【摘要】：目的:篩查并確證碳青霉烯異質(zhì)性耐藥大腸埃希菌(Carbpenem-Heteroresistant Escherichia coli,CHEC),探究異質(zhì)性耐藥的分子機(jī)制;評估六種碳青霉烯酶表型方法的檢測效果。方法:收集我院2011年6月至2015年12月無菌體液中分離的大腸埃希菌532株,使用紙片擴(kuò)散法(Kirby-Bauer disk diffusion test,K-B test)篩檢CHEC菌株,菌落譜型分析(Population Analysis Profile,PAP)實(shí)驗(yàn)進(jìn)一步證實(shí)CHEC菌株,微量肉湯稀釋法檢測CHEC菌株對亞胺培南的MIC值;PCR及測序方法檢測CHEC菌株碳青霉烯酶基因、超廣譜β-內(nèi)酰胺酶(Extended Spectrum Beta-Lactamases,ESBLs)基因、膜孔蛋白(Outer membrane proteins,OMPs)基因和青霉素結(jié)合蛋白2(penicillin-binding protein 2,PBP2)基因mrd A;體外IPM誘導(dǎo)實(shí)驗(yàn)驗(yàn)證異質(zhì)性耐藥的進(jìn)展過程;RT-q PCR方法檢測原代及各誘導(dǎo)亞群ESBLs和膜孔蛋白的表達(dá)水平;改良霍金實(shí)驗(yàn)(MHT)、Triton Hodge實(shí)驗(yàn)(THT)、Carba NP實(shí)驗(yàn)(CNPt)、simplified Carba NP實(shí)驗(yàn)(CNPt-direct)、Blue-Carba NP實(shí)驗(yàn)(BCT)和碳青霉烯滅活實(shí)驗(yàn)(CIM)檢測256株碳青霉烯耐藥革蘭陰性菌產(chǎn)酶情況,麥克尼馬爾試驗(yàn)對六種表型方法進(jìn)行方法學(xué)評估。結(jié)果:1.2011-2015年,K-B法篩選出IPM異質(zhì)性耐藥菌株分別為41株(68.33%)、41株(34.17%)、37株(32.17%)、70株(59.32%)和84株(70.58%);ETP異質(zhì)性耐藥菌株分別為31株(51.67%)、29株(24.17%)、17株(14.78%)、30株(25.42%)和56株(47.58%);MEM異質(zhì)性耐藥菌株分別為2株(3.33%)、8株(6.67%)、4株(3.48%)、12株(10.17%)和8株(6.72%);PAP實(shí)驗(yàn)證實(shí)36株表型異質(zhì)性耐藥菌株均為異質(zhì)性耐藥菌株。2.耐藥基因檢測結(jié)果顯示,36株CHEC菌株中共檢出bla CTX-M 33株(91.67%)和bla TEM 12株(33.3%)。其中10株(27.8%)聯(lián)合表達(dá)bla CTX-M和bla TEM;僅一株omp C基因缺失;未檢測到碳青霉烯酶基因。3.體外IPM誘導(dǎo)實(shí)驗(yàn)結(jié)果顯示,長時間低濃度抗生素誘導(dǎo)條件下,隨著誘導(dǎo)亞群的MIC值升高,PAP曲線右移,說明異質(zhì)性耐藥是進(jìn)化至碳青霉烯耐藥的中間階段。4.RT-q PCR結(jié)果顯示,隨著誘導(dǎo)亞群MIC值的增高,bla TEM-1表達(dá)顯著上調(diào),膜孔蛋白表達(dá)顯著下調(diào),P值有統(tǒng)計(jì)學(xué)差異。5.PCR及測序方法檢出256株碳青霉烯耐藥菌株中產(chǎn)碳青霉烯酶139株,非產(chǎn)碳青霉烯酶菌株117株。MHT、THT、CNPt、CNPt-direct、BCT和CIM分別檢出產(chǎn)酶菌株105株(75.5%)、118株(84.9%)、92株(66.2%)、129株(92.8%)、114株(82.0%)和130(93.5%)。CNPt-direct和CIM的靈敏度均高于MHT、CNPt和BCT,有統(tǒng)計(jì)學(xué)差異(P0.003);CNPt-direct和CIM比較,靈敏度無統(tǒng)計(jì)學(xué)差異(92.8%vs93.5%,P=1);THT的特異度(91.5%)低于CNPt,CNPt-direct,BCT和CIM特異度(100%),有統(tǒng)計(jì)學(xué)差異(P0.003)。結(jié)論:1.無菌體液中分離的大腸埃希菌存在普遍的碳青霉烯異質(zhì)性耐藥現(xiàn)象,近三年CHEC菌株對IPM和ETP異質(zhì)性耐藥率呈上升趨勢,對MEM異質(zhì)性耐藥率較低較穩(wěn)定。2.體外IPM誘導(dǎo)實(shí)驗(yàn)證實(shí)碳青霉烯異質(zhì)性耐藥是大腸埃希菌由敏感進(jìn)展至碳青霉烯耐藥的中間階段。3.bla TEM-1表達(dá)上調(diào)聯(lián)合膜孔蛋白表達(dá)下調(diào)可能是我院CHEC菌株進(jìn)展至耐藥的主要機(jī)制。4.CNPt-direct和CIM特異度和靈敏度高,操作簡單,能夠快速檢測碳青霉烯酶,具有重要的臨床實(shí)用價值。
[Abstract]:Objective: to screen and confirm Carbpenem-Heteroresistant Escherichia coli (CHEC), to investigate the molecular mechanism of hetero resistance, and to evaluate the detection effect of six carbapenem phenotypes. Methods: 532 strains of Escherichia coli isolated from the aseptic fluid of our hospital from June 2011 to December 2015 were collected. The Kirby-Bauer disk diffusion test (K-B test) was used to screen the CHEC strain, and the colony spectral analysis (Population Analysis Profile, PAP) further confirmed the CHEC strain. The microdilution method was used to detect the value of the strain to imipenem. Extended Spectrum Beta-Lactamases (ESBLs) gene, membrane pore protein (Outer membrane proteins, OMPs) gene and penicillin binding protein 2 (penicillin-binding protein 