离子对大肠杆菌生长的影响以及大肠杆菌生物被膜的初步研究

发布时间：2018-03-06 13:54

本文选题：大肠杆菌　切入点：生物被膜　出处：《山东中医药大学》2010年硕士论文　论文类型：学位论文

【摘要】：目的：通过分光光度法及麦氏比浊等方法对大肠杆菌菌液浓度进行测定,了解大肠杆菌不同生长时期生长曲线的特征,分析不同浓度的铁、锌离子对大肠杆菌生长状况的影响,为微量元素用于大肠杆菌的体外培养和生物被膜的形成提供参考数值；通过药敏试验了解不同浓度的铁、锌离子对阿奇霉素抗菌活性的影响；通过生物被膜银染色观察、细菌计数等方法并以硅胶为载体建立大肠杆菌生物被膜的体外模型,了解大肠杆菌生物被膜的形成,并研究生物被膜的形成鉴定标准及阿奇霉素与黄连素的联合用药效果,为阿奇霉素和黄连素联合应用于大肠杆菌生物被膜感染的治疗提供依据。方法：1分别测量大肠杆菌不同生长时期的菌液的OD值,以时间为横坐标,以OD值为纵坐标建立坐标系,绘制出大肠杆菌生长曲线；给MH培养基中加入不同浓度的锌、铁离子,混匀接种之后,利用分光光度法测定的OD值反应大肠杆菌菌液浓度,建立时间---OD值坐标系,通过曲线的走向来分析铁、锌离子对大肠杆菌生长的影响；2通过纸片扩散法的药敏试验,比较不同铁离子和锌离子浓度下阿奇霉素对大肠杆菌抑菌圈直径的影响；3以硅胶为载体建立大肠杆菌生物被膜模型,用细菌计数法比较阿奇霉素与黄连素的联合用药和单独用药的药效。结果：1大肠杆菌的生长曲线基本符合微生物的生长特性,并且呈现二次生长曲线的特征。从生长曲线图来看,加入铁离子浓度分别为11,17,27,32μmol/L,各组加入铁离子的大肠杆菌菌液的OD值都比对照组高,其中27μmol/L铁离子浓度下大肠杆菌菌液的OD值显著高于对照组,但当铁离子浓度达到32μmol/L时,其OD值要比铁离子浓度为27μmol/L时的OD值要小；即通过试验比较,在设计的不同铁离子浓度中,铁离子浓度为27μmol/L时大肠杆菌菌液的OD值最大。锌离子浓度分别为6,12,18,52μmol/L时,与对照组相比,各浓度锌离子组大肠杆菌菌液的OD值都低于对照组；但锌离子浓度在12-18μmol/L浓度范围内菌液OD值几乎没有明显改变,数值比较稳定。2铁离子浓度分别为11,17,27,32μmol/L的培养基中大肠杆菌的抑菌圈直径进一步证明了铁离子对大肠杆菌生长的促进作用；同样锌离子浓度为6,12,18,52μmol/L的培养基中抑菌圈直径也再次说明锌离子对大肠杆菌生长有抑制作用；各浓度铁、锌离子对阿奇霉素的抑菌活性均无明显影响。3利用银染色方法鉴定,成功建立大肠杆菌生物被膜模型,配制0.15,0.3,0.6 mg/L的阿奇霉素溶液与0.2,0.4,0.8mg/L的黄连素溶液,通过单独用药和两两联合用药后的大肠杆菌细菌计数值分析比较,当黄连素浓度达到0.8mg/L才出现明显的协同抑菌作用,且阿奇霉素的协同作用与其浓度无关。结论：铁离子对大肠杆菌的生长有一定的促进作用,但与其浓度有很大关系。低浓度时几乎无促进作用,随着铁离子浓度的增加其作用开始增强,但到达一定浓度后其作用不但不会增强反而有所减弱。锌离子在正常血清浓度11-23μmol/L范围内对大肠杆菌的生长有抑制作用,在机体缺乏锌离子的情况下,较为容易感染大肠杆菌。锌离子在12-18μmol/L浓度范围内作用没有明显改变,比较稳定；随着锌离子浓度的增加,到体内最高浓度的两倍多时,抑制作用才比较显著。不同浓度的铁、锌离子对阿奇霉素的抑菌活性无明显影响。当黄连素浓度达到0.8mg/L时,阿奇霉素与黄连素联合用药对生物被膜内大肠杆菌的抑制具有协同效应。
[Abstract]:Objective: to Escherichia coli bacteria concentration were determined by spectrophotometry and Maxwell turbidimetric method, to understand the characteristics of Escherichia coli in different growth stages of growth curve, analysis of different concentrations of iron, effect of zinc ions on the growth of Escherichia coli, for trace elements for in vitro culture and biological Escherichia coli is provided reference values of film formation; understand the different concentrations of iron through drug sensitivity test, effect of zinc ions on the antibacterial activity of Azithromycin; biofilm by silver staining, the bacterial count method with silica gel as the carrier to establish an in vitro model of Escherichia coli biofilm, understanding of biofilm formation of Escherichia coli, and research biofilm formation and identification standard of azithromycin and berberine combination effect, provide for the treatment of azithromycin and berberine on Escherichia coli biofilm infection Basis.
Methods: 1 od bacteria Escherichia coli were measured at different growth periods, with time as the abscissa, the OD value of the vertical axis coordinate system, draw the growth curve of Escherichia coli; to MH medium added different concentration of zinc, iron, mixing after inoculation, using Escherichia coli od reaction the concentration of spectrophotometry, the establishment of time ---OD coordinates, through the curve to analysis of iron, effect of zinc ions on the growth of Escherichia coli; drug sensitivity test of 2 by disk diffusion method, comparing the different effects of iron ion and zinc ion concentration of azithromycin on Escherichia coli inhibition zone diameter; 3 with silica gel as a vector of Escherichia coli biofilm model, comparison with the bacterial count of azithromycin in combination with berberine and individual drug efficacy.
