本文選題：腸桿菌科菌　切入點(diǎn)：碳青霉烯類(lèi)藥物　出處：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：[目的]研究碳青霉烯耐藥腸桿菌科菌的感染危險(xiǎn)因素、臨床預(yù)后、分子流行病學(xué)以及耐藥機(jī)制。[方法]收集2004-2012年我國(guó)34家教學(xué)醫(yī)院的342株碳青霉烯耐藥腸桿菌科菌。采用病例-對(duì)照研究分析碳青霉烯耐藥腸桿菌科菌感染的危險(xiǎn)因素和臨床預(yù)后。采用對(duì)倍瓊脂稀釋法測(cè)定抗菌藥物的最低抑菌濃度；多位點(diǎn)序列分析(MLST)和脈沖場(chǎng)凝膠電泳(PFGE)分析CRE的同源性。采用改良的Hodge試驗(yàn)檢測(cè)菌株產(chǎn)的碳青霉烯酶；聚合酶鏈?zhǔn)椒磻?yīng)(PCR)檢測(cè)包括碳青霉烯酶基因在內(nèi)的多種p內(nèi)酰胺酶基因；對(duì)PCR陽(yáng)性產(chǎn)物進(jìn)行序列測(cè)定以確定基因型別；接合試驗(yàn)檢測(cè)耐藥基因的可移動(dòng)性；質(zhì)粒提取試驗(yàn)分析菌株攜帶的質(zhì)粒；Southern雜交進(jìn)行碳青霉烯酶基因定位；全質(zhì)粒序列測(cè)序和分析進(jìn)行碳青霉烯酶基因環(huán)境研究：十二烷基磺酸鈉一聚丙烯酰胺凝膠電泳(SDS-PAGE)分析菌株的外膜孔通道蛋白。基因克隆技術(shù)研究marR基因突變對(duì)大腸埃希菌碳青霉烯耐藥的影響。[結(jié)果]CRE感染的臨床治療失敗率76.7%�！案腥綜RE前2個(gè)月內(nèi)廣譜抗生素使用大于7天”是CRE感染的獨(dú)立危險(xiǎn)因素(OR= 19.088, P=0.006)。CRE敏感性較高的藥物包括多粘菌素B(總體敏感率96.1%),替加環(huán)素(總體敏感率84.2%)和阿米卡星(總體敏感率55.5%)。342株CRE菌株中241株(70.5%)攜帶碳青霉烯酶基因,其中173株攜帶blaKPC-2基因,26株攜帶blaNDM-1基因,42株攜帶blaIMP-type基因。產(chǎn)KPC-2酶的菌株在近年間的數(shù)量上升迅速,產(chǎn)NDM-1酶的菌株在2010年開(kāi)始出現(xiàn),近年來(lái)也呈現(xiàn)增長(zhǎng)趨勢(shì)。產(chǎn)KPC-2酶的CRE菌株主要分布于浙江省、北京市和江蘇省。多位點(diǎn)序列分析發(fā)現(xiàn)152株產(chǎn)碳青霉烯酶的肺炎克雷伯菌中105株(69.1%)為ST11型。29株產(chǎn)碳青霉烯酶的大腸埃希菌屬于17種ST型別。不同的碳青霉烯酶基因經(jīng)Southern雜交證實(shí)位于不同大小的質(zhì)粒上。全質(zhì)粒測(cè)序的結(jié)果顯示轉(zhuǎn)座子、插入序列以及整合子等多種可移動(dòng)元件在碳青霉烯酶基因的傳播中扮演重要作用。外膜蛋白分析顯示外膜蛋白缺失為不產(chǎn)碳青霉烯酶CRE的主要耐藥機(jī)制。MarR突變分析研究顯示MarR的Gln42Arg突變可引起MarR功能失活,從而提高M(jìn)arA的表達(dá),進(jìn)而提升大腸埃希菌對(duì)碳青霉烯類(lèi)藥物的耐藥性。[結(jié)論]本研究是一項(xiàng)全國(guó)多中心的連續(xù)9年CRE流行病學(xué)和耐藥機(jī)制研究。闡明了CRE感染的高治療失敗率和危險(xiǎn)因素,分子流行病學(xué)顯示產(chǎn)碳青霉烯酶菌株是主要的CRE類(lèi)型,且ST11型為中國(guó)主流的碳青霉烯耐藥肺炎克雷伯菌克隆型。產(chǎn)碳青酶烯酶以及外膜蛋白缺失在CRE耐藥機(jī)制中起主要作用。質(zhì)粒、轉(zhuǎn)座子、插入序列以及整合子等多種可移動(dòng)元件在碳青霉烯酶基因的傳播中扮演重要作用。marR基因突變?cè)诖竽c埃希菌中能導(dǎo)致碳青霉烯耐藥。
[Abstract]:[objective] to study the risk factors, clinical prognosis, molecular epidemiology and drug resistance mechanism of carbapenem resistant Enterobacteriaceae.Methods 342 strains of carbapenem resistant Enterobacteriaceae were collected from 34 educational hospitals in China from 2004 to 2012.Case-control study was used to analyze the risk factors and clinical prognosis of carbapenem-resistant Enterobacteriaceae infection.The minimal inhibitory concentration of antimicrobial agents was determined by para-Agar dilution method, and the homology of CRE was analyzed by multilocus sequence analysis and pulsed field gel electrophoresis (PFGE).The improved Hodge test was used to detect carbapenase, polymerase chain reaction (PCR) was used to detect many kinds of plactamases including carbapenem genes, and the positive products of PCR were sequenced to determine the genotypes.The conjugation test was used to detect the mobility of drug resistance genes, and the plasmids carried by the strains were analyzed by Southern blotting to locate the carbapenem gene.Sequencing and analysis of the whole plasmid sequence for carbapenem gene environment: sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the outer membrane pore channel protein of the strain.The effect of marR gene mutation on carbapenem resistance of Escherichia coli was studied by gene cloning technique.