南京地区临床分离大肠杆菌耐药性基因多样性及相关特性的研究

发布时间：2018-08-27 20:15

【摘要】： 细菌感染曾严重威胁着人类的生命和健康。自Fleiming发现青霉素后60余年来,人们不断地从微生物次级代谢产物中提取出众多的抗生素,并开发出半合成抗生素。近年来,抗生素药物的广泛和不合理应用所形成的强选择压力使细菌耐药性逐年上升,特别是多重耐药菌株的出现和耐药性的快速传播已经成为临床感染性疾病治疗的难题。细菌的耐药性可由染色体或质粒上相关耐药性基因介导,耐药性质粒可通过接合方式在不同菌种之间传递,使受体细菌成为耐药性菌株,这是细菌获得耐药性的主要方式。本实验将从接合基因水平转移角度研究南京临床大肠杆菌耐药性产生机制及相关基因的多样性。选取2008年3月到8月南京军区总院临床分离培养的200株大肠杆菌,采用平板涂布方法测定对8种不同抗生素耐药情况,结果显示94%的分离菌株具有超过4种抗生素耐药性,未得到不耐受或仅耐受1种抗生素菌株。其中超过90%的菌株对Amp, Nal耐药,Chi, Spe耐药率相对较低,但也分别超过了30%和50%。接合实验来研究耐药基因的水平情况,筛选其中KmS或StrS的菌株75株,通过与SM10λpir (KmR)或Bw20676 (StrR)接合实验发现,大部分菌株耐药基因可通过接合方式发生水平转移,其中Amp, Tet, Nal, Str耐药基因发生水平转移概率较Chl, Spe,Gm高,分别占可接合菌株的91.69%,54.70%,53.08%,33.77%。并且多种不同的耐药基因可转移至同一受体菌中。对可发生水平转移菌株,其耐药基因水平转移方式通过电击转化实验来确定,得到的接合子提质粒后电击转化实验发现,90%以上Amp耐药基因通过质粒方式转移,其它的Tc, Str耐药基因也部分通过质粒方式转移,其比率为可发生水平转移菌株的20.62%和5.52%。对可同时转移多种耐药基因至同一受体菌的转化子进一步实验发现,其多种耐药基因可同时携带于同一质粒,或多个不同质粒分别携带不同的耐药性。并在实验中发现一株同时携带有Amp, Tet, Chl, Str多种耐药性质粒菌株。由于大部分的Amp耐药基因可通过质粒方式转移,实验设计通过PCR的方法检测菌株TEM型,SHV型及CTX-M型β-内酰胺酶基因分型及耐药基因携带情况。结果显示我们研究的南京地区大部分大肠杆菌对氨苄青霉素耐药的机制主要是由TEM和CTX-M型β-内酰胺酶的产生,对200株大肠杆菌PCR发现,其中TEM, CTX-M检出率分别为36.5%和35.5%。SHV型PCR扩增未发现阳性标本。对耐受Amp抗生素程度不同的菌株序列比对发现,其基因型相同,推测其耐药程度与β-内酰胺酶表达量有关。
[Abstract]:Bacterial infection has been a serious threat to human life and health. Since the discovery of penicillin by Fleiming for more than 60 years, many antibiotics have been extracted from microbial secondary metabolites and semi-synthetic antibiotics have been developed. In recent years, the strong selective pressure caused by the extensive and irrational use of antibiotic drugs has led to the increase of bacterial resistance year by year, especially the emergence of multidrug resistant strains and the rapid spread of drug resistance, which has become a difficult problem in the treatment of clinical infectious diseases. Drug resistance of bacteria can be mediated by related genes on chromosomes or plasmids. Drug resistant plasmids can be transferred between different strains by conjugation, which makes the receptor bacteria become resistant strains, which is the main way for bacteria to obtain drug resistance. In this study, the mechanism of drug resistance and the diversity of related genes in clinical Escherichia coli in Nanjing were studied from the perspective of horizontal transfer of conjugate genes. From March to August 2008, 200 strains of Escherichia coli were isolated and cultured in Nanjing military region General Hospital, and the resistance to 8 different antibiotics was determined by plate coating method. The results showed that 94% of the isolates had more than 4 antibiotic resistance. No intolerance or only one antibiotic strain was obtained. More than 90% of the strains were resistant to Amp, Nal and the rate of Spe resistance was relatively low, but more than 30% and 50%, respectively. The level of drug resistance gene was studied by conjugation test. 75 strains of KmS or StrS were screened. It was found that most of the drug resistance genes could be transferred horizontally by conjugation with SM10 位 pir (KmR) or Bw20676 (StrR). The probability of horizontal transfer of Amp, Tet, Nal, Str resistance gene was higher than that of Chl, Spe,Gm, accounting for 91.69% of the conjugable strains, 54.70% and 53.08%, 33.77% respectively. And many different drug resistance genes can be transferred to the same receptor bacteria. The way of horizontal transfer of drug resistance genes was determined by electroporation experiment. After the plasmids were extracted from the conjugate, it was found that more than 90% of Amp resistance genes were transferred by plasmid. Other Tc, Str resistance genes were also partially transferred by plasmid, the rates of which were 20.62% and 5.52% of those that could be transfered horizontally. Further experiments on transformants that can transfer multiple drug resistance genes to the same receptor bacteria at the same time showed that their multidrug resistance genes could be carried in the same plasmid at the same time, or different plasmids could carry different drug resistance at the same time. One strain carrying multiple Amp, Tet, Chl, Str resistance plasmids was also found in the experiment. Because most of the Amp resistant genes can be transferred by plasmid, PCR was designed to detect the genotyping of TEM and CTX-M 尾 -lactamases and the carrying of drug resistance genes. The results showed that the mechanism of ampicillin resistance of most Escherichia coli in Nanjing was mainly produced by TEM and CTX-M 尾 -lactamases, and 200 strains of Escherichia coli PCR were found. The positive rate of TEM, CTX-M was 36.5% and 35.5%.SHV type PCR was not found. It was found that the genotypes of the strains with different antibiotic resistance to Amp were the same, and the degree of drug resistance was related to the expression of 尾 -lactamases.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

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