养殖场分离的耐氟喹诺酮类药物的大肠杆菌基因突变研究

【目的】探讨从养殖场动物、环境和饲养员分离的大肠杆菌的gyrA 和parC 基因突变特征。【方法】用琼脂稀释法测定环丙沙星和恩诺沙星对菌株的最小抑菌浓度。PCR扩增gyrA 和parC 基因的喹诺酮耐药决定区，扩增的片段长度分别为525 bp和487 bp，PCR产物直接测序。【结果】在63株突变株中，在GyrA 亚基发生的氨基酸替代有Ser83→Leu（62株）和Asp87→Asn（52株）、Asp87→Tyr（2株）、Asp87→His（2株）；ParC 亚基的氨基酸替代有Ser80→Ile（47株）、Ser80→Arg（2株）和Glu84→Val（3株）、Glu84→Lys（4株）、Glu84→Gly（5株）、Glu84→Ala（1株）。环丙沙星对菌株的MIC小于0.125μg·ml-1时，GyrA和ParC亚基均没有任何变异；环丙沙星的MIC为0.125～0.25 μg·ml-1时，GyrA亚基出现单一氨基酸替代；环丙沙星的MIC为0.5～32μg·ml-1时，出现GyrA 83位和87位双替代或者GyrA83和ParC80位双替代；环丙沙星的MIC为4～128μg·ml-1，发生GyrA 双替代和ParC单替代；环丙沙星的MIC在16～128μg·ml-1，发生GyrA双替代和ParC 双替代。【结论】不同来源的耐氟喹诺酮类药物的大肠杆菌GyrA和ParC具有多种氨基酸替代类型，而且GyrA和ParC突变位点的数量与菌株对氟喹诺酮类耐药水平呈正相关。

Mutations of gyrA and parC in Fluoroquinolones-Resistant Escherichia coli Strains Isolated from Animals, Farmers and Environment

【Objective】To detect mutations of gyrA and parC in 72 Escherichia coli strains isolated from human feces, animal feces and the environment.【Method】MICs of ciprofloxacin or enrofloxacin for 72 isolates were determined by an agar dilution test with standard strain ATCC25922 as the control. The mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed by PCR and sequencing. 【Result】Amino acid substitutions (number of strains) found in 63 mutants were Ser83→Leu（62）and Asp87→Asn（52），Asp87→Tyr（2），Asp87→His（2）in GyrA and Ser80→Ile（47），Ser80→Arg（2）and Glu84→Val（3），Glu84→Lys（4），Glu84→Gly（5），Glu84→Ala（1）in ParC．No amino acid changes in GyrA or ParC were detected when strains MIC of ciprofloxacin were under 0.125 μg·ml-1．A single substitution in GyrA was found in the strains whose MIC of ciprofloxacin ranged from 0.125 to 0.25μg·ml-1．Double substitutions in GyrA or one in GyrA with one in ParC were found in the strains with MICof ciprofloxacin ranging from 0.5 to 32 μg·ml-1．A double changes in GyrA and a single change in ParC were found in strains with MIC of ciprofloxacin ranging from 4 to 128 μg·ml-1．Double changes in GyrA and double changes in ParC were found in strains with MIC of ciprofloxacin ranging from 16 to 128 μg·ml-1．【Conclusion】A variety of amino acid changes in GyrA and ParC were detected in the fluoroquinolone-resistant E. coli isolates from different origin．A correlation was found between the number of changes in the QRDR of GyrA and ParC and the resistance level to fluoroquinolone in E. coli strains.