昆明地区产ESBLs肠杆菌科细菌对喹诺酮类质粒介导的耐药机制研究

发布时间：2018-03-14 07:39

本文选题：肠杆菌科细菌　切入点：质粒介导　出处：《昆明理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：喹诺酮类药物(Quinolones)是一类人工合成的广谱、强效抗菌药物,是目前临床应用最广泛的抗菌药物。肠杆菌科细菌(Enterobacteriaceae bacteria)是临床最常见的分离菌,其中又以大肠埃希菌和肺炎克雷伯菌为主,近年来肠杆菌科细菌对喹诺酮类的耐药性越来越严重,常常导致临床治疗延迟或失败。之前的观点认为肠杆菌科的喹诺酮耐药是由染色体介导的,包括细胞膜通透性改变、主动外排系统和靶基因的改变三种途径。后来研究发现了质粒介导的喹诺酮类耐药(plasmid-mediated quinolone resistance,PMQR)途径,这很好的解释了耐药基因水平转移的问题。质粒介导的喹诺酮类的耐药基因,包括qnr基因家族和aac(6')-Ib-cr基因等,常与超广谱 β-内酰胺酶(extended-spectrum β-lactamases.ESBLs)协同传播,且这些基因的分布有地域性差异,研究本地区质粒介导喹诺酮类耐药机制研究对临床的经验性用药和制定适宜的院感控制措施显得尤为重要。本文的主要研究工作是昆明地区产超广谱β-内酰胺酶的肠杆菌科大肠埃希氏菌和肺炎克雷伯菌质粒介导的喹诺酮类耐药的机制,包括以下三个方面:(1)检测PMQR的流行基因2015年5月—2016年7月从云南省第一人民医院不同科室、不同年龄段病人,不同感染部位分离出的50株喹诺酮类耐药的产ESBLs的肠杆菌科细菌,其中包括26株大肠埃希氏菌和24株肺炎克雷伯菌。设计特异的质粒耐药基因引物,对实验菌株提取基因组后,利用PCR技术检测不同菌株质粒耐药基因的携带情况,结果发现在大肠埃希氏菌和肺炎克雷伯菌中流行的耐药基因有所差异,大肠埃希氏菌最流行的是qnrB基因,检出率高达88.4%,其次是aac(6')-Ib-cr基因,检出率为61.5%,qnrD和qnrS基因都只检测到了一例。肺炎克雷伯菌中都携带qnrB、qnrS和aac(6')-Ib-cr基因,检出率都为100%。我们还发现在同一株菌中可能有携带几种不同的质粒耐药基因,大肠埃希氏菌中42.3%菌株携带有两种耐药基因,7.6%的菌株携带有三种耐药基因。所有的肺炎克雷伯菌都携带了三种相同质粒耐药基因。(2)多重耐药性研究对携带有PMQR基因的肠杆菌科细菌进行质粒接合实验,大肠埃希氏菌全部接合成功,肺炎克雷伯菌除两株外,也全部接合成功。这说明携带有喹诺酮耐药基因的质粒具有很高的水平转移能力,没有接合成功的样品可能是因为携带耐药基因的质粒缺乏接合相关的转移操纵子。用E-test的方法检测喹诺酮类、β-内酰胺类和氨基糖苷类药物对接合供体菌、受体菌、接合子的MIC值。我们发现供体菌和接合子对喹诺酮类药物都具有耐药性,这间接说明耐药基因是位于质粒上的。绝大部分的供体菌和接合子对氨基糖苷类药物有耐药作用,只有一株E.coli供体菌有耐药作用,而其接合子对药物敏感,说明其耐药基因位于基因组上。供体菌和接合子对除碳青霉烯类外大多数的β-内酰胺药物耐药,说明质粒上同样携带了这些药物的耐药基因。综合这些数据我们可以得出携带有喹诺酮类药物耐药基因的质粒上,同样携带了氨基糖苷类和β-内酰胺类药物的耐药基因,造成了肠杆菌科细菌的多重耐药作用。(3)肠杆菌科细菌同源性分析用脉冲电泳技术分析携带有PMQR基因的肠杆菌科细菌的同源性,有14株大肠埃希氏菌的同源性为85%以上判定为相同菌株来源,占到了总数的58.3%(14/24),其中有一组两个菌株的同源性为100%;肺炎克雷伯菌同源性达到85%以上的菌株有16株,占到了总数的66.6%(16/24),其中有5组十株基因组条带完全一致是为近期传播的同一菌株,占比为41.6%(10/24),这五组菌株中有四组来自于重症监护室,说明在免疫力低下病人中,这种耐药菌传播更迅速。综合这些数据可以得出质粒介导的喹诺酮类耐药,在肺炎克雷伯菌中的流行爆发相对于大肠埃希氏菌更集中,耐药菌在免疫力低下病人中传播更迅速。综上所述,本研究发现在昆明地区临床分离肠杆菌科菌中最流行的PMQR基因是qnrB、qnrS和aac(6')-Ib-cr,且这些基因水平传播能力很强,携带有PMQR基因的质粒上同时携带有其他多种药物的耐药基因,同源性分析发现肺炎克雷伯菌的流行暴发相对于大肠埃希氏菌更集中,免疫力低下病人中传播更迅速,这些结果对本地区耐药基因库的建立,临床耐药菌的控制提供了实验依据。
[Abstract]:Quinolones (Quinolones) is a kind of synthetic broad-spectrum, potent antibacterial drugs, is currently the most widely used clinical antibiotics. Enterobacteriaceae (Enterobacteriaceae bacteria) is the most common clinical isolates, including Escherichia coli and Klebsiella pneumoniae, drug resistance and Enterobacteriaceae in recent years bacteria to quinolones is more serious, often leads to clinical treatment delay or failure. Before the view of quinolone resistant Enterobacteriaceae is mediated by the chromosome, including cell membrane permeability, three ways of active efflux system and target genes. Then the study found that the plasmid mediated quinolone resistance (plasmid-mediated quinolone resistance, PMQR), which is a good explanation of the horizontal transfer of antibiotic resistance genes. The plasmid mediated quinolone resistance genes, including qnr gene family And group AAC (6') -Ib-cr gene, often amides with extended spectrum beta enzyme (extended-spectrum beta -lactamases.ESBLs) collaborative communication, and regional differences in the distribution of these genes, the research region of plasmid mediated quinolone resistance mechanism is particularly important for clinical empiric therapy and formulating appropriate control measures is the hospital. The main research work of this paper is the mechanism of quinolone resistance in Kunming producing ESBLs Enterobacteriaceae Escherichia coli and Klebsiella pneumoniae plasmid mediated, including the following three aspects: (1) May 2015 - detection of PMQR genotype in July 2016 from the first people's Hospital of Yunnan province from different departments. Patients of different ages, 50 strains of quinolone resistance in different infection sites of the isolated ESBLs producing Enterobacteriaceae strains, including 26 strains of Escherichia