本文選題：陰溝腸桿菌 + 碳青霉烯類非敏感　； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:分析我院2012-2014年碳青霉烯非敏感陰溝腸桿菌(CNS E.cloacae)的耐藥情況,初步探討CNS E.cloacae的耐藥機(jī)制和分子流行病學(xué)特點(diǎn)。方法:收集重慶醫(yī)科大學(xué)附屬一院2012年3月至2014年12月分離的CNS E.cloacae非重復(fù)菌株,經(jīng)全自動(dòng)細(xì)菌鑒定和藥物檢測(cè)系統(tǒng)進(jìn)行檢測(cè),并通過(guò)微量肉湯稀釋法檢測(cè)菌株對(duì)碳青霉烯類抗生素的最低抑菌濃度(MIC);設(shè)計(jì)合成碳青霉烯酶、超廣譜β-內(nèi)酰胺酶(Extended spectrumβ-lactamases,ESBLs)、喹諾酮(Fluoroquinolones resistance determinants,QRDs)、氨基糖苷(Aminoglycosides resistance determinants,ARDs)、整合子Intl-1和膜孔蛋白o(hù)mpF、ompC基因的引物,采用PCR方法進(jìn)行擴(kuò)增檢測(cè);Carba NP法與改良Hodge實(shí)驗(yàn)分別對(duì)產(chǎn)碳青霉烯酶E.cloacae進(jìn)行檢測(cè);同時(shí)采用脈沖場(chǎng)凝膠電泳(Pulsed Field Gel Electrophoresis,PFGE)和多位點(diǎn)序列分型(Multiple Locus Sequence Typing,MLST)實(shí)驗(yàn)進(jìn)行分子流行病學(xué)分析。結(jié)果:1、我院2012年3月-2014年12月共分離到非重復(fù)CNS E.Cloacae101株,主要來(lái)源于泌尿外科(19.8%)、肝膽外科(15.8%)和神經(jīng)內(nèi)科(9.9%);標(biāo)本分布以尿液(27.7%)和痰標(biāo)本(23.8%)所占比例較高,另有16(15.8%)株菌分離自分泌物標(biāo)本,14(13.9%)株菌分離自膽汁。2、體外藥物敏感性分析結(jié)果顯示,101株實(shí)驗(yàn)菌株(100%)對(duì)厄他培南(etp)均表現(xiàn)為非敏感,耐藥率達(dá)88.12%,24株(23.76%)對(duì)亞胺培南(ipm)非敏感,25株(24.75%)對(duì)美羅培南(mem)非敏感;對(duì)三代頭孢菌素耐藥率較高(97%),對(duì)四代頭孢菌素耐藥率為57.43%;對(duì)喹諾酮類和氨基糖苷類抗生素的耐藥率分別為58.42%和55.45%。3、耐藥基因分析結(jié)果顯示,101株實(shí)驗(yàn)菌株中,碳青霉烯酶基因陽(yáng)性e.cloacae共有14株(13.86%),其中7株分別表達(dá)blandm-1、3株blaimp-8、2株blaimp-4、1株blakpc-2,有一株菌同時(shí)表達(dá)blandm-1和blaimp-8基因;62株(61.39%)菌株esbls基因陽(yáng)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基因陽(yáng)性,其中qnra陽(yáng)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基因陽(yáng)性菌株中有43株(42.57%)表達(dá)aac(6')-ib,15株(14.85%)菌表達(dá)arma基因,5株(4.95%)菌表達(dá)rmtb;49株檢測(cè)到intl-1基因,陽(yáng)性率為48.51%;膜孔蛋白基因缺失率達(dá)28.71%(29/101),ompc缺失21株(20.79%)ompf缺失5株(4.95%),3株(2.97%)菌表現(xiàn)為ompf、ompc基因共同缺失。4、檢測(cè)實(shí)驗(yàn)菌株碳青霉烯酶的方法比較顯示:carbanp需1-2個(gè)小時(shí)即可讀取結(jié)果,而改良hodge試驗(yàn)則需16-24個(gè)小時(shí);carbanp試驗(yàn)檢測(cè)14株產(chǎn)碳青霉烯酶E.cloacae和14株非產(chǎn)酶菌株的靈敏度、特異度均為100%,而改良Hodge試驗(yàn)檢測(cè)上述菌株的靈敏度和特異度均為85.71%。5、PFGE結(jié)果顯示:14株產(chǎn)碳青霉烯酶E.cloacae可分為12個(gè)型別,F、H型各包括2個(gè)亞型,分別來(lái)源于泌尿外科和骨科;MLST結(jié)果顯示14株菌共有8種ST型別,不存在菌株的爆發(fā)流行,但部分菌株有相同型別。結(jié)論:1、我院分離的CNS E.cloacae主要以厄他培南耐藥為主,菌株多重耐藥現(xiàn)象明顯;2、碳青霉烯酶基因陽(yáng)性率不高,ESBLs基因高表達(dá)合并膜孔蛋白基因缺失比率較高,多種耐藥基因共同表達(dá)和Intl-1高表達(dá)可能與多重耐藥有關(guān),首次發(fā)現(xiàn)一株blaNDM-1與blaIMP-8共表達(dá)的E.cloacae菌株;3、菌株來(lái)源以尿液和痰液標(biāo)本為主,且主要分離自泌尿外科、肝膽外科和神經(jīng)內(nèi)科;分子流行病學(xué)顯示菌株間沒(méi)有存在爆發(fā)流行現(xiàn)象。4、Carba NP法較改良Hodge試驗(yàn)靈敏度和特異度高,且易于操作,可用于臨床實(shí)驗(yàn)室快速檢測(cè)產(chǎn)碳青霉烯酶腸桿菌科細(xì)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R446.5

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