[results] the clinical treatment failure rate of CRE infection was 76. 7%."use of broad-spectrum antibiotics more than 7 days before CRE infection" is an independent risk factor for CRE infection, OR = 19.088. The drugs with higher P=0.006).CRE sensitivity include polymyxin B (total sensitivity rate 96.1g), tegicycline (overall sensitivity rate 84.2%) and amitine (total sensitivity rate 84.2%).Caraxine (total sensitivity rate of 55.5%, #number0# strains of CRE strain, 70.5%) carried carbapenase gene,Among them, 173 strains carried blaKPC-2 gene, 26 strains carried blaNDM-1 gene and 42 strains carried blaIMP-type gene.The number of strains producing KPC-2 enzyme increased rapidly in recent years. The strains producing NDM-1 enzyme began to appear in 2010 and also showed an increasing trend in recent years.KPC-2-producing CRE strains were mainly distributed in Zhejiang, Beijing and Jiangsu provinces.Multilocus sequence analysis showed that 105 strains of Klebsiella pneumoniae producing carbapenem belonged to 17 types of St type, which belonged to ST11 type. 29 strains of Escherichia coli.Different carbapenase genes were identified to be located on plasmids of different sizes by Southern hybridization.The results of whole plasmid sequencing showed that transposon, insertion sequence and integron play an important role in the transmission of carbapenase gene.The analysis of outer membrane protein showed that the absence of outer membrane protein was the main drug resistance mechanism of non-carbapenase CRE. Marr mutation analysis showed that the Gln42Arg mutation of MarR could cause MarR function inactivation and thus increase the expression of MarA.Furthermore, the resistance of Escherichia coli to carbapenems was enhanced.[conclusion] this study is a national multi-center 9-year CRE epidemiology and drug resistance mechanism study.The high treatment failure rate and risk factors of CRE infection were elucidated. Molecular epidemiology showed that carbapenem producing strain was the main type of CRE, and ST11 type was the dominant clone of Klebsiella pneumoniae in China.Carbogenase production and absence of outer membrane proteins play a major role in the mechanism of drug resistance of CRE.Plasmid, transposon, insertion sequence and integron play an important role in the transmission of carbapenase gene. The mutation of Marr gene can lead to carbapenem resistance in Escherichia coli.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R446.5

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