粉防己甲素逆转多重耐药大肠埃希菌外排喹诺酮类药物机制的研究

发布时间：2018-07-09 10:00

本文选题：大肠埃希菌 + 粉防己甲素　；参考：《西南大学》2017年硕士论文

【摘要】：抗生素和其他抗菌药物的发展可以说是现代医学最伟大的成就,它增加了人类的平均寿命。但是,过度使用和滥用抗生素及抗菌药已导致了微生物的耐药性。喹诺酮类抗菌药在人类和动物的临床使用上有着极其重要的作用。然而细菌对喹诺酮类药物的耐药性却日趋严重,特别是大肠埃希菌对喹诺酮类药物的耐药率呈逐年增高的趋向。现如今,喹诺酮类药物的临床治疗效果低下,使用剂量大、治疗成本高等诸多问题摆在我们面前。如果能控制或解决细菌耐药性的问题,无疑是医学界的一重大成果,可以大大提高现有抗菌药的抗菌效果。当前,为应对细菌耐药性问题,研究比较多的是关于细菌外排泵抑制剂的研究,如利血平、维拉帕米、碳酰氰基-对-氯苯腙(CCCP)等,但由于其毒副作用很难应用于临床。近年来发现的天然植物活性成分如小檗碱、胡椒碱等也被证实拥有抑制外排泵的作用。中药有效成分相对安全、残留小、无耐药性危害等,在产品研发与临床应用上具有明显优势。本试验通过结合前期实验室的研究,选择中药防己的主要有效成分粉防己甲素,研究其是否具有抑制多重耐药大肠埃希菌外排喹诺酮类药物的效果,为后期外排泵抑制剂的确认奠定基础;同时通过对耐药菌逆转机制的初步探究,为粉防己甲素外排抑制剂的研制提供理论依据。1、粉防己甲素对临床分离耐药大肠埃希菌的逆转作用(1)采用联合药敏试验测定粉防己甲素对乳酸环丙沙星抑菌效果的影响;(2)使用不同浓度的乳酸环丙沙星HM肉汤进行耐药菌73-2杀菌试验,绘制乳酸环丙沙星以及联合粉防己甲素和CCCP对耐药菌73-2的杀菌曲线;(3)使用含粉防己甲素和不同浓度乳酸环丙沙星的含药MH琼脂平皿测定在粉防己甲素存在下大肠埃希菌ATCC25922的突变率;(4)采用高效液相色谱法分别检测耐药菌73-2和大肠埃希菌ATCC25922在粉防己甲素条件下,细菌体内乳酸环丙沙星的蓄积量,试验以CCCP作为药物对照;(5)使用含粉防己甲素的LB肉汤反复培养耐药菌73-2,以获得敏感性恢复的逆转细菌。结果:乳酸环丙沙星联合粉防己甲素对大肠埃希菌73-2的FIC指数为0.1875,具有较强的协同作用,使用160μg/mL的粉防己甲素和10μg/m L的CCCP与乳酸环丙沙星联合应用,乳酸环丙沙星对耐药菌73-2的最小抑菌浓度(MIC)均由联合前的320μg/m L降低到10μg/m L,而对大肠埃希菌ATCC25922的MIC值无影响;粉防己甲素可以提高1/2MIC浓度的乳酸环丙沙星对耐药菌73-2的杀灭作用,抑制细菌的生长;在4MIC浓度的乳酸环丙沙星条件下粉防己甲素可以有效降低大肠埃希菌ATCC25922的突变,突变率从正常的10-7降低到10-9;在粉防己甲素存在条件下,耐药菌73-2细胞体内乳酸环丙沙星的蓄积量明显比正常情况明显增加,约为4.2倍,而对大肠埃希菌ATCC25922几乎无影响,在CCCP存在条件下,也出现相似情况,耐药菌73-2乳酸环丙沙星的蓄积量约是正常条件下的3.9倍;通过使用含粉防己甲素的肉汤培养耐药菌得到了乳酸环丙沙星对其MIC下降的菌株B(Tet)。结论:粉防己甲素具有增强乳酸环丙沙星杀灭细菌的作用,可以抑制细菌的突变,增加乳酸环丙沙星在耐药菌细胞内的蓄积量。粉防己甲素具有外排抑制剂的活性。2、粉防己甲素对临床分离耐药大肠埃希菌的逆转机制研究检测正常生长的耐药菌73-2和在粉防己甲素条件下生长的耐药菌73-2及正常生长的耐药逆转菌B(Tet)外排泵基因acrA、acrB、tolC、acrZ及其调控基因marA、rob、acrR mRNA的表达量;检测逆转菌B(Tet)在逆转前后外排泵基因acrA/B/Z、tolC和调控基因marA、acrR、rob的碱基序列。结果:在粉防己甲素存在下生长的耐药菌73-2(Tet)和耐药逆转的细菌B(Tet)和耐药菌73-2外排泵基因acrA/B/Z mRNA的表达量比较都极显著下降;tolC mRNA的表达量73-2(Tet)只是下降,B(Tet)表现为极显著下降。对外排泵起正调控作用的marA、rob mRNA表达量都极显著减少;B(Tet)表现出负调控基因acrR mRNA表达量显著上升,而73-2(Tet)acrR mRNA表达量的只是上升。耐药逆转菌B(Tet)的相关基因碱基序列和耐药菌73-2相比均未发生改变。结论:粉防己甲素对耐药大肠埃希菌的外排泵基因acrA/B/Z和正调控基因marA、rob的表达具有显著的抑制作用,可以促进负调控基因acrR的表达。不能使耐药菌的外排泵基因acrA/B/Z、tolC和调控基因marA、rob、acr R的碱基序列再次发生突变。粉防己甲素可以通过影响外排泵的表达逆转细菌的耐药性。3、粉防己甲素对体外诱导耐药菌AcrAB-TolC外排泵的作用采取多步法,以1/2MIC浓度的乳酸环丙沙星体外人工诱导大肠埃希菌ATCC25922产生耐药性;将乳酸环丙沙星与粉防己甲素联合应用,检测粉防己甲素是否具备逆转诱导菌耐药性的作用;检测在粉防己甲素条件下生长的诱导菌A(Cip)外排泵基因acrA/B/Z、tolC mRNA的表达量。结果:以1/2MIC为起点人工体外诱导获得了能稳定遗传的耐药菌A(Cip);粉防己甲素可以使乳酸环丙沙星对耐药菌A(Cip)的MIC从640μg/m L下降到80μg/mL;在粉防己甲素条件下外排泵基因acrA/B/Z、tolC mRNA表达量极显著减少。结论:粉防己甲素对体外诱导的耐药菌也能很好的进行耐药逆转,其作用方式主要是通过减少外排泵AcrAB-TolC的表达量。
[Abstract]:The development of antibiotics and other antimicrobials can be said to be the greatest achievement in modern medicine, which increases the life expectancy of human beings. However, excessive use and abuse of antibiotics and antibiotics have led to microbial resistance. Quinolones have an extremely important role in the clinical use of humans and animals. The drug resistance of quinolones is becoming more and more serious, especially the resistance rate of Escherichia coli to quinolones is increasing year by year. Nowadays, the clinical treatment effect of quinolones is low, the dosage is large and the cost of treatment is high in front of our face. If we can control or solve the problem of bacterial resistance, No doubt it is a major achievement in the medical field, which can greatly