本文選題：肺炎克雷伯菌 + 環(huán)丙沙星��； 參考：《寧波大學》2013年碩士論文

【摘要】：目的外排泵系統(tǒng)是肺炎克雷伯耐藥的重要機制，外排泵抑制劑的研究和開發(fā)是解決細菌耐藥問題的重要途徑之一。本研究擬了解抗生素誘導過程對肺炎克雷伯菌外排泵的影響，闡明連翹抑菌作用的有效成分，詮釋連翹有效成分對外排泵活性的作用機制，為連翹在抗菌治療中的應用提供詳細的實驗資料，從而為連翹的開發(fā)應用提供科學依據(jù)。 方法收集臨床分離的20株對環(huán)丙沙星敏感且ESBLs為陰性的肺炎克雷伯菌，經(jīng)法國梅里埃VITEK2微生物分析系統(tǒng)進行菌種鑒定及藥敏分析，測定在加入外排泵抑制劑羰基氰氯苯腙（CCCP）前后環(huán)丙沙星對菌株的最低抑菌濃度（MIC）,用逐步遞增濃度的環(huán)丙沙星誘導肺炎克雷伯菌至最高耐藥獲得誘導后菌株，采用外排泵抑制實驗和溴化乙錠（EB）蓄積實驗兩種方法比較誘導前后細菌的外排泵活性變化，比較連翹三種單體的抑菌效果，選取連翹酯苷B作用于10株誘導后細菌，采用EB蓄積實驗比較連翹酯苷B作用前后細菌外排泵活性的變化。 結果環(huán)丙沙星梯度誘導后的細菌經(jīng)鑒定并篩選出16株ESBLs為陰性的肺炎克雷伯菌，外排泵抑制結果顯示誘導后菌株MIC值比誘導前增高16-256倍不等，CCCP對4株誘導前菌株有2倍抑制，而對一半的誘導后菌株有2或4倍抑制。CCCP對7株誘導前菌株無抑制作用，而對其各自誘導后菌株有2或4倍抑制，CCCP對3株誘導前菌株有2倍抑制作用，而對其各自誘導后菌株無抑制作用。EB蓄積實驗結果顯示誘導前后各菌株在加入CCCP的胞內熒光量均明顯高于各自未加CCCP的熒光量。無論CCCP加入與否，誘導后菌株的EB蓄積量均低于誘導前菌株的EB蓄積量。連翹酯苷A和連翹酯苷B的抑菌作用強于連翹苷。連翹酯苷A或連翹酯苷B與抗生素具有協(xié)同作用。連翹酯苷B的亞抑菌濃度是512mg/L。連翹酯苷B作用前后細菌的外排泵活性水平比較結果顯示：2株經(jīng)連翹酯苷B作用后的細菌熒光量均高于作用前菌株，并且高于連翹酯苷B作用前加CCCP的熒光量。6株經(jīng)連翹酯苷B作用后的細菌熒光量均高于作用前菌株，但低于連翹酯苷B作用前加CCCP的熒光量。2株連翹酯苷B作用前后菌株的熒光值差別不大。 結論外排泵機制普遍存在于肺炎克雷伯菌中，環(huán)丙沙星逐步誘導使肺炎克雷伯菌外排泵活性增高，外排泵抑制實驗不能很好地反映細菌外排泵活性水平，EB蓄積實驗比外排泵抑制實驗更敏感，，連翹酯苷A和連翹酯苷B抑菌作用強于連翹苷，連翹酯苷A或連翹酯苷B與抗生素具有協(xié)同作用，連翹酯苷B對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.
【學位授予單位】：寧波大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R563.1

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