碳青霉烯非敏感阴沟肠杆菌耐药机制及分子流行病学研究

发布时间：2018-05-07 06:51

本文选题：阴沟肠杆菌 + 碳青霉烯类非敏感　；参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:分析我院2012-2014年碳青霉烯非敏感阴沟肠杆菌(CNS E.cloacae)的耐药情况,初步探讨CNS E.cloacae的耐药机制和分子流行病学特点。方法:收集重庆医科大学附属一院2012年3月至2014年12月分离的CNS E.cloacae非重复菌株,经全自动细菌鉴定和药物检测系统进行检测,并通过微量肉汤稀释法检测菌株对碳青霉烯类抗生素的最低抑菌浓度(MIC);设计合成碳青霉烯酶、超广谱β-内酰胺酶(Extended spectrumβ-lactamases,ESBLs)、喹诺酮(Fluoroquinolones resistance determinants,QRDs)、氨基糖苷(Aminoglycosides resistance determinants,ARDs)、整合子Intl-1和膜孔蛋白ompF、ompC基因的引物,采用PCR方法进行扩增检测;Carba NP法与改良Hodge实验分别对产碳青霉烯酶E.cloacae进行检测;同时采用脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)和多位点序列分型(Multiple Locus Sequence Typing,MLST)实验进行分子流行病学分析。结果:1、我院2012年3月-2014年12月共分离到非重复CNS E.Cloacae101株,主要来源于泌尿外科(19.8%)、肝胆外科(15.8%)和神经内科(9.9%);标本分布以尿液(27.7%)和痰标本(23.8%)所占比例较高,另有16(15.8%)株菌分离自分泌物标本,14(13.9%)株菌分离自胆汁。2、体外药物敏感性分析结果显示,101株实验菌株(100%)对厄他培南(etp)均表现为非敏感,耐药率达88.12%,24株(23.76%)对亚胺培南(ipm)非敏感,25株(24.75%)对美罗培南(mem)非敏感;对三代头孢菌素耐药率较高(97%),对四代头孢菌素耐药率为57.43%;对喹诺酮类和氨基糖苷类抗生素的耐药率分别为58.42%和55.45%。3、耐药基因分析结果显示,101株实验菌株中,碳青霉烯酶基因阳性e.cloacae共有14株(13.86%),其中7株分别表达blandm-1、3株blaimp-8、2株blaimp-4、1株blakpc-2,有一株菌同时表达blandm-1和blaimp-8基因;62株(61.39%)菌株esbls基因阳性,其中blatem(40/101,39.60%)、blashv(28/101,27.72%)、blactx-m(29/101,28.71%);64株(63.37%)菌株qrds基因阳性,其中qnra阳性基因11株(10.89%)、qnrb基因38株(37.62%)、qnrs基因29株(28.71%)、aac(6')-ib-cr基因37株(36.63%);ards基因阳性菌株中有43株(42.57%)表达aac(6')-ib,15株(14.85%)菌表达arma基因,5株(4.95%)菌表达rmtb;49株检测到intl-1基因,阳性率为48.51%;膜孔蛋白基因缺失率达28.71%(29/101),ompc缺失21株(20.79%)ompf缺失5株(4.95%),3株(2.97%)菌表现为ompf、ompc基因共同缺失。4、检测实验菌株碳青霉烯酶的方法比较显示:carbanp需1-2个小时即可读取结果,而改良hodge试验则需16-24个小时;carbanp试验检测14株产碳青霉烯酶E.cloacae和14株非产酶菌株的灵敏度、特异度均为100%,而改良Hodge试验检测上述菌株的灵敏度和特异度均为85.71%。5、PFGE结果显示:14株产碳青霉烯酶E.cloacae可分为12个型别,F、H型各包括2个亚型,分别来源于泌尿外科和骨科;MLST结果显示14株菌共有8种ST型别,不存在菌株的爆发流行,但部分菌株有相同型别。结论:1、我院分离的CNS E.cloacae主要以厄他培南耐药为主,菌株多重耐药现象明显;2、碳青霉烯酶基因阳性率不高,ESBLs基因高表达合并膜孔蛋白基因缺失比率较高,多种耐药基因共同表达和Intl-1高表达可能与多重耐药有关,首次发现一株blaNDM-1与blaIMP-8共表达的E.cloacae菌株;3、菌株来源以尿液和痰液标本为主,且主要分离自泌尿外科、肝胆外科和神经内科;分子流行病学显示菌株间没有存在爆发流行现象。4、Carba NP法较改良Hodge试验灵敏度和特异度高,且易于操作,可用于临床实验室快速检测产碳青霉烯酶肠杆菌科细菌。
[Abstract]:Objective: to analyze the drug resistance of non sensitive Enterobacter cloacae (CNS E.cloacae) in our hospital for 2012-2014 years. The drug resistance mechanism and molecular epidemiological characteristics of CNS E.cloacae were preliminarily discussed. Methods: the non repeated CNS E.cloacae strains isolated from the Affiliated Hospital of Medical University Of Chongqing from March 2012 to December 2014 were collected and identified by automatic bacterial identification. And the drug detection system was tested, and the minimal broth dilution method was used to detect the minimum inhibitory concentration (MIC) of carbapenems, and the synthesis of carbapenem, Extended spectrum beta -lactamases (ESBLs), quinolone (Fluoroquinolones resistance determinants, QRDs), and aminoglycoside (Aminogly) were designed. Cosides resistance determinants, ARDs), the integron Intl-1 and the membrane pore protein ompF, the primers of the ompC gene were amplified by PCR method, and the Carba NP method and the improved Hodge experiment were used to detect the carbon penicylenes E.cloacae, respectively. Molecular epidemiological analysis of the Multiple Locus Sequence Typing (MLST) experiment. Results: 1, the non repeated CNS E.Cloacae101 strains were isolated in December March 2012 in our hospital, mainly from the Department of Urology (19.8%), the Department of hepatobiliary surgery (15.8%) and