2, PBP2) gene MRD. The improved Hocking experiment (MHT), the Triton Hodge experiment (THT), the Carba NP experiment (CNPt), the simplified Carba NP experiment (CNPt-direct), the Blue-Carba experiments and the carbapenem inactivation test were used to detect the enzyme production of 256 carbapenem resistant gram-negative bacteria. The McNemar test conducted a methodological assessment of the six phenotypic methods. Results: in 1.2011-2015, 41 strains (68.33%), 41 (34.17%), 37 (32.17%), 70 (59.32%) and 84 (70.58%) were screened by K-B method, respectively. The ETP hetero resistant strains were respectively, 41 strains (51.67%), 29 strains, and MEM strains, respectively. 67%), 4 strains (3.48%), 12 (10.17%) and 8 (6.72%), and PAP experiments showed that 36 strains of hetero resistant strains were heterogeneous resistant strains.2. resistance gene detection results, and 36 strains of CHEC strains detected bla CTX-M 33 (91.67%) and Bla TEM 12 (33.3%). The results of IPM induction in vitro of carbapenem gene.3. showed that the PAP curve shifted rightward with the increase of MIC value of the induced subgroup, indicating that heterogeneity resistance was the intermediate stage of evolution to carbapenene resistance,.4.RT-q PCR results showed, with the increase of MIC value of the induced subgroup, BLA T. The expression of EM-1 was significantly up-regulated, and the expression of membrane pore protein was significantly down, and P values were statistically different.5.PCR and sequencing methods detected 256 carbapenem resistant strains of carbapenems, 117 strains of.MHT, THT, CNPt, CNPt-direct, BCT and CIM, 105 (75.5%), 118 (84.9%), 92 (66.2%), 129, 129. The sensitivity of plant (92.8%), 114 (82%) and 130 (93.5%).CNPt-direct and CIM were higher than that of MHT, CNPt and BCT (P0.003). There was no statistical difference between CNPt-direct and CIM (92.8%vs93.5%, P=1), and THT specificity (91.5%) was lower than that of CNPt, 100%, 100%, and there was a statistical difference. Conclusion: 1. no The heterogenous resistance to carbapenems existed in the Escherichia coli isolated from the bacteria solution. The resistance rate of CHEC to IPM and ETP was increasing in the last three years. The resistance rate of MEM heterogeneity was lower than that of stable.2. in vitro. It was confirmed that the hetero resistance of carbapenems was the sensitive progress of Escherichia coli to carbapenems. The down regulation of the up regulation of.3.bla TEM-1 expression in the intermediate stage of the drug may be the main mechanism of the progression to drug resistance of CHEC strain in our hospital,.4.CNPt-direct and CIM with high specificity and sensitivity, simple operation and rapid detection of carbapenems, which has important clinical value.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.5

【參考文獻(xiàn)】

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1 陳晶;鄭紹同;李紅林;朱家斌;于亮;;大腸埃希菌感染的臨床分布與耐藥性研究[J];中華醫(yī)院感染學(xué)雜志;2015年01期

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