Abstract:

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 0.03125μg/mL to 128μg/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.

Key words: Escherichia coli; Antiinfective agents, fluoroquinolone; Serial passage; Point mutation