大肠埃希菌碳青霉烯异质性耐药分子机制研究及碳青霉烯酶表型检测方法评价
发布时间：2018-07-17 20:30
【摘要】：目的:筛查并确证碳青霉烯异质性耐药大肠埃希菌(Carbpenem-Heteroresistant Escherichia coli,CHEC),探究异质性耐药的分子机制;评估六种碳青霉烯酶表型方法的检测效果。方法:收集我院2011年6月至2015年12月无菌体液中分离的大肠埃希菌532株,使用纸片扩散法(Kirby-Bauer disk diffusion test,K-B test)筛检CHEC菌株,菌落谱型分析(Population Analysis Profile,PAP)实验进一步证实CHEC菌株,微量肉汤稀释法检测CHEC菌株对亚胺培南的MIC值;PCR及测序方法检测CHEC菌株碳青霉烯酶基因、超广谱β-内酰胺酶(Extended Spectrum Beta-Lactamases,ESBLs)基因、膜孔蛋白(Outer membrane proteins,OMPs)基因和青霉素结合蛋白2(penicillin-binding protein 2,PBP2)基因mrd A;体外IPM诱导实验验证异质性耐药的进展过程;RT-q PCR方法检测原代及各诱导亚群ESBLs和膜孔蛋白的表达水平;改良霍金实验(MHT)、Triton Hodge实验(THT)、Carba NP实验(CNPt)、simplified Carba NP实验(CNPt-direct)、Blue-Carba NP实验(BCT)和碳青霉烯灭活实验(CIM)检测256株碳青霉烯耐药革兰阴性菌产酶情况,麦克尼马尔试验对六种表型方法进行方法学评估。结果:1.2011-2015年,K-B法筛选出IPM异质性耐药菌株分别为41株(68.33%)、41株(34.17%)、37株(32.17%)、70株(59.32%)和84株(70.58%);ETP异质性耐药菌株分别为31株(51.67%)、29株(24.17%)、17株(14.78%)、30株(25.42%)和56株(47.58%);MEM异质性耐药菌株分别为2株(3.33%)、8株(6.67%)、4株(3.48%)、12株(10.17%)和8株(6.72%);PAP实验证实36株表型异质性耐药菌株均为异质性耐药菌株。2.耐药基因检测结果显示,36株CHEC菌株中共检出bla CTX-M 33株(91.67%)和bla TEM 12株(33.3%)。其中10株(27.8%)联合表达bla CTX-M和bla TEM;仅一株omp C基因缺失;未检测到碳青霉烯酶基因。3.体外IPM诱导实验结果显示,长时间低浓度抗生素诱导条件下,随着诱导亚群的MIC值升高,PAP曲线右移,说明异质性耐药是进化至碳青霉烯耐药的中间阶段。4.RT-q PCR结果显示,随着诱导亚群MIC值的增高,bla TEM-1表达显著上调,膜孔蛋白表达显著下调,P值有统计学差异。5.PCR及测序方法检出256株碳青霉烯耐药菌株中产碳青霉烯酶139株,非产碳青霉烯酶菌株117株。MHT、THT、CNPt、CNPt-direct、BCT和CIM分别检出产酶菌株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的灵敏度均高于MHT、CNPt和BCT,有统计学差异(P0.003);CNPt-direct和CIM比较,灵敏度无统计学差异(92.8%vs93.5%,P=1);THT的特异度(91.5%)低于CNPt,CNPt-direct,BCT和CIM特异度(100%),有统计学差异(P0.003)。结论:1.无菌体液中分离的大肠埃希菌存在普遍的碳青霉烯异质性耐药现象,近三年CHEC菌株对IPM和ETP异质性耐药率呈上升趋势,对MEM异质性耐药率较低较稳定。2.体外IPM诱导实验证实碳青霉烯异质性耐药是大肠埃希菌由敏感进展至碳青霉烯耐药的中间阶段。3.bla TEM-1表达上调联合膜孔蛋白表达下调可能是我院CHEC菌株进展至耐药的主要机制。4.CNPt-direct和CIM特异度和灵敏度高,操作简单,能够快速检测碳青霉烯酶,具有重要的临床实用价值。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5
本文编号：2130850
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5
【参考文献】
相关期刊论文 前1条
1 陈晶;郑绍同;李红林;朱家斌;于亮;;大肠埃希菌感染的临床分布与耐药性研究[J];中华医院感染学杂志;2015年01期
，本文编号：2130850
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