Results: 1 Escherichia coli growth curve accords with the characteristics of microbial growth, and the growth curve showed two characteristics. From the growth curve, adding iron ion concentration were 11,17,27,32 mol/L, each adding iron ions of Escherichia coli bacteria OD values than the control group, the OD value of 27 mol/L of iron ion the concentration of Escherichia coli was significantly higher than the control group, but when the iron ion concentration reached 32 mol/L, the OD value than the iron ion concentration was 27 mol/L smaller; by comparison, in different concentration of iron ion in the design, the iron ion concentration for Escherichia coli bacteria od the maximum of 27 mol/L. The zinc ion concentration was 6,12,18,52 mol/L, compared with the control group, the OD value of the concentration of zinc ion group of Escherichia coli bacteria were lower than the control group; but the zinc ion concentration at 12-18 mol/L concentration range of bacteria OD The value of almost did not change significantly,.2 values are stable iron ion concentration respectively. The antibacterial circle diameter medium 11,17,27,32 of Escherichia coli mol/L in further proved that the promoting effect of iron ion on the growth of Escherichia coli; the same concentration of zinc ions is bacteriostatic medium 6,12,18,52 mol/L in diameter also showed that zinc ions inhibited again on the growth of Escherichia coli; the concentration of iron and zinc ions on the antibacterial activity of azithromycin were not significantly influenced by.3 silver staining, successfully established Escherichia coli biofilm model, berberine solution of azithromycin solution with 0.2,0.4,0.8mg/L preparation of 0.15,0.3,0.6 mg/ L, by E. coli bacteria alone or the combination of the 22 meter comparative analysis numerical, when berberine concentration of 0.8mg/L apparent only synergistic inhibitory effect, and the synergistic effect of azithromycin and Concentration has nothing to do with it.
Conclusion: there is a certain growth promoting effect of iron ions on Escherichia coli, but a great relationship with its concentration. Low concentration of almost no promotion, with the iron ion concentration started to increase its role, but its role to reach a certain concentration not only will not strengthen but weaken. Some zinc ions on Escherichia coli growth in the normal serum concentration of 11-23 mol/L in the range of inhibition, lack of zinc in the body of the ion, the more susceptible to Escherichia coli. Zinc ions in 12-18 mol/L concentration range function did not change significantly, relatively stable; with the increase of the concentration of zinc ions, to two times for a long time in the highest concentration, inhibition was more significant. Different concentrations of iron, no obvious effect of zinc ions on the antibacterial activity of azithromycin. When berberine concentration of 0.8mg/L, berberine Azithromycin combined with the biofilm The inhibition of internal Escherichia coli has a synergistic effect.

【学位授予单位】：山东中医药大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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