因)发生水平转移的能力。三、质粒的水平转移情况研究。首先,我们对不同多抗菌中15Kb大小质粒进行胶回收后酶切。酶切后的电泳图谱显示,这些质粒很可能是同一个质粒。接下来,我们根据该质粒的全序列设计12对引物,对其进行PCR扩增测序与拼接。结果显示,存在于干燥棒状杆菌Corynebacterium xerosis、河流漫游球菌Vagococcus fluvialis、大肠杆菌Escherichia coli、变形杆菌Proteus sp.、小鳟鱼大洋芽孢杆菌Oceanobacillus oncorhynchi、柠檬酸杆菌Citrobacter sp.中的15Kb大小质粒确为同一质粒。因此,这一15Kb大小质粒是环境中介导抗生素抗性基因发生水平转移的一个重要载体,它可能携带多种抗性基因在不同种属细菌中进行水平转移。
[Abstract]:Due to the extensive use of antibiotics in livestock breeding and clinical medicine, a large number of multidrug resistant bacteria have appeared in recent years, but the mechanism of multidrug resistance bacteria is not completely clear. Studies have shown that plasmids often carry some mobile elements (such as transposons) to capture different resistance genes to mediate the diffusion of antibiotic resistance genes, which is considered to be one of the main mechanisms for the production of multidrug resistant bacteria. In this paper, the role of plasmids in the horizontal transmission of environmental antibiotic resistance genes was studied in the following aspects: 1. Isolation, identification and detection of multidrug resistant bacteria in chicken manure. With gentamicin, chloramphenicol, amoxicillin, cefalexin, ciprofloxacin, tetracycline, erythromycin, Kanamycin (LB medium / SS medium) was used to isolate 35 strains of multidrug resistant bacteria from chicken dung of a large scale chicken farm. These multidrug resistant bacteria were distributed in 13 different genera, mainly Gram-negative bacteria. In LB medium, G- bacteria and G bacteria accounted for 63% and 37%, respectively. In SS medium, G- bacteria and G bacteria accounted for 87% and 13%, respectively. Among these bacteria, the most resistant to erythromycin and the least to amoxicillin are found in Proteus, actinomycetes and phylum thunbergiformis. Antibiotic resistance genes are often located on the same genetic element as heavy metal resistance genes. Two strains of Escherichia coli with no antibiotic resistance were compared with two strains of antibiotic resistant bacteria. The results showed that the resistance of these multidrug resistant bacteria to ZnPbCU mn was stronger than that of bacteria with no antibiotic resistance. Second, the detection and genetic characteristics of multidrug resistant bacteria plasmids. We extracted various antimicrobial resistant plasmids and transformed them into model strain DH 5a. Further detection showed that one of the plasmids about 15kb was successfully transferred into DH 5a. DH 5a carrying the plasmid could grow on LB solid medium containing tetracycline, kanamycin and cefalexin, which suggested that the plasmid might carry these three antibiotic resistance genes. The results of sequencing showed that the size of the plasmid was 14591bp.The homology analysis between the plasmid and the existing plasmid in NCBI showed that the similarity between the plasmid and a plant shuttle expression vector pZGA22 was 99%. By bioinformatics analysis, we found that it carries the tet (tet), phosphotransferases (NTPII), NTPIII resistance genes and a superfamily domain GloEDI _ (BRP), which can control both antibiotic resistance and heavy metal resistance. These results suggest that the plasmid may be capable of mediating horizontal transfer of many drug resistant genes (including multiple heavy metal resistance genes) in the environment. Third, the horizontal transfer of plasmids. Firstly, 15 KB plasmids of different antimicrobial species were recovered by gel and digested by enzyme. The electrophoretic patterns of the digested plasmids showed that the plasmids were probably the same plasmids. Then, we designed 12 pairs of primers according to the whole sequence of the plasmid, sequenced and sequenced them by PCR. The results showed that it existed in Corynebacterium xerosis, Vagococcus fluvialisi, Escherichia coli, Proteus sp., Oceanobacillus oncorhynchiand Citrobacter sp. The size of 15 kb plasmids was the same. Therefore, this 15Kb plasmid is an important vector that mediates the horizontal transfer of antibiotic resistance genes in the environment, and it may carry a variety of resistance genes for horizontal transfer in different species of bacteria.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172;Q78

【参考文献】