肠杆菌科细菌替加环素耐药机制研究

发布时间：2018-06-12 08:08

本文选题：肠杆菌科细菌 + 替加环素　；参考：《浙江大学》2017年博士论文

【摘要】：肠杆菌科细菌,包括肺炎克雷伯菌和大肠埃希菌,是当今世界范围内出现的引起院内感染的重要病原体。替加环素因其良好的抗菌活性和临床使用的安全性,是目前国内外用于应对碳青霉烯耐药肠杆菌科细菌的首选抗菌药物。近年来随着替加环素的广泛使用,关于肠杆菌科细菌替加环素耐药的报道逐年增多,尤其是肺炎克雷伯菌和大肠埃希菌对替加环素的耐药率也呈逐年上升趋势,而现有的研究并不能合理解释肠杆菌科细菌对替加环素耐药的原因。本研究通过两部分内容,探讨了肠杆菌科细菌对替加环素耐药的主要机制,并深入探索了肠杆菌科细菌替加环素耐药的新机制。第一部分,主要研究产KPC肺炎克雷伯菌临床菌株对替加环素的敏感性以及探索RND型外排泵在肺炎克雷伯菌替加环素耐药中所起的作用。我们共收集了215株产KPC肺炎克雷伯菌临床菌株,通过微量肉汤稀释法测定这些菌株的替加环素最低抑菌浓度(MIC);挑选替加环素耐药菌株,使用外排泵抑制剂探讨外排泵在这些菌株替加环素耐药中所起的作用;通过荧光定量PCR检测RND型外排泵基因acrB和oqxB以及它们的转录调节因子(ramA、marA、soxS和rarA)的表达并分析这些基因与替加环素MIC的相关性。结果显示:215株产KPC肺炎克雷伯菌中有24株对替加环素耐药(MIC≥4mg/L,EUCAST标准),耐药率为11.2%。而外排泵抑制剂NMP可有效恢复上述菌株对替加环素的敏感性(91.7%的菌株恢复敏感)。荧光定量PCR结果显示外排泵基因acrB的表达量与替加环素的MIC呈正相关关系(P0.05)。此外,我们还在3株耐药菌株中发现ramA高表达现象,进一步研究发现这些菌株均存在ramR突变。对其中一株ramR突变引起基因表达终止的菌株进行野生型ramR回补,回补后的菌株恢复对替加环素的敏感性,同时ramA和acrB的表达均受到抑制。本研究结果表明:RND型外排泵AcrAB-TolC在肺炎克雷伯菌临床菌株替加环素耐药中起着关键的作用,它的表达量和细菌替加环素MIC呈正相关关系(P0.05);同时我们发现ramR基因的突变进而引起ramA基因和外排泵AcrAB的高表达是肺炎克雷伯菌临床菌株替加环素耐药的主要机制之一。第二部分,通过基因敲除、体外诱导等技术探索了大肠埃希菌替加环素耐药的新机制。我们利用Red重组系统,进行基因敲除。以标准菌株大肠埃希菌ATCC 25922为亲本菌株,构建了大肠埃希菌外排泵AcrAB敲除株25922△acrAB,然后以ATCC 25922和25922△acrAB为亲本菌株进行替加环素体外诱导实验,获得耐药菌株25922△acrAB-TGC8和25922-TGC8。对耐药菌株和亲本菌株进行全基因组测序,并通过比较基因组学数据分析方法寻找突变基因,发现在25922△acrAB-TGC8和25922-TGC8中均存在mlaA基因突变,而随后的基因敲除和回补实验亦证明mlaA基因的突变可以导致大肠埃希菌替加环素的MIC升高8倍。进一步研究表明,在菌株25922-TGC8中该细菌对替加环素耐药是Mla系统、外排泵AcrAB和核糖体蛋白变异三种机制共同作用的结果,最终使菌株对替加环素的MIC从0.125mg/L上升到8mg/L(FDA耐药标准)。因为Mla系统、外排泵AcrAB和核糖体蛋白均由细菌染色体编码,当细菌在替加环素药物选择压力的作用下,上述基因易发生突变,进而导致替加环素耐药的发生,可能是对肠杆菌科细菌在替加环素治疗过程中容易从敏感发展成为耐药较为合理的解释。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R446.5

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