【摘要】： 目的利用分子生物學(xué)的方法檢測太原地區(qū)雞源性大腸埃希氏菌對氟喹諾酮類藥物的耐藥情況,旨在探討喹諾酮類藥物耐藥決定區(qū)(QRDR)的促旋酶、拓?fù)洚悩?gòu)酶以及孔道蛋白OmpC、OmpF基因變異情況。 方法采集山西省畜牧研究所種雞場30株雞源性大腸桿菌,用紙片法篩選出12株耐氟喹諾酮類藥菌的大腸桿菌,分別用gyrA、gyrB、parC、parE、ompC、ompF基因相應(yīng)的引物對12株耐氟喹諾酮類藥菌的大腸桿菌進(jìn)行PCR擴(kuò)增,并選擇其中4株有代表性菌株進(jìn)行基因序列的測定;用SDS-PAGE電泳對12株耐藥菌的外膜蛋白進(jìn)行分析;用脈沖場凝膠電泳對12株耐藥菌進(jìn)行了流行情況分析。 結(jié)果所有檢測株中,其中12株菌對環(huán)丙沙星耐藥,占所采集標(biāo)本的40%;PCR實(shí)驗(yàn)gyrA、gyrB、parC、parE、ompC、ompF都有清晰的目的條帶;SDS-PAGE電泳結(jié)果顯示雞4、雞5號菌株外膜蛋白OmpC的表達(dá)缺失;脈沖場凝膠電泳結(jié)果分析顯示,存在小范圍的流行,爆發(fā)流行的幾率較小;測序結(jié)果顯示,送檢的測序株都存在GyrA在83或87位氨基酸的變異(Ser-83→Leu;Asp-87→Asn);雞5號菌株P(guān)arC在80位由由絲氨酸變位亮氨酸(Ser-80→Leu),雞6號菌株P(guān)arC在第84位由谷氨酸變?yōu)橘嚢彼?Glu-84→Lys);雞5號菌株P(guān)arE在89位絲氨酸變?yōu)楸彼?Ser-89→Ala)。 結(jié)論山西省畜牧研究所養(yǎng)雞場的大腸桿菌對氟喹諾酮耐藥的主要機(jī)制是由于促旋酶在第83氨基酸、87位氨基酸發(fā)生一點(diǎn)或同時(shí)發(fā)生變異造成的;另外,部分菌株伴隨parC和ParE的突變可能是造成氟喹諾酮類藥物高水平耐藥的主要因素。部分菌株存在外膜蛋白OmpC的缺失,但是OmpC的缺失不是造成以上菌株對氟喹諾酮類抗菌素耐藥的主要原因。
[Abstract]:Objective to detect the resistance of Escherichia coli from chicken to fluoroquinolones in Taiyuan area by molecular biology, and to investigate the protopolase, topoisomerase and pore protein OmpC, of quinolone drug resistance determining region (QRDR). Variation of OmpF gene. Methods 30 strains of Escherichia coli were collected from chicken farm of Shanxi Animal Husbandry Institute. Twelve strains of Escherichia coli resistant to fluoroquinolones were screened by disk method. GyrA,gyrB,parC,parE,ompC, was used respectively. The corresponding primers of ompF gene were used to amplify 12 strains of Escherichia coli resistant to fluoroquinolones and 4 representative strains were selected for gene sequencing. The outer membrane proteins of 12 strains of drug-resistant bacteria were analyzed by SDS-PAGE electrophoresis, and the epidemic situation of 12 strains of resistant bacteria was analyzed by pulsed field gel electrophoresis. Results among all the tested strains, 12 strains were resistant to ciprofloxacin, accounting for 40% of the collected samples. The results of SDS-PAGE electrophoresis showed that the expression of outer membrane protein (OmpC) of chicken strain 4 and chicken 5 was absent, the results of pulse field gel electrophoresis showed that there was a small epidemic, and the probability of outbreak was lower. The sequencing results showed that the Ser-83 Leu;Asp-87 Asn); existed in the GyrA at 83 or 87 amino acids in all the tested strains. Chicken 5 strain ParC changed from Ser-80 Leu), chicken 6 ParC to Glu-84 Lys); at 84th position from Ser-80 serine to Glu-84 Lys); The serine of chicken 5 strain ParE was transformed into Ser-89 Ala). At 89 position. Conclusion the main mechanism of Escherichia coli resistance to fluoroquinolone in chicken farm of Shanxi Animal Husbandry Research Institute is due to the mutation of a little or both amino acids at 83 amino acid and 87 amino acids. In addition, the mutation of parC and ParE in some strains may be the main factor of high level resistance of fluoroquinolones. The absence of outer membrane protein (OmpC) was found in some strains, but the deletion of OmpC was not the main reason for the resistance to fluoroquinolones.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R378

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