本文選題：腸桿菌科細(xì)菌 + 替加環(huán)素�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：腸桿菌科細(xì)菌,包括肺炎克雷伯菌和大腸埃希菌,是當(dāng)今世界范圍內(nèi)出現(xiàn)的引起院內(nèi)感染的重要病原體。替加環(huán)素因其良好的抗菌活性和臨床使用的安全性,是目前國(guó)內(nèi)外用于應(yīng)對(duì)碳青霉烯耐藥腸桿菌科細(xì)菌的首選抗菌藥物。近年來(lái)隨著替加環(huán)素的廣泛使用,關(guān)于腸桿菌科細(xì)菌替加環(huán)素耐藥的報(bào)道逐年增多,尤其是肺炎克雷伯菌和大腸埃希菌對(duì)替加環(huán)素的耐藥率也呈逐年上升趨勢(shì),而現(xiàn)有的研究并不能合理解釋腸桿菌科細(xì)菌對(duì)替加環(huán)素耐藥的原因。本研究通過(guò)兩部分內(nèi)容,探討了腸桿菌科細(xì)菌對(duì)替加環(huán)素耐藥的主要機(jī)制,并深入探索了腸桿菌科細(xì)菌替加環(huán)素耐藥的新機(jī)制。第一部分,主要研究產(chǎn)KPC肺炎克雷伯菌臨床菌株對(duì)替加環(huán)素的敏感性以及探索RND型外排泵在肺炎克雷伯菌替加環(huán)素耐藥中所起的作用。我們共收集了215株產(chǎn)KPC肺炎克雷伯菌臨床菌株,通過(guò)微量肉湯稀釋法測(cè)定這些菌株的替加環(huán)素最低抑菌濃度(MIC);挑選替加環(huán)素耐藥菌株,使用外排泵抑制劑探討外排泵在這些菌株替加環(huán)素耐藥中所起的作用;通過(guò)熒光定量PCR檢測(cè)RND型外排泵基因acrB和oqxB以及它們的轉(zhuǎn)錄調(diào)節(jié)因子(ramA、marA、soxS和rarA)的表達(dá)并分析這些基因與替加環(huán)素MIC的相關(guān)性。結(jié)果顯示:215株產(chǎn)KPC肺炎克雷伯菌中有24株對(duì)替加環(huán)素耐藥(MIC≥4mg/L,EUCAST標(biāo)準(zhǔn)),耐藥率為11.2%。而外排泵抑制劑NMP可有效恢復(fù)上述菌株對(duì)替加環(huán)素的敏感性(91.7%的菌株恢復(fù)敏感)。熒光定量PCR結(jié)果顯示外排泵基因acrB的表達(dá)量與替加環(huán)素的MIC呈正相關(guān)關(guān)系(P0.05)。此外,我們還在3株耐藥菌株中發(fā)現(xiàn)ramA高表達(dá)現(xiàn)象,進(jìn)一步研究發(fā)現(xiàn)這些菌株均存在ramR突變。對(duì)其中一株ramR突變引起基因表達(dá)終止的菌株進(jìn)行野生型ramR回補(bǔ),回補(bǔ)后的菌株恢復(fù)對(duì)替加環(huán)素的敏感性,同時(shí)ramA和acrB的表達(dá)均受到抑制。本研究結(jié)果表明:RND型外排泵AcrAB-TolC在肺炎克雷伯菌臨床菌株替加環(huán)素耐藥中起著關(guān)鍵的作用,它的表達(dá)量和細(xì)菌替加環(huán)素MIC呈正相關(guān)關(guān)系(P0.05);同時(shí)我們發(fā)現(xiàn)ramR基因的突變進(jìn)而引起ramA基因和外排泵AcrAB的高表達(dá)是肺炎克雷伯菌臨床菌株替加環(huán)素耐藥的主要機(jī)制之一。第二部分,通過(guò)基因敲除、體外誘導(dǎo)等技術(shù)探索了大腸埃希菌替加環(huán)素耐藥的新機(jī)制。我們利用Red重組系統(tǒng),進(jìn)行基因敲除。以標(biāo)準(zhǔn)菌株大腸埃希菌ATCC 25922為親本菌株,構(gòu)建了大腸埃希菌外排泵AcrAB敲除株25922△acrAB,然后以ATCC 25922和25922△acrAB為親本菌株進(jìn)行替加環(huán)素體外誘導(dǎo)實(shí)驗(yàn),獲得耐藥菌株25922△acrAB-TGC8和25922-TGC8。對(duì)耐藥菌株和親本菌株進(jìn)行全基因組測(cè)序,并通過(guò)比較基因組學(xué)數(shù)據(jù)分析方法尋找突變基因,發(fā)現(xiàn)在25922△acrAB-TGC8和25922-TGC8中均存在mlaA基因突變,而隨后的基因敲除和回補(bǔ)實(shí)驗(yàn)亦證明mlaA基因的突變可以導(dǎo)致大腸埃希菌替加環(huán)素的MIC升高8倍。進(jìn)一步研究表明,在菌株25922-TGC8中該細(xì)菌對(duì)替加環(huán)素耐藥是Mla系統(tǒng)、外排泵AcrAB和核糖體蛋白變異三種機(jī)制共同作用的結(jié)果,最終使菌株對(duì)替加環(huán)素的MIC從0.125mg/L上升到8mg/L(FDA耐藥標(biāo)準(zhǔn))。因?yàn)镸la系統(tǒng)、外排泵AcrAB和核糖體蛋白均由細(xì)菌染色體編碼,當(dāng)細(xì)菌在替加環(huán)素藥物選擇壓力的作用下,上述基因易發(fā)生突變,進(jìn)而導(dǎo)致替加環(huán)素耐藥的發(fā)生,可能是對(duì)腸桿菌科細(xì)菌在替加環(huán)素治療過(guò)程中容易從敏感發(fā)展成為耐藥較為合理的解釋。
[Abstract]:Enterobacteriaceae, including Klebsiella pneumoniae and Escherichia coli, is an important pathogen causing nosocomial infection in the world today. Due to its good antibacterial activity and clinical safety, tegacycline is the first choice to deal with carbapenem resistant Enterobacteriaceae at home and abroad. With the widespread use of tegacycline, reports on the resistance of tegacycline to Enterobacteriaceae are increasing year by year, especially the resistance rate of Klebsiella pneumoniae and Escherichia coli to tegacycline is also increasing year by year. The current study does not explain the cause of resistance to tegacycline by the Enterobacteriaceae. Two In part, the main mechanisms of Enterobacteriaceae resistance to tegacycline were discussed, and a new mechanism of tigacycline resistance in Enterobacteriaceae was explored. The first part was mainly to study the sensitivity of KPC producing Klebsiella pneumoniae strains to tegacycline and to explore the resistance of RND type efflubsiella pneumoniae to tegacycline. 