本文關(guān)鍵詞：大腸埃希菌臨床株質(zhì)粒介導的喹諾酮類耐藥機制的研究　出處：《天津醫(yī)科大學》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 大腸埃希菌 質(zhì)粒介導 喹諾酮類耐藥 qnr aac(6’)-Ib-cr β-內(nèi)酰胺酶

【摘要】：研究目的：隨著喹諾酮類抗菌藥物的廣泛應用,我國臨床分離的大腸埃希菌對該類藥物的耐藥性明顯上升。以往的觀點認為喹諾酮耐藥主要由于細菌染色體上藥物靶蛋白的突變造成,近年來發(fā)現(xiàn)了質(zhì)粒介導的喹諾酮耐藥基因,包括qnr、aac(6’)-Ib-cr和qepA基因。這些基因雖然只能引起低水平的喹諾酮耐藥,但它們可以通過質(zhì)粒接合、轉(zhuǎn)座等途徑和其它常位于質(zhì)粒上的耐藥基因特別是超廣譜p-內(nèi)酰胺酶(ESBLs)基因一起快速傳播給臨床用藥帶來巨大威脅。本實驗通過分析大腸埃希菌臨床分離株的耐藥情況,分子水平研究質(zhì)粒介導的喹諾酮類耐藥機制和探討攜帶質(zhì)粒介導的喹諾酮耐藥基因的大腸埃希菌臨床株感染的臨床特征,從而為指導臨床合理使用抗菌藥物、減少細菌耐藥性的出現(xiàn)或傳播及新型抗菌藥物的研制提供理論依據(jù)。 研究方法： 1、菌株收集、藥敏試驗：收集自天津醫(yī)科大學第二醫(yī)院2008年7月-2009年9月期間臨床分離的大腸埃希菌共143株,經(jīng)常規(guī)生化鑒定確認。剔除從同一病人同一部位分離的重復菌株。采用微量肉湯稀釋法測定臨床菌株對12種常用抗菌藥物(哌拉西林、頭孢氨噻肟、頭孢哌酮、頭孢他啶、頭孢曲松、頭孢吡肟、頭孢哌酮/舒巴坦、阿米卡星、慶大霉素、左氧氟沙星、環(huán)丙沙星、氧氟沙星)的MIC值,分析耐藥率。 2、質(zhì)粒介導的喹諾酮耐藥基因的檢測：應用聚合酶鏈式反應(PCR)擴增環(huán)丙沙星耐藥的大腸埃希菌臨床株的qnrA、qnrB、qnrS、aac(6’)-Ib及qepA基因,對aac(6’)-Ib基因陽性菌株用Fok I酶切確認aac(6’)-Ib-cr基因。 3、接合實驗驗證-qnr的轉(zhuǎn)移性,受體菌為E coli J53(Azide resistant),對接合子的選擇采用含頭孢噻肟(3mg/L)和疊氮化鈉(100mg/L)的LB瓊脂平皿,采用微量肉湯稀釋法比較受體菌、供體菌及接合子的MIC值,包括8種常用抗菌藥物。 4、ESBLs表型的篩選,分析其與質(zhì)粒介導的喹諾酮類耐藥基因的關(guān)系,采用PCR法檢測攜帶質(zhì)粒介導的喹諾酮類耐藥基因的陽性菌株ESBLs基因型：包括SHV、CTX-M-1、CTX-M-2、CTX-M-9型超廣譜p-內(nèi)酰胺酶。 5、收集攜帶質(zhì)粒介導的喹諾酮耐藥基因的陽性菌株感染患者的臨床資料,進行回顧性分析：包括基本資料、臨床資料、感染相關(guān)資料及微生物相關(guān)資料。 結(jié)果： 1、143株大腸埃希菌臨床株的藥敏結(jié)果：對12種抗菌藥物的耐藥率為哌拉西林(70.63%),氧氟沙星(58.04%),頭孢哌酮(55.24%),左氧氟沙星(54.54%),環(huán)丙沙星(52.45%),慶大霉素(51.05%),頭孢吡肟(49.65%),頭孢曲松(48.95%),頭孢氨噻肟(47.55%),頭孢他啶(35.66%),阿米卡星(12.59%),頭孢哌酮/舒巴坦(6.29%),多重耐藥菌株60株(41.96%)。 2、質(zhì)粒介導喹諾酮耐藥基因的檢測：75株環(huán)丙沙星耐藥的大腸埃希菌中共有18株(24%)攜帶qnr基因和(或)aac(6’)-Ib-cr基因,其中qnr和aac(6’)-Ib-cr檢出率分別為6.7%和21.3%,未檢出qnrA和qepA。 3、接合實驗：5株qnr陽性菌株均接合傳遞成功,各種抗菌藥物對接合子的MIC值較受體菌均有不同程度提高。 4、75株耐環(huán)丙沙星的大腸埃希菌臨床株有39株(52%)產(chǎn)ESBLs,不攜帶qnr/aac(6’)-Ib-cr的菌株產(chǎn)酶率為36.84%,18株qnr/aac(6')-Ib-cr陽性的大腸埃希菌中均產(chǎn)ESBLs,主要以CTX-M-1型、CTX-M-9型、SHV型ESBLs為主。 5、陽性菌株的臨床特征分析：陽性菌株對喹諾酮類和頭孢菌素類藥物高度耐藥且主要來源于泌尿系感染,感染患者均為中老年人且絕大多數(shù)患者同時患有一種及其以上的基礎(chǔ)疾病。 結(jié)論 1、大腸埃希菌是臨床最常見的病原菌,我院臨床分離的大腸埃希菌對多種抗菌藥物的耐藥率較高,尤其是頭孢菌素和氟喹諾酮類抗生素。 2、天津地區(qū)臨床存在qnr和aac(6’)-Ib-cr陽性菌株的流行。包含qnr和aac(6')-Ib-cr的菌株多為產(chǎn)ESBLs的臨床分離菌株,且qnr基因可在患者體外水平傳播。 3、攜帶質(zhì)粒介導的喹諾酮耐藥基因的大腸埃希菌多為多重耐藥菌,感染患者多為有慢性基礎(chǔ)疾病和(或)手術(shù)外傷史的免疫力低下的中老年人,而先前多有喹諾酮類和頭孢菌素類藥物抗生素使用史,抗生素選擇壓力在一定程度上促進了包含質(zhì)粒介導的喹諾酮類藥物耐藥機制的細菌的存在和流行。
[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錛，

本文編號：1400899