利用重建祖先基因方法研究连续诱导培养大肠杆菌对恩诺沙星抗药性突变的规律

The law of the resistant mutation of Escherichia coli strains continuously induced with Enrofloxacin by ancestral gene reconstruction

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 mutation law of fluoroquinolone resistance of E.coli,and the correspondence between genotype and phenotype.Methods The quality control strain Escherichia coli ATCC 25922 was used as the initial strain to continuously induce with increasing concentrations of enrofloxacin from 0.031 25 μg/mL to 128 μg/mL,and 13 stable enrofloxacin-resistant strains were obtained.The mutation sites were detected by complete sequencing fluoroquinolone target gene gyrA,gyrB,parC,and parE.while the corresponding MICs and MIC by efflux pump inhibition of each strain were measured.Then the relationships between resistance phenotypes and the genotypes,protein structures,biochemical properties and efflux pump activities were analyzed.Results Low concentrations of fluoroquinolone did not induce high resistance mutations.A single locus of mutation Ser83-Leu in GyrA,and even combined with mutation Glu84-Lys in ParC could cause low-level resistance.However,Ser83-Leu gradually combined with Asp87-Gly and Glu84-Lys mutations resulting in the changes of polar and steric effects of mutation amino acids in the ＂Quinolones resistance determining region＂,which induced high resistance mutations by precluding the combination of enrofloxacin,target proteins and DNA.In addition,Efflux pumps combined with gene mutations led to bacterial tolerance to the gradually increasing drug concentrations.MIC had about twice the inducing drug concentrations.Conclusion An increasing dose of fluoroquinolone can induce high resistance,and there are regular corresponding relationships between phenotypes and genotypes of enrofloxacin resistance.