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本文通过微量肉汤稀释法筛选出了2株耐环丙沙星(CIP)、头孢噻肟(CTX)、阿米卡星(AN)、氨苄西林(AM)药物的多重耐药性大肠杆菌,测定了苦豆子碱对大肠埃希菌多重耐药菌株的MIC值为12.5mg/mL,MH肉汤稀释棋盘式法测定环丙沙星联合苦豆子碱、CCCP MIC值,通过计算部分抑菌浓度指数判断环丙沙星联合苦豆子碱呈协同作用,优化试验确定最适宜的逆转条件对耐药性大肠埃希菌进行逆转,对编码外排泵系统中AcrA、AcrB、AcrR、ToLC蛋白基因进行克隆、测序,同时应用实时定量PCR检测苦豆子碱对多重耐药菌株作用前后AcrA、AcrB、AcrR、ToLC蛋白的mRNA表达量影响。数据分析发现作用前后编码大肠杆菌外排泵系统AcrA、AcrB、AcrR、ToLC蛋白基因碱基序列未发生变化,但苦豆子碱作用后外排泵系统中AcrA、AcrB蛋白mRNA表达量下降,负调控蛋白AcrR mRNA表达量上升。苦豆子碱从整体上可以抑制耐多药大肠埃希氏菌AcrAB-ToLC的蛋白mRNA表达量,使大肠杆菌多药外排泵AcrAB-ToLC的蛋白mRNA表达量下降,从而恢复大肠杆菌对抗生素的相对敏感性。实验结果为深入研究苦豆子碱逆转耐药性大肠杆菌其它机制提供了依据和参考.
In this paper, two multi-drug resistant Escherichia coli strains resistant to ciprofloxacin (CIP), cefotaxime (CTX), amikacin (AN) and ampicillin (AM) The MIC value of multi-drug resistant strains of aconitine to bentinine was determined to be 12.5 mg / mL. The MHC broth was diluted by the checkerboard method to determine the ciprofloxacin combined with the aconitine and CCCP MIC values. By calculating the partial inhibitory concentration Index ciprofloxacin combined with the synergistic effect of aconitine, the optimal test to determine the most appropriate reversal conditions of drug-resistant Escherichia coli reverse the encoding efflux pump system AcrA, AcrB, AcrR, ToLC protein gene for Cloning, sequencing, and real-time quantitative PCR were used to detect the mRNA expression of AcrA, AcrB, AcrR and ToLC before and after treatment with multi-drug resistant strains. Data analysis showed that the nucleotide sequences of AcrA, AcrB, AcrR and ToLC did not change before and after E. coli efflux pump system. However, AcrA and AcrB mRNA expression decreased after aconitum efflux pump system, Protein AcrR mRNA expression increased. As a whole, the inhibition of the protein mRNA expression of AcrAB-ToLC against Escherichia coli multi-drug efflux pump decreased protein expression of AcrAB-ToLC in Escherichia coli, thereby restoring the antibiotic resistance of E. coli to antibiotics Relative sensitivity. The experimental results provide the basis and reference for the further study on the mechanism of abasective reversal of drug-resistant Escherichia coli.