大肠埃希菌临床株质粒介导的喹诺酮类耐药机制的研究

发布时间：2018-01-09 09:36

  本文关键词：大肠埃希菌临床株质粒介导的喹诺酮类耐药机制的研究　出处：《天津医科大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： 大肠埃希菌 质粒介导 喹诺酮类耐药 qnr aac(6’)-Ib-cr β-内酰胺酶

【摘要】：研究目的：随着喹诺酮类抗菌药物的广泛应用,我国临床分离的大肠埃希菌对该类药物的耐药性明显上升。以往的观点认为喹诺酮耐药主要由于细菌染色体上药物靶蛋白的突变造成,近年来发现了质粒介导的喹诺酮耐药基因,包括qnr、aac(6’)-Ib-cr和qepA基因。这些基因虽然只能引起低水平的喹诺酮耐药,但它们可以通过质粒接合、转座等途径和其它常位于质粒上的耐药基因特别是超广谱p-内酰胺酶(ESBLs)基因一起快速传播给临床用药带来巨大威胁。本实验通过分析大肠埃希菌临床分离株的耐药情况,分子水平研究质粒介导的喹诺酮类耐药机制和探讨携带质粒介导的喹诺酮耐药基因的大肠埃希菌临床株感染的临床特征,从而为指导临床合理使用抗菌药物、减少细菌耐药性的出现或传播及新型抗菌药物的研制提供理论依据。 研究方法： 1、菌株收集、药敏试验：收集自天津医科大学第二医院2008年7月-2009年9月期间临床分离的大肠埃希菌共143株,经常规生化鉴定确认。剔除从同一病人同一部位分离的重复菌株。采用微量肉汤稀释法测定临床菌株对12种常用抗菌药物(哌拉西林、头孢氨噻肟、头孢哌酮、头孢他啶、头孢曲松、头孢吡肟、头孢哌酮/舒巴坦、阿米卡星、庆大霉素、左氧氟沙星、环丙沙星、氧氟沙星)的MIC值,分析耐药率。 2、质粒介导的喹诺酮耐药基因的检测：应用聚合酶链式反应(PCR)扩增环丙沙星耐药的大肠埃希菌临床株的qnrA、qnrB、qnrS、aac(6’)-Ib及qepA基因,对aac(6’)-Ib基因阳性菌株用Fok I酶切确认aac(6’)-Ib-cr基因。 3、接合实验验证-qnr的转移性,受体菌为E coli J53(Azide resistant),对接合子的选择采用含头孢噻肟(3mg/L)和叠氮化钠(100mg/L)的LB琼脂平皿,采用微量肉汤稀释法比较受体菌、供体菌及接合子的MIC值,包括8种常用抗菌药物。 4、ESBLs表型的筛选,分析其与质粒介导的喹诺酮类耐药基因的关系,采用PCR法检测携带质粒介导的喹诺酮类耐药基因的阳性菌株ESBLs基因型：包括SHV、CTX-M-1、CTX-M-2、CTX-M-9型超广谱p-内酰胺酶。 5、收集携带质粒介导的喹诺酮耐药基因的阳性菌株感染患者的临床资料,进行回顾性分析：包括基本资料、临床资料、感染相关资料及微生物相关资料。 结果： 1、143株大肠埃希菌临床株的药敏结果：对12种抗菌药物的耐药率为哌拉西林(70.63%),氧氟沙星(58.04%),头孢哌酮(55.24%),左氧氟沙星(54.54%),环丙沙星(52.45%),庆大霉素(51.05%),头孢吡肟(49.65%),头孢曲松(48.95%),头孢氨噻肟(47.55%),头孢他啶(35.66%),阿米卡星(12.59%),头孢哌酮/舒巴坦(6.29%),多重耐药菌株60株(41.96%)。 2、质粒介导喹诺酮耐药基因的检测：75株环丙沙星耐药的大肠埃希菌中共有18株(24%)携带qnr基因和(或)aac(6’)-Ib-cr基因,其中qnr和aac(6’)-Ib-cr检出率分别为6.7%和21.3%,未检出qnrA和qepA。 3、接合实验：5株qnr阳性菌株均接合传递成功,各种抗菌药物对接合子的MIC值较受体菌均有不同程度提高。 4、75株耐环丙沙星的大肠埃希菌临床株有39株(52%)产ESBLs,不携带qnr/aac(6’)-Ib-cr的菌株产酶率为36.84%,18株qnr/aac(6')-Ib-cr阳性的大肠埃希菌中均产ESBLs,主要以CTX-M-1型、CTX-M-9型、SHV型ESBLs为主。 5、阳性菌株的临床特征分析：阳性菌株对喹诺酮类和头孢菌素类药物高度耐药且主要来源于泌尿系感染,感染患者均为中老年人且绝大多数患者同时患有一种及其以上的基础疾病。 结论 1、大肠埃希菌是临床最常见的病原菌,我院临床分离的大肠埃希菌对多种抗菌药物的耐药率较高,尤其是头孢菌素和氟喹诺酮类抗生素。 2、天津地区临床存在qnr和aac(6’)-Ib-cr阳性菌株的流行。包含qnr和aac(6')-Ib-cr的菌株多为产ESBLs的临床分离菌株,且qnr基因可在患者体外水平传播。 3、携带质粒介导的喹诺酮耐药基因的大肠埃希菌多为多重耐药菌,感染患者多为有慢性基础疾病和(或)手术外伤史的免疫力低下的中老年人,而先前多有喹诺酮类和头孢菌素类药物抗生素使用史,抗生素选择压力在一定程度上促进了包含质粒介导的喹诺酮类药物耐药机制的细菌的存在和流行。
[Abstract]:Objective: with the wide application of quinolones, Escherichia coli clinical isolates of China's resistance to the drugs increased significantly. The previous view mainly due to mutations in quinolone resistant bacterial chromosome drug target protein found in recent years, the plasmid mediated quinolone resistance gene, including qnr, AAC (6 ") -Ib-cr and qepA genes. Although these genes can cause quinolone resistance at low level, but they can through transposition plasmid