improve the antibacterial effect of existing antiseptic drugs. At present, in order to deal with the problem of bacterial resistance, there are many studies on bacterial efflux pump inhibitors, such as reserpine, Vera Pammy, carbonyl chlorobenzene hydrazone (CCCP) and so on, but it is difficult to apply their toxic and side effects in clinical practice in recent years. The natural plant active ingredients, such as berberine and piperine, have also been proved to have the effect of inhibiting the efflux pump. The effective components of the Chinese medicine are relatively safe, small, and have no drug resistance, and have obvious advantages in the development and clinical application of the products. The effect of tetrandrine on the inhibition of multidrug-resistant Escherichia coli was studied, which lay the foundation for the confirmation of late efflux pump inhibitors. At the same time, a theoretical basis for the development of the inhibitor of tetrandrine was provided by the preliminary exploration of the reversal mechanism of drug resistant bacteria..1, tetrandrine, powder tetrandrine, was used in clinical practice. The reversal effect of the isolated Escherichia coli (1) the effect of tetrandrine on the bacteriostasis effect of ciprofloxacin lactate was determined by combined drug sensitivity test. (2) the bactericidal test of drug resistant bacteria 73-2 with ciprofloxacin HM broth with different concentrations was carried out, and the bactericidal effect of ciprofloxacin lactate and tetrandrine and CCCP on drug resistant bacteria 73-2 was plotted. (3) the mutation rate of Escherichia coli ATCC25922 in the presence of tetrandrine in the presence of tetrandrine containing powder tetrandrine and ciprofloxacin with different concentrations was measured. (4) high performance liquid chromatography was used to detect the drug resistant bacteria 73-2 and Escherichia coli ATCC25922 in the Bacillus tetrandrine, and the bacterial Lactobacillus lactate in the bacteria was found in the MH. The volume of the star was tested with CCCP as the drug control; (5) the resistant bacteria 73-2 were repeatedly cultured with the LB broth containing tetrandrine containing tetrandrine. The results showed that the FIC index of ciprofloxacin combined with tetrandrine to Escherichia coli 73-2 was 0.1875, with a stronger synergistic effect and 160 micron g/mL. The minimum inhibitory concentration (MIC) of ciprofloxacin (ciprofloxacin) of lactic acid (ciprofloxacin) to ciprofloxacin (ciprofloxacin) of 10 g/m L was reduced from 320 mu g/m L to 10 mu g/m L, but no effect on the MIC value of Escherichia coli ATCC25922; tetrandrine powder could improve the killing of ciprofloxacin against drug resistant bacteria 73-2 by 1/ 2MIC. In the condition of 4MIC concentration of ciprofloxacin, tetrandrine could effectively reduce the mutation of Escherichia coli ATCC25922, and the mutation rate was reduced from 10-7 to 10-9. Under the presence of tetrandrine, the accumulation of ciprofloxacin in the 73-2 cells of the drug resistant bacteria was obviously more obvious than that of the normal condition. The increase was about 4.2 times, but there was almost no effect on Escherichia coli ATCC25922. In the presence of CCCP, the accumulation of ciprofloxacin was about 3.9 times that of the normal condition. The strain of ciprofloxacin lactate to its MIC decreased B (Tet) was obtained by using the broth containing tetrandrine containing powder. Conclusion: tetrandrine can enhance the bactericidal effect of ciprofloxacin lactate, inhibit the mutation of bacteria and increase the accumulation of ciprofloxacin in the cell of drug resistant bacteria. Tetrandrine has the activity of.2, and the reverse mechanism of tetrandrine for clinical isolation of Escherichia coli in clinical isolation and detection of normal growth Drug resistant bacteria 73-2 and resistant bacteria 73-2 and normal growth resistant reverse bacteria B (Tet) outer row pump gene acrA, acrB, tolC, acrZ and the expression of marA, rob, acrR mRNA of the regulated genes, and the detection of reverse bacteria B (Tet) gene sequence before and after reversal. Results: in the presence of tetrandrine, the expression of drug resistant bacteria 73-2 (Tet), resistance reverse bacteria B (Tet) and drug resistant bacteria 73-2 efflux pump gene acrA/B/Z mRNA were all significantly decreased; tolC mRNA expression 73-2 (Tet) only declined, B (Tet) showed a significant decline. The positive regulation of the external pump was marA, rob expression was expressed. B (Tet) showed a significant increase in the expression of acrR mRNA in the negative regulation gene, while the expression of 73-2 (Tet) acrR mRNA was only rising. The gene base sequence of the drug-resistant reverse bacteria B (Tet) was not changed compared with the drug resistant bacteria 73-2. Conclusion: tetrandrine powder against Escherichia coli of the drug resistant Escherichia coli gene acrA/B/Z and tuning The expression of the controlled gene marA, rob has a significant inhibitory effect and can promote the expression of the negative regulatory gene acrR. It can not make the outer row pump gene acrA/B/Z, tolC and the regulatory gene marA, rob, ACR R base sequence again mutation. Tetrandrine can reverse the expression of the efflux pump to reverse the bacterial resistance.3, tetrandrine The multistep method was adopted to induce the drug resistance of the AcrAB-TolC efflux pump in vitro, and the drug resistance of Escherichia coli ATCC25922 was induced by ciprofloxacin lactate in vitro at the concentration of 1/2MIC, and the combination of ciprofloxacin and tetrandrine was used to detect the effect of tetrandrine to reverse the resistance of the inducible bacteria. The expression of the efflux A (Cip) efflux pump gene acrA/B/Z, tolC mRNA under the condition of tetrandrine. Results: a stable genetic resistant strain A (Cip) was obtained from 1/2MIC as the starting point. Tetrandrine could reduce the MIC from 640 micron g/m to 80 mu under the condition of tetrandrine. The expression of the efflux pump gene acrA/B/Z and tolC mRNA decreased significantly. Conclusion: tetrandrine can also reverse the resistance of drug resistant bacteria in vitro, mainly by reducing the expression of AcrAB-TolC in the efflux pump.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S859.7

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