【作者】 苑青艳；

【导师】 李艳华；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 猪的链球菌病是由多种溶血性链球菌引起的一种多型性疫病,已成为现代养猪业生产经济损失重要的疫病之一。最常见的致病类型为血清型2型,人感染猪链球菌主要引起脑膜炎、败血症等严重疾患。根据近些年来的报道,猪链球菌对氟喹诺类酮药物的耐药性也在日益增强。氟喹诺酮类药物的作用机制是抑制细菌拓扑异构酶Ⅱ及拓扑异构酶Ⅳ的活性,导致细菌无法正常分裂而死亡。目前报道的猪链球菌对氟喹诺酮类药物的耐药机制是靶位改变和外排增强。氟喹诺酮类药物作用靶位拓扑异构酶Ⅳ由ParC及ParE组成,分别由parC、parE基因编码。DNA旋转酶为三磷酸腺苷能量依赖性Ⅱ类拓扑异构酶,由两对亚单位GyrA和GyrB组成,分别由gyrA、gyrB基因编码。突变点位多发生于ParC及GyrA的67和106残基间这一狭窄区域,因此该区域被称作gyrA及parC基因的喹诺酮耐药决定区(Quinolone resistance determining region,QRDR)。目前国内还未见对猪链球菌parC、gyrA基因点突变与耐药性的相关报道。本研究将对东北地区分离的耐氟喹诺酮类药物猪链球菌的gyrA、parC基因的点突变及氨基酸突变进行研究,阐明gyrA、parC基因的点突变及氨基酸突变与耐药水平的关系,为预防猪链球菌耐药性提供科学的理论基础。利用东北地区不同猪场健康猪及发病猪的鼻腔棉拭子为样品,利用生化试验、兰氏分群的方法分离出100株猪链球菌并对其进行致病性试验;通过纸片扩散法和微量稀释法测定猪链球菌对九种常用的抗菌药物以及11种氟喹诺酮类药物的敏感性;通过PCR方法扩增28株猪链球菌氟喹诺酮类药物耐药株的parC和gyrA基因的QRDR区并测序分析。猪链球菌生化试验的结果表明:马尿酸盐和高盐肉汤均为100%阴性,棉籽糖和菊糖57%为阴性,甘露醇和乳糖98%为阳性,山梨醇90%为阳性,七叶苷、水杨苷和蕈糖均为100%阳性;兰氏分群98%为D群;猪链球菌对氯霉素、庆大霉素、红霉素、青霉素G、复方新诺明、氨苄青霉素、苯唑青霉素、环丙沙星、四环素的耐药率分别为69%、75%、96%、82%、91%、98%、100%、48%、97%;对乳酸环丙沙星、甲磺酸加替沙星、盐酸沙拉沙星、盐酸左氧氟沙星、盐酸洛美沙星、氧氟沙星、氟罗沙星、乳酸诺氟沙星、恩诺沙星、双氟沙星、培氟沙星耐药率分别为:48%、45%、51%、34%、85%、30%、86%、69%、35%、53%、75%。parC和gyrA基因扩增测序结果为:28株猪链球菌的同源性分别为89.3%和91.6%,parC和gyrA基因与网上发布的标准序列的同源性为74%和80%,突变位点多发生在parC基因核苷酸33~264位点和gyrA基因核苷酸的57~369位点;有18株耐药菌发生了parC基因的S80→I突变,其中的11株高度耐药菌(MIC均≥32μg/ml)发生了gyrA基因的S81→I、F或Y的突变。当菌株对环丙沙星的MIC值≤1μg/ml时,parC和gyrA基因的QRDR区均未检测出突变,而当MIC≥2μg/ml时,parC基因发生了S80→I的突变,同时发生gyrA基因Ser81突变的菌株,耐药水平很高;耐药基因的扩增和测序分析发现的低水平氟喹诺酮类耐药是由于拓扑异构酶Ⅳ改变引起,而高水平耐药是由拓扑异构酶Ⅳ、DNA旋转酶共同改变引起的。突变位点越多,耐药性越强。更多还原

【Abstract】 Streptococcus suis infection is a kind of polytypism plague caused by Streptococcus suis which is now one of the most important factor on pig husbandry loss. SerotypeⅡhas been discovered to be the most frequently associated with zoonosis. Meningitis and septicemia of human can also be caused by S.suis. It is crucial and inevitable for pig industry of many countries about how to prevent and cure S.suis infection effectively. According to recent reports, isolations of S.suis strain resistant to fluoroquinolones is getting commonplace.The action mechanism of fluoroquinolones is to inhibit the activity of topoisomeraseⅡand topoismeraseⅣby creating a Quinolone-DNA-topoisomerase complex so that bacteria might die because its normal cleavage can not be completed. Resistance mechanism reported about S.suis against fluoroquinolones is mutations of target site and efflux pump. TopoisomeraseⅣis made of two subunit: ParC and ParE, encoded by parC and parE. DNA gyrase which catalyzes ATP-dependent DNA supercoiling is made of GyrA and GyrB, encoded by gyrA and gyrB gene. With GyrA and ParC subunits of resistant bacteria, amino acid changes are generally localized to a region of the enzyme in the amino terminus that contains the active site that is the mutation sites which are often found in a certain stegnotic segment between residue 67 and 106 of ParC and GyrA, named QRDR of the two genes. There isn’t any report about the relationship between S.suis resistance to fluoroquinolones and the mutation of parC and gyrA genes in China until now. This experiment will study on the mutation of the two genes of S.suis isolated from the Northeast of China in order to provide useful basic scientific information for further research on S.suis and the proventation of S.suis disease.Srains of S.suis were isolated from hogpens of northeast area using nasal swabs. 100 srains were identified after biochemical reaction and lancefield group experiments. Pathogenicity was tested on Kunming mice. Resistant strains against 9 kinds of common used antibiotics and 11 kinds of fluoroquinolones drugs were selected by disk susceptibility test and microdilution susceptibility test. The mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes from 28 strains of resistant bacteria were analyzed by polymerase chain reaction (PCR), the production of PCR was purified and sequenced and analyzed. The results of biochemical reaction are like these: all strains didn’t live in 6.5% NaCl broth and hippurate, 57% strains did not live in the condition of raffinose or alantin; 98% strains could make use of manicol and lactose; 90% of strains decomposed sorbitol; all strains could live with esculin, salicin, Trehalose; 98% strains are identified as D type with the test of lancefield group. The results of antibiotic sensibility against nine common drugs are like these: 69% strains were resistant to alficetin, and 75% to cidomycin, 96% to erythromycin, 82% to penicilin G, 91% to trimethoprim- sulfamethoxazole, 98% to ampicillin, 100% to oxacillin, 97% to tetracycline, 48% to ciprofloxacin. The results of antibiotic sensitivity tests of S.suis against 11 kinds of fluoroquinolones are like these: 48% strains were resistant to ciprofloxacin, 45% to gatifloxacin, 51% to sarafloxacin, 34% to levofloxacin, 85% to lomefloxacin, 30% to ofloxacin, 86% to fleroxacin, 69% to norfloxacin, 35% to enrofloxacin, 53% to difloxacin, 75% to pefloxacin;Homology analysis indicated that parC and gyrA genes of 28 strains have the homology of 89.3% and 91.6% respectively, and the homology with those standard srains submitted online were 74% and 80% respectively. Nucleotide mutaion mostly occurred among the 33~264 