体外诱导大熊猫大肠埃希菌对普多沙星的耐药性及其机制研究

发布时间：2018-03-17 04:33

本文选题：大熊猫　切入点：大肠埃希菌　出处：《西北农林科技大学学报(自然科学版)》2017年03期 　论文类型：期刊论文

【摘要】：【目的】探究大熊猫源大肠埃希菌对新型动物专用氟喹诺酮类药物普多沙星的耐药机制。【方法】以从大熊猫体液分离的对氟喹诺酮类药物敏感的大肠埃希菌株为研究对象,采用环丙沙星、麻保沙星和普多沙星通过体外逐渐递增药物浓度的方法诱导其成为耐药菌株。采用PCR扩增和基因测序的方法,分析诱导耐药菌株的靶位基因gyrA和parC耐药决定区(QRDR)的基因突变情况。采用外排泵抑制剂苯丙氨酸-精氨酸-β-萘酰胺(PAβA)和实时荧光定量PCR方法探讨外排泵在体外耐药中的作用。【结果】采用体外浓度递增法可诱导大熊猫大肠埃希菌对3种氟喹诺酮类药物产生耐药性,其中环丙沙星最易产生耐药性,其次是麻保沙星,普多沙星相对较慢。普多沙星诱导的耐药菌首先在gyrA基因发生点突变,随着MIC的升高,靶基因突变位点也逐渐增多。环丙沙星和麻保沙星诱导的4株耐药菌的gyrA基因出现Ala 119Glu突变,而该突变在普多沙星所诱导的突变体中未发现。acrB,emrE和mdfA 3种外排泵基因在所有诱导的耐药菌株中均出现不同程度的过量表达,特别是在环丙沙星和麻保沙星诱导突变体中的表达明显高于在普多沙星诱导突变体中的表达。3种受试外排泵基因的表达均随MIC的升高而增加。【结论】普多沙星引起的大熊猫致病性大肠埃希菌多重耐药性,主要由靶基因的点突变和外排泵基因的过量表达所导致。
[Abstract]:[objective] to investigate the mechanism of resistance of Escherichia coli to prodofloxacin, a novel animal specific fluoroquinolone drug. [methods] A strain of Escherichia coli, which was sensitive to fluoroquinolones, was isolated from the body fluid of giant panda. Ciprofloxacin, eplofloxacin and profloxacin were induced to become drug-resistant strains by increasing drug concentration in vitro. PCR amplification and gene sequencing were used. The gene mutations of target gene gyrA and parC resistance determinant region (parC) in drug-resistant strains were analyzed. The efflux pump inhibitor, phenylalanine-arginine-尾-naphthalamide PA 尾-A) and real-time fluorescence quantitative PCR were used to study the resistance of efflux pump in vitro. [results] the drug resistance of Escherichia coli to three fluoroquinolones was induced by increasing concentration in vitro. Ciprofloxacin was the most susceptible to drug resistance, followed by eplofloxacin, which was relatively slow. Profloxacin induced drug resistant bacteria first mutated in the gyrA gene, and with the increase of MIC. The mutation sites of target gene also increased gradually. The gyrA gene of 4 drug-resistant strains induced by ciprofloxacin and epofloxacin showed Ala 119Glu mutation. However, in the pudofloxacin induced mutants, three efflux pump genes, acrBnemrE and mdfA, were not found to be overexpressed to varying degrees in all induced drug-resistant strains. Especially in ciprofloxacin and epofloxacin induced mutants, the expression of 3.3-species efflux pump genes increased with the increase of MIC. [conclusion] the expression of profloxacin induced mutants was significantly higher than that of profloxacin induced mutants. [conclusion] the expression of profloxacin induced by profloxacin increased with the increase of MIC. The pathogenic Escherichia coli from the giant panda has multiple drug resistance. It is mainly caused by point mutation of target gene and overexpression of efflux gene.
【作者单位】：西北农林科技大学动物医学院;陕西省富平县畜牧兽医局;
【基金】：中央高校基本科研业务费项目(2452016044) 西北农林科技大学博士科研启动基金项目(2013BSJJ012);西北农林科技大学大学生创新创业训练计划项目(2014068)
【分类号】：S852.61

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