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目的研究大肠杆菌对氟喹诺酮抗药性突变的规律及基因型与表型的对应关系。方法以质控菌株Escherichia coli ATCC 25922为初始菌株,恩诺沙星浓度由0.031 25μg/mL增加到128μg/mL,分步诱导得到13株稳定的抗药性菌株,检测氟喹诺酮靶酶基因gyrA、gyrB和parC、parE的序列及其突变位点,检测恩诺沙星的最低抑菌浓度(MIC)及抑制外排泵后MIC,分析抗药性表型与基因型、蛋白结构、外排泵及生化特性的关系。结果低浓度氟喹诺酮可诱导GyrA的Ser83-Leu和ParC的Glu84-Lys单一位点突变,致低水平抗药性,而Ser83-Leu逐步与Asp87-Gly、Glu84-Lys组合,导致氟喹诺酮突变决定区突变位点氨基酸的极性、空间位阻变化,影响靶蛋白与DNA和恩诺沙星的结合,可致高水平抗药性。外排泵与基因突变共同作用,使细菌耐受逐步升高药物浓度,M IC基本平行高出培养浓度1倍。结论增加恩诺沙星的使用剂量,能诱导高抗菌株产生,恩诺沙星抗药性表型与基因型具有对应关系和规律性变化。
Objective To study the rule of fluoroquinolone resistance mutation in Escherichia coli and the corresponding relationship between genotypes and phenotypes. Methods Escherichia coli ATCC 25922 was used as the initial strain. The concentration of enrofloxacin was increased from 0.031 25 μg / mL to 128 μg / mL, and 13 stable strains were obtained in a stepwise manner. The gyrA, gyrB And parC, parE and their mutated sites. The minimum inhibitory concentration (MIC) of enrofloxacin and inhibition of MIC after efflux pump were determined. The resistant phenotype and genotype, protein structure, efflux pump and biochemical Characteristics of the relationship. Results Low concentrations of fluoroquinolone induced a single site mutation in Ser83-Leu in GyrA and Glu84-Lys in ParC, resulting in a low level of drug resistance. Ser83-Leu was gradually combined with Asp87-Gly and Glu84-Lys, leading to fluoroquinolone mutation-determining region Amino acid mutation site mutation, steric hindrance changes affect the target protein and DNA and enrofloxacin combination, can lead to a high level of resistance. Excretory pump and gene mutation work together, so that bacterial resistance and gradually increase the drug concentration, M IC substantially parallel to the culture concentration 1 times. Conclusion Increasing the dose of enrofloxacin can induce the production of highly resistant strains. The enrofloxacin resistant phenotype has a corresponding relationship with the genotype.