215 clinical strains of Klebsiella pneumoniae produced by KPC were collected, and the minimum inhibitory concentration (MIC) of tegagin was measured by the broth dilution method; the drug resistant strains of tegagin were selected and the efflux pump inhibitor was used to explore the role of the efflux pump in the resistance of these strains to tegicine resistance. The expression of acrB and oqxB and their transcriptional regulators (ramA, marA, soxS and rarA) were detected by quantitative PCR, and the correlation between these genes and tegocyclin MIC was analyzed. The results showed that 24 of 215 strains of Klebsiella pneumoniae were resistant to tegatin (MIC > 4mg/L, standard), and the resistance rate was suppressed. The strain NMP could effectively restore the sensitivity of the strain to tegatocycline (sensitivity to 91.7% of the strain). Fluorescence quantitative PCR showed that the expression of the efflux pump gene acrB was positively correlated with the MIC of tegatin (P0.05). In addition, we found the high expression of ramA in the 3 strains of resistant strains, and further studies found that these strains were all strains. There was a ramR mutation. The strain of one of the ramR mutations that caused the termination of gene expression was retraced with wild type ramR, and the strain recovered to tigocycline, while the expression of ramA and acrB were inhibited. The results of this study showed that the RND type efflux pump AcrAB-TolC was resistant to tigocycline resistance of Klebsiella pneumoniae clinical strain. There is a positive correlation between the expression and the bacterial tegagin MIC (P0.05), and we find that the mutation of the ramR gene and the high expression of the ramA gene and the AcrAB of the efflux pump is one of the main mechanisms of the drug resistance of the clinical strain of Klebsiella pneumoniae. The second part, by gene knockout, in vitro induction, and so on. A new mechanism for the resistance of Escherichia coli to tegocycline was explored. We used the Red recombinant system to perform gene knockout. The standard strain of Escherichia coli ATCC 25922 was used as a parent strain, and the AcrAB knockout strain of Escherichia coli 25922 Delta acrAB was constructed, and then ATCC 25922 and 25922 Delta acrAB were used as parental strains to induce in vitro induction of tegotin. The whole genome sequencing of drug-resistant strains 25922 Delta acrAB-TGC8 and 25922-TGC8. was obtained in the experiment. The mutation gene was found by comparative genomics data analysis method. It was found that there were mlaA gene mutations in 25922 Delta acrAB-TGC8 and 25922-TGC8, and the subsequent gene knockout and supplementation tests also proved mlaA Mutations in the gene can cause a 8 fold increase in the MIC of the Escherichia coli tegagin. Further studies suggest that the bacterial resistance to tegagin in strain 25922-TGC8 is a Mla system, a result of the three mechanisms of the displacement of the pump AcrAB and ribosome protein, which eventually makes the strain of the strain of tegagin from 0.125mg/L to 8mg/L (FDA resistant). Drug standards). Because the Mla system, the AcrAB and ribosome proteins are encoded by the bacterial chromosomes. When the bacteria are selected under the action of tegacycline, the above gene is prone to mutation, which leads to the occurrence of tegacycline resistance. It may be susceptible to the susceptibility of Enterobacteriaceae in the treatment of tegacycline. A more reasonable explanation for resistance.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.5

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