连翘酯苷B对肺炎克雷伯菌外排泵活性的影响

发布时间：2018-05-31 04:39

本文选题：肺炎克雷伯菌 + 环丙沙星　；参考：《宁波大学》2013年硕士论文

【摘要】：目的外排泵系统是肺炎克雷伯耐药的重要机制，外排泵抑制剂的研究和开发是解决细菌耐药问题的重要途径之一。本研究拟了解抗生素诱导过程对肺炎克雷伯菌外排泵的影响，阐明连翘抑菌作用的有效成分，诠释连翘有效成分对外排泵活性的作用机制，为连翘在抗菌治疗中的应用提供详细的实验资料，从而为连翘的开发应用提供科学依据。方法收集临床分离的20株对环丙沙星敏感且ESBLs为阴性的肺炎克雷伯菌，经法国梅里埃VITEK2微生物分析系统进行菌种鉴定及药敏分析，测定在加入外排泵抑制剂羰基氰氯苯腙（CCCP）前后环丙沙星对菌株的最低抑菌浓度（MIC）,用逐步递增浓度的环丙沙星诱导肺炎克雷伯菌至最高耐药获得诱导后菌株，采用外排泵抑制实验和溴化乙锭（EB）蓄积实验两种方法比较诱导前后细菌的外排泵活性变化，比较连翘三种单体的抑菌效果，选取连翘酯苷B作用于10株诱导后细菌，采用EB蓄积实验比较连翘酯苷B作用前后细菌外排泵活性的变化。结果环丙沙星梯度诱导后的细菌经鉴定并筛选出16株ESBLs为阴性的肺炎克雷伯菌，外排泵抑制结果显示诱导后菌株MIC值比诱导前增高16-256倍不等，CCCP对4株诱导前菌株有2倍抑制，而对一半的诱导后菌株有2或4倍抑制。CCCP对7株诱导前菌株无抑制作用，而对其各自诱导后菌株有2或4倍抑制，CCCP对3株诱导前菌株有2倍抑制作用，而对其各自诱导后菌株无抑制作用。EB蓄积实验结果显示诱导前后各菌株在加入CCCP的胞内荧光量均明显高于各自未加CCCP的荧光量。无论CCCP加入与否，诱导后菌株的EB蓄积量均低于诱导前菌株的EB蓄积量。连翘酯苷A和连翘酯苷B的抑菌作用强于连翘苷。连翘酯苷A或连翘酯苷B与抗生素具有协同作用。连翘酯苷B的亚抑菌浓度是512mg/L。连翘酯苷B作用前后细菌的外排泵活性水平比较结果显示：2株经连翘酯苷B作用后的细菌荧光量均高于作用前菌株，并且高于连翘酯苷B作用前加CCCP的荧光量。6株经连翘酯苷B作用后的细菌荧光量均高于作用前菌株，但低于连翘酯苷B作用前加CCCP的荧光量。2株连翘酯苷B作用前后菌株的荧光值差别不大。结论外排泵机制普遍存在于肺炎克雷伯菌中，环丙沙星逐步诱导使肺炎克雷伯菌外排泵活性增高，外排泵抑制实验不能很好地反映细菌外排泵活性水平，EB蓄积实验比外排泵抑制实验更敏感，，连翘酯苷A和连翘酯苷B抑菌作用强于连翘苷，连翘酯苷A或连翘酯苷B与抗生素具有协同作用，连翘酯苷B对部分肺炎克雷伯菌的外排泵活性有抑制作用。
[Abstract]:Objective efflux pump system is an important mechanism of Klebsiella pneumoniae resistance, the research and development of the efflux pump inhibitors is one of the important ways to solve the problem of bacterial resistance. This study is to understand the effect of antibiotic induction on Klebsiella pneumoniae efflux pump, to clarify the effective components of the Bacteriostasis of Forsythia forsythia, and to explain the effective components of Forsythia Forsythia The mechanism of pump activity provides detailed experimental data for the application of Forsythia in antibacterial treatment, so as to provide scientific basis for the development and application of Forsythia suspense.
Methods 20 clinical isolates of Klebsiella pneumoniae, which were sensitive to ciprofloxacin and ESBLs negative, were identified and analyzed by the French mil VITEK2 microbiological analysis system. The minimum inhibitory concentration (MIC) of ciprofloxacin against the carbonyl cyanobenzene hydrazone (CCCP) before and after the addition of the pump inhibitor was measured and increased gradually. The concentration of ciprofloxacin induced Klebsiella pneumoniae to the highest drug resistance to obtain the induced strain. Two methods were used to compare the activity of the efflux pump activity before and after the induction of the efflux pump inhibition test and the ethidium bromide (EB) accumulation experiment. The bacteriostasis effect of the three monomers of Forsythia Forsythia was compared, and the forsythia glycoside B was selected to act on the 10 strains of induced bacteria and used EB The activity of bacterial efflux pump before and after the action of forsythoside B was compared.
Results 16 strains of Klebsiella pneumoniae were identified and screened after the ciprofloxacin gradient induction. The inhibition results of ESBLs were 16-256 times higher than before induction, and CCCP was 2 times more inhibited for 4 strains of pre induced strains, while half of the induced strains had 2 or 4 times the inhibition of.CCCP to 7 pre induced strains. The strain had no inhibitory effect, but the strain was inhibited 2 or 4 times after its induction, and CCCP had a 2 times inhibitory effect on the 3 strains of the pre induced strain. The results of the no inhibition effect of.EB accumulation after the induction of the strains showed that the intracellular fluorescence of each strain before and after the induction was higher than that of the unadded CCCP, no matter CCCP added. Or not, the EB accumulation of the induced strain was lower than the EB accumulation of pre induced strain. The Bacteriostasis of Forsythia glycoside A and forsythia glycoside B was stronger than that of forsythia. Forsythia glycoside A or Forsythia glycoside B had synergism with antibiotics. The subinhibitory concentration of Forsythia glycoside B was the comparison of the activity level of the efflux pump of the 512mg/L. Forsythia glycoside before and after the B use. The results showed that the fluorescence of 2 strains of bacteria after the action of Forsythia glycoside B were higher than that of the pre action strain, and the fluorescence of B before the action of Forsythia glycoside and CCCP was higher than that of the pre action strain after the action of Forsythia glycoside B, but it was lower than the B effect of the forsythia glycoside and B before and before and before and after the B action of the CCCP. There is little difference in the fluorescence value.
Conclusion the mechanism of the efflux pump is common in Klebsiella pneumoniae, and ciprofloxacin is gradually induced to increase the activity of Klebsiella pneumoniae, and the inhibition experiment of the outer row pump can not reflect the activity level of the bacterial efflux pump well. The EB accumulation experiment is more sensitive than the efflux pump inhibition test. The bacteriostasis of alsythiin A and the forsythia glycoside B are stronger than the forsythia glycoside Forsythoside A or forsythoside B has synergistic effect with antibiotics. Forsythoside B has inhibitory effect on the efflux pump activity of some Klebsiella pneumoniae.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.1

【参考文献】