the neurology department (9.9%); the specimens were distributed in the urine (27.7%) and sputum specimens (23.8%). High, 16 (15.8%) isolates were isolated from the secretions and 14 (13.9%) strains were isolated from the bile.2. In vitro drug sensitivity analysis showed that 101 strains (100%) were not sensitive to ETP, the drug resistance rate was 88.12%, 24 (23.76%) was not sensitive to imipenem (IPM), 25 (24.75%) was not sensitive to Mei Lopez Nan (MEM); The resistance rate of the three generation cephalosporins was higher (97%), the resistance rate to four generation cephalosporins was 57.43%, and the resistance rates to quinolones and aminoglycoside antibiotics were 58.42% and 55.45%.3 respectively. The results of resistance gene analysis showed that among 101 experimental strains, 14 (13.86%) were positive for carbapenem gene positive e.cloacae, of which 7 strains expressed blandm-1 respectively. 3 strains of blaimp-8,2 strain blaimp-4,1 blakpc-2, one strain expressed blandm-1 and blaimp-8 gene simultaneously, and 62 strains (61.39%) were positive for ESBLs gene, including blatem (40/101,39.60%), blashv (28/101,27.72%), blactx-m (29/101,28.71%), and 64 (63.37%) strains positive, including 11 (10.89%) positive gene and 38 (37.62%) gene. RS Gene 29 (28.71%), AAC (6') -ib-cr gene 37 strain (36.63%); 43 (42.57%) of ARDS gene positive strains expressed AAC (6') -ib, 15 (14.85%) strains expressed ARMA gene, 5 (4.95%) bacteria expressed rmtb, 49 strains detected intl-1 gene, the positive rate was 48.51%, and the loss rate of membrane foramen gene was 28.71% (29/101). (4.95%) 3 strains (2.97%) showed OmpF and OmpC gene co deletion.4. The method of detecting the experimental strain carbapenem showed that carbanp needed 1-2 hours to read the result, while the modified Hodge test was 16-24 hours, and the sensitivity and specificity of 14 strains of carbapenem E.cloacae and 14 non producing enzyme strains were detected by carbanp test. For 100%, the sensitivity and specificity of the modified Hodge test were 85.71%.5. The results of PFGE showed that 14 strains of carbapenem producing enzyme were divided into 12 types, F and H included 2 subtypes, respectively from Department of Urology and Department of Orthopedics; MLST results showed that there were 8 kinds of ST types in 14 strains, but there was no outbreak of strain. The strains had the same types. Conclusion: 1, the CNS E.cloacae isolated in our hospital is mainly eamapenem resistance, and the multidrug resistance of strains is obvious. 2, the positive rate of carbapenem gene is not high, the high expression of ESBLs gene is higher than that of the membrane pore protein gene, and the common expression of multiple resistance genes and the high expression of Intl-1 may be associated with multidrug resistance. For the first time, a strain of E.cloacae co expressed by blaNDM-1 and blaIMP-8 was found. 3, the main source of the strain was urine and sputum, and it was mainly isolated from Department of Urology, Department of hepatobiliary surgery and neurology. Molecular Epidemiology showed that there was no outbreak of.4 among the strains, and Carba NP method was more sensitive and highly specific than the modified Hodge test. It is easy to operate and can be used for rapid detection of carbapenem producing Enterobacteriaceae bacteria in clinical laboratories.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.5

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