conjugation, and other ways are often located on the plasmid resistance genes especially the ultra broad spectrum p- lactamases (ESBLs) gene with the rapid spread of the enormous threat to the experiment through the analysis of clinical medication. Drug resistance in clinical isolates of Escherichia coli, studies on the molecular mechanism of plasmid mediated quinolone resistance and study of plasmid mediated quinolone resistance gene The clinical characteristics of Escherichia coli clinical strains, so as to guide clinical rational use of antibiotics, reduce the occurrence and spread of bacterial resistance, and provide a theoretical basis for the development of new antibacterial drugs.
Research methods:
1 strains were collected, drug sensitivity test: collected from Second Hospital Affiliated to Tianjin Medical University in July 2008 -2009 year in September during the period of Escherichia coli clinical isolates of 143 strains, confirming routine biochemical identification. Repetitive strains removed from the same patient the same parts of separation. Determination of isolates to 12 antimicrobial agents by broth microdilution method (piperacillin, cefotaxime, cefoperazone, ceftriaxone, ceftazidime, cefepime, Cefoperazone / sulbactam, Amikacin, gentamicin, levofloxacin, ciprofloxacin, ofloxacin) MIC value analysis, the rate of drug resistance.
2, detection of quinolone resistance gene in plasmid mediated: polymerase chain reaction (PCR) amplification of ciprofloxacin resistant clinical isolates of Escherichia coli qnrA, qnrB, qnrS, AAC (6) -Ib and qepA gene of AAC (6 ') -Ib gene positive strains by Fok I enzyme digestion confirmed AAC (6') -Ib-cr gene.
3, transfer joint experimental verification of -qnr, E coli J53 receptor (Azide, resistant) exconjugants choice with cefotaxime (3mg/L) and sodium azide (100mg/L) LB agar and broth microdilution method comparison of donor and recipient strains, zygote MIC value. Including 8 kinds of antibacterial drugs.
4, screening for ESBLs phenotype, analyzing its relationship with plasmid mediated quinolone resistance genes, and using PCR method to detect ESBLs genotype of plasmid positive quinolone resistant genes: SHV, CTX-M-1, CTX-M-2, CTX-M-9 extended spectrum p- lactamase.
5, the clinical data of patients with positive strains collected with quinolone resistance gene in plasmid mediated infection were retrospectively analyzed, including basic data, clinical data, infection related data and microbial related data.
Result锛，

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