【作者】 雷连成；

【导师】 王兴龙； 韩文瑜；

【作者基本信息】 中国人民解放军军需大学 ， 预防兽医学， 2001， 硕士

【摘要】 氟喹诺酮药物（即第三代喹诺酮类药物）自1978年发现以来由于其疗效显著得到了国内外的广泛重视。但是细菌对氟喹诺酮类药物极易产生耐药性，而且传播广泛、迅速，据推测，氟喹诺酮药物的抗性质粒可能是耐药性传递的重要因素之一，但是在耐药机制复杂、对氟喹诺酮类耐药率极高的大肠杆菌中尚未报道有此类质粒的存在。本课题以研究大肠杆菌对氟喹诺酮等常用药物的耐药现状为基础，以危害严重的多重、超强耐药大肠杆菌为研究对象，研究了氟喹诺酮抗性质粒及其与染色体gyrA基因喹诺酮耐药决定区（QRDR）突变共同作用的耐药机制。 1996年10月至1999年月4月分离鉴定了146株动物源性致病性大肠杆菌，采用Kinby-Bayer（KB）琼脂弥散法测定对18种抗生素的耐药性，测定标准按美国NCCLS标准。结果最少耐药3种，最多耐药15种，其中对氟喹诺酮的耐药率猪源大肠杆菌为56％，鸡源大肠杆菌为20.5％。 筛选出32株以氟喹诺酮耐药为主的多重耐药大肠杆菌，质粒提取、纯化后转化DH5α、JM109大肠杆菌感受态，进行转化，用含20μg/ml OFL的LB平板筛选转化子，在菌株CE01质粒获得转化子。 EB、BS作为耐药性消除剂对32株耐氟喹诺酮药物的致病性大肠杆菌分别处理24h，48h，其中4株对氟喹诺酮的耐药水平明显降低，提取其质粒进行电泳检测，结果只有大肠杆菌CE01在转化试验中可随氟喹诺酮抗性转移的质粒带随耐药水平的降低而减弱或消失，其余3株耐药水平降低的菌株质粒带无变化。 用聚合酶链式反应（PCR）扩增CE01菌株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668bp的DNA片段。经测序分析，两者基因序列相同率为98.17％。与基因数据库Swanberg，S.L.and Wang,J.C.报告的大肠杆菌gyrA基因相应序列比较，该菌质粒DNA PCR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DNA PCR产物同源率98.00％，有12个位点发生突变，2个氨基酸被替换。 放射性同位素α-³²P分别标记CE01菌株染色体DNA和质粒DNA的PCR扩增片段制成探针，用DNA斑点杂交法对CE01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。将CE01菌株质粒DNA于0.7％琼脂糖凝胶电泳后转移至硝酸纤维素膜，采用奶粉杂交体系与α-³²P标记的CE01菌株质粒DNA的PCR扩增片段进行Southern杂交，CE01菌株质粒谱中可随氟喹诺酮抗性转移的质粒带与质粒探针出现杂交阳性。证实CE01菌株含有氟喹诺多重迟强耐荷大肠抨官耐派喳话酮质粒及gw篡因的研宠 中文折昙酮抗性质粒，并在质粒和染色体上同时存在耐喳诺酮gyrA突变基因，该菌对氧氟沙星高水平耐药是质粒介导和染色体突变共同作用的结果。更多还原

【Abstract】 DNA gyrase of Escherichia co/i consists of subunits A and B,which are the products of the gyrA and gyrB genes,respectively. Mutations in gyrA gene can cause quinolone resistance. DNA sequence analysis previously done has disclosed that these mutations were located within a relatively small region of the A subunit, which is called a quinlolne resistance-determining region (QRDR). and these mutations are basic and important for quinoloneresistance. Over the last decade there have been sporadic reports of plasmidmediated quinolone-resistance, which described when a mutator plasmid was present .the frequency of resistance for selecting antibiotic-resistant strains always went up,but no quinolone-resistance plasmid was found in E.coli. To study on the mechanisms of Fluoroquinolone resistance of E.coli. the drug susceptibility of E.coli clinical isolates from Jilin province during the past 3year period (1996-1999) were tested. 32 strains of pathogenic E. co/i, which were superhigh fluoroquinolone-resistant and multi-drug resistant, were further studied. After the plasmid DNA extraction, purification and transformation. only one multi-drug resistant strain CEO1 whose fluoroquinolone resistance was associated with its plasmid DNA was obtained, and after drug-resistant plasmid elimination, there were only four strains which levels of fluoroquinolone resistance lowered clearly after elimination . Then plasmids of these four strains were extracted and electrophoresized, a plasmid of strain CEO 1, which can be transferred with fluoroquinolone resistance, had been eliminated by BS or EB while there were no changes of plasmids associated with eliminators in the others. Therefore, the fluoroquinolone-resistant plasmid was found in the strain CEO 1. And the quinolone resistance-determining region (QRDR) of the gyrA gene v%as amplified by PCR with the plasmid DNA and chromosomal DNA templates from the strain CEO 1. The expected 668-bp gy抮A fragments were both obtained from them by PCR. The nucleotide sequences of PCR products58from plsmid DNA and chromosomal DNA of strain CEOI showed significant homology (98.17% identity). When compared to the corresponding sequences of gyrA of F. coil from the nucleotide sequence data reported by Swanberg,S.L. and Wang,J.C., 13 mutant sites were found in the nucleotide sequence of PCR product from plasmid DNA, and 3 amino acids changed:while 12 mutant sites were found in that from chromosomal DNA, and 2 amino acids changed. The amplified products from plasmid DNA and chromosomal DNA of the strain CEO1 were labelled with a -32P dCTP by nick translation reaction. and were used as probes to detect plasmid DNA and chromosomal DNA of the strain CEO1 by southern hybridization and dot-blot hybridization, respectively . Plasmid DNA and chromosomal DNA of the strain CEOI all obtained positive dot-blot to plasmid probe and chromosomal probe correspondingly. As a result, fluoroquinolone-resistant rate of E.coli strains isolated from swine was 56%, that from chicken was 20.5%. Strain CEOI has a plasmid associated with fluoroquinolone resistance. and fluoroquinolone resistant mutant gy抮A gene existed surely both in the plasmid and chromosome of strain CEO 1. Both the plasmid and chromosome are associated with fluoroquinolone resistance of strain CEO 1.更多还原