coli and 24 strains of Klebsiella pneumonia Strain. Plasmid resistant gene specific primer design, the strain was extracted after detecting different plasmid carrying resistance genes by using PCR technology, the difference has popular resistance gene in Escherichia coli and Klebsiella pneumoniae, Escherichia coli and Salmonella is the most popular qnrB gene, detection rate up to 88.4%, followed by AAC (6') -Ib-cr gene, the detection rate was 61.5%, qnrD and qnrS genes were detected only one case. Klebsiella pneumoniae carried qnrB, qnrS and AAC (6') -Ib-cr gene, the detection rate is 100%. we also found in the same strains may there are several different plasmids carrying resistance genes, 42.3% strains of Escherichia coli carrying two resistant genes, 7.6% strains carrying three resistant genes. All Klebsiella pneumoniae had three different plasmid resistance genes. (2) multi drug resistance research To carry the Enterobacteriaceae PMQR gene plasmid conjugation experiments, Escherichia coli engages all successful, Klebsiella pneumoniae in two strains, all joined successfully. This shows that carrying the plasmid of quinolone resistance gene has a high level of ability to transfer, no joint success is possible because the plasmid carrying samples the lack of resistance genes related to quinolones. Conjugative transfer operon detection using E-test method, beta lactam and aminoglycoside drugs on the donor receptor bacteria, bacteria, zygote MIC value. We found that donor and TRANSCONJUGANT are resistant to quinolones, which indirectly indicated that the resistance gene is located on plasmids the vast majority of bacteria. The donor and TRANSCONJUGANT resistant to aminoglycosides, only a E.coli donor drug resistance, and the zygote is sensitive to medicine and instructions The resistance gene in genome. The donor and TRANSCONJUGANT of carbapenem most beta lactam drug resistance, plasmid that also carry resistance genes of these drugs. The combination of these data we can conclude that carrying the plasmid quinolone resistance gene, also carry aminoglycoside resistance gene and beta lactam drugs, caused by the multiple drug resistance of Enterobacteriaceae. (3) analysis with Enterobacteriaceae homology of PMQR gene by pulsed field gel electrophoresis, a bacterial homologue of Enterobacteriaceae, 14 strains of Escherichia coli was 85% homologous to the above decision for the same strain the source, accounted for 58.3% of the total (14/24), including a group of two strains was 100% homology; Klebsiella pneumoniae homology more than 85% strains 16 strains accounted for 66.6% of the total (16/24), the There are 5 groups of ten genomic bands identical for recent spread of the same strain, accounted for 41.6% (10/24), the five groups of strains in four group from the ICU, in immunocompromised patients, the resistant bacteria spread more quickly. These data can be obtained by quinolone resistance plasmid mediated, in Klebsiella pneumoniae in Escherichia coli outbreaks compared to the more concentrated, drug resistant bacteria in immunocompromised patients spread more quickly. In summary, this study found that the most popular PMQR gene in clinical isolates of Enterobacteriaceae in Kunming area is qnrB, qnrS and AAC (6') -Ib-cr. And these gene horizontal transmission ability is very strong, carrying the plasmid PMQR gene and carrying resistance genes in a variety of other drugs, the homology analysis showed that Klebsiella pneumoniae epidemic outbreaks of Escherichia coli with more concentrated immune These results provide an experimental basis for the establishment of drug-resistant gene banks in the region and the control of clinical drug resistant bacteria.

【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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