一株多重耐药大肠杆菌耐喹诺酮gyrA基因突变的研究

Quinolone-resistant Mutaions of the gurA Gene of a Strain of E.coli with Multiple Drug Resistance

临床获得 1株鸡致病性大肠杆菌CE0 1,进行药敏、质粒转化、耐药质粒消除试验 ,结果该菌对 11种受试抗菌药物全部呈高度耐药 ,其中氧氟沙星的MIC≥ 5 0 μg/ml,且含有喹诺酮抗性质粒 ,溴化乙淀和黄连素作为消除剂作用 48小时可使其喹诺酮抗性质粒消除 ,耐药水平显著下降。聚合酶链反应 (PCR)扩增该菌gyrA基因喹诺酮耐药决定区 (QRDR) ,质粒DNA和染色体DNA均可扩增出长度为 6 6 8bp的PCR产物片段。经测序分析 ,两者基因序列相同率为 98.17%,相同位点突变相同的碱基有 5个 ,相同位点突变不同的碱基有 2个 ;与基因数据库Swanberg等报道的大肠杆菌gyrA基因相应序列比较 ,该菌质粒DNAPCR产物同源率 97.8%,有 13个位点发生突变 ,3个氨基酸被替换 ;染色体DNAPCR产物同源率 98%,有 12个位点发生突变 ,2个氨基酸被替换。用放射性同位素α_3 2 P分别标记CE0 1菌株染色体DNA和质粒DNA的PCR扩增片段制备探针 ,对CE0 1菌株进行检测。质粒DNA和染色体DNA分别与质粒探针和染色体探针杂交出现典型的阳性杂交斑。证实该菌株质粒和染色体上同时存在耐喹诺酮gyrA突变基因 ,对喹诺酮的耐药性是质粒介导和染色体突变共同作用的结果。

A Quinolone resistant strain CE01 of chicken pathogenic E.coli is collected from clinical isolates.After the plasmid DNA extraction,purification,transformation and elimination,the Quinolone resistant plasmid is found in the strain CE01,the quinolone resistance determining region (QRDR) of the gyrA gene is amplified by PCR with the plasmid DNA and chromosomal DNA templates from the strain CE01.The expected 668 bp gyrA fragments are both obtained from them by PCR.The nucleotide sequences of PCR product from plsmid DNA and chromosomal DNA of strain CE01 shows significant homology (98.17% identity).When compared to the corresponding sequences of gyrA of E.coli from the nucleotide sequence data reported by Swanberg,S.L. and Wang,J.C.,13 mutant sites are found in the nucleotide sequence of PCR product from plasmid DNA,and 3 amino acids changed;while 12 mutant sites are found in which from chromosomal DNA,and 2 amino acids changed.And the amplified products from plasmid DNA and chromosomal DNA of the strain CE01 are labelled with ɑ_ 3cp dCTP by nick translation reaction as probe to detect plsmid DNA and chromosomal DNA of the strain CE01 by dot_blot hybridization.Plasmid DNA and chromosomal DNA of the strain CE01 all obtained positive dot_blot to plasmid probe and chromosomal probe correspondingly.As a result,quinolone resistant gene exists surely both in the plasmid and chromosome of strain CE01.And the plasmid and chromosome are associated with quinolone resistance of strain CE01.