本文選題：大腸埃希菌 + 粉防己甲素�。� 參考：《西南大學》2017年碩士論文

【摘要】：抗生素和其他抗菌藥物的發(fā)展可以說是現(xiàn)代醫(yī)學最偉大的成就,它增加了人類的平均壽命。但是,過度使用和濫用抗生素及抗菌藥已導致了微生物的耐藥性。喹諾酮類抗菌藥在人類和動物的臨床使用上有著極其重要的作用。然而細菌對喹諾酮類藥物的耐藥性卻日趨嚴重,特別是大腸埃希菌對喹諾酮類藥物的耐藥率呈逐年增高的趨向。現(xiàn)如今,喹諾酮類藥物的臨床治療效果低下,使用劑量大、治療成本高等諸多問題擺在我們面前。如果能控制或解決細菌耐藥性的問題,無疑是醫(yī)學界的一重大成果,可以大大提高現(xiàn)有抗菌藥的抗菌效果。當前,為應對細菌耐藥性問題,研究比較多的是關于細菌外排泵抑制劑的研究,如利血平、維拉帕米、碳酰氰基-對-氯苯腙(CCCP)等,但由于其毒副作用很難應用于臨床。近年來發(fā)現(xiàn)的天然植物活性成分如小檗堿、胡椒堿等也被證實擁有抑制外排泵的作用。中藥有效成分相對安全、殘留小、無耐藥性危害等,在產品研發(fā)與臨床應用上具有明顯優(yōu)勢。本試驗通過結合前期實驗室的研究,選擇中藥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藥大腸埃希菌外排喹諾酮類藥物的效果,為后期外排泵抑制劑的確認奠定基礎;同時通過對耐藥菌逆轉機制的初步探究,為粉防己甲素外排抑制劑的研制提供理論依據(jù)。1、粉防己甲素對臨床分離耐藥大腸埃希菌的逆轉作用(1)采用聯(lián)合藥敏試驗測定粉防己甲素對乳酸環(huán)丙沙星抑菌效果的影響;(2)使用不同濃度的乳酸環(huán)丙沙星HM肉湯進行耐藥菌73-2殺菌試驗,繪制乳酸環(huán)丙沙星以及聯(lián)合粉防己甲素和CCCP對耐藥菌73-2的殺菌曲線;(3)使用含粉防己甲素和不同濃度乳酸環(huán)丙沙星的含藥MH瓊脂平皿測定在粉防己甲素存在下大腸埃希菌ATCC25922的突變率;(4)采用高效液相色譜法分別檢測耐藥菌73-2和大腸埃希菌ATCC25922在粉防己甲素條件下,細菌體內乳酸環(huán)丙沙星的蓄積量,試驗以CCCP作為藥物對照;(5)使用含粉防己甲素的LB肉湯反復培養(yǎng)耐藥菌73-2,以獲得敏感性恢復的逆轉細菌。結果:乳酸環(huán)丙沙星聯(lián)合粉防己甲素對大腸埃希菌73-2的FIC指數(shù)為0.1875,具有較強的協(xié)同作用,使用160μg/mL的粉防己甲素和10μg/m L的CCCP與乳酸環(huán)丙沙星聯(lián)合應用,乳酸環(huán)丙沙星對耐藥菌73-2的最小抑菌濃度(MIC)均由聯(lián)合前的320μg/m L降低到10μg/m L,而對大腸埃希菌ATCC25922的MIC值無影響;粉防己甲素可以提高1/2MIC濃度的乳酸環(huán)丙沙星對耐藥菌73-2的殺滅作用,抑制細菌的生長;在4MIC濃度的乳酸環(huán)丙沙星條件下粉防己甲素可以有效降低大腸埃希菌ATCC25922的突變,突變率從正常的10-7降低到10-9;在粉防己甲素存在條件下,耐藥菌73-2細胞體內乳酸環(huán)丙沙星的蓄積量明顯比正常情況明顯增加,約為4.2倍,而對大腸埃希菌ATCC25922幾乎無影響,在CCCP存在條件下,也出現(xiàn)相似情況,耐藥菌73-2乳酸環(huán)丙沙星的蓄積量約是正常條件下的3.9倍;通過使用含粉防己甲素的肉湯培養(yǎng)耐藥菌得到了乳酸環(huán)丙沙星對其MIC下降的菌株B(Tet)。結論:粉防己甲素具有增強乳酸環(huán)丙沙星殺滅細菌的作用,可以抑制細菌的突變,增加乳酸環(huán)丙沙星在耐藥菌細胞內的蓄積量。粉防己甲素具有外排抑制劑的活性。2、粉防己甲素對臨床分離耐藥大腸埃希菌的逆轉機制研究檢測正常生長的耐藥菌73-2和在粉防己甲素條件下生長的耐藥菌73-2及正常生長的耐藥逆轉菌B(Tet)外排泵基因acrA、acrB、tolC、acrZ及其調控基因marA、rob、acrR mRNA的表達量;檢測逆轉菌B(Tet)在逆轉前后外排泵基因acrA/B/Z、tolC和調控基因marA、acrR、rob的堿基序列。結果:在粉防己甲素存在下生長的耐藥菌73-2(Tet)和耐藥逆轉的細菌B(Tet)和耐藥菌73-2外排泵基因acrA/B/Z mRNA的表達量比較都極顯著下降;tolC mRNA的表達量73-2(Tet)只是下降,B(Tet)表現(xiàn)為極顯著下降。對外排泵起正調控作用的marA、rob mRNA表達量都極顯著減少;B(Tet)表現(xiàn)出負調控基因acrR mRNA表達量顯著上升,而73-2(Tet)acrR mRNA表達量的只是上升。耐藥逆轉菌B(Tet)的相關基因堿基序列和耐藥菌73-2相比均未發(fā)生改變。結論:粉防己甲素對耐藥大腸埃希菌的外排泵基因acrA/B/Z和正調控基因marA、rob的表達具有顯著的抑制作用,可以促進負調控基因acrR的表達。不能使耐藥菌的外排泵基因acrA/B/Z、tolC和調控基因marA、rob、acr R的堿基序列再次發(fā)生突變。粉防己甲素可以通過影響外排泵的表達逆轉細菌的耐藥性。3、粉防己甲素對體外誘導耐藥菌AcrAB-TolC外排泵的作用采取多步法,以1/2MIC濃度的乳酸環(huán)丙沙星體外人工誘導大腸埃希菌ATCC25922產生耐藥性;將乳酸環(huán)丙沙星與粉防己甲素聯(lián)合應用,檢測粉防己甲素是否具備逆轉誘導菌耐藥性的作用;檢測在粉防己甲素條件下生長的誘導菌A(Cip)外排泵基因acrA/B/Z、tolC mRNA的表達量。結果:以1/2MIC為起點人工體外誘導獲得了能穩(wěn)定遺傳的耐藥菌A(Cip);粉防己甲素可以使乳酸環(huán)丙沙星對耐藥菌A(Cip)的MIC從640μg/m L下降到80μg/mL;在粉防己甲素條件下外排泵基因acrA/B/Z、tolC mRNA表達量極顯著減少。結論:粉防己甲素對體外誘導的耐藥菌也能很好的進行耐藥逆轉,其作用方式主要是通過減少外排泵AcrAB-TolC的表達量。
[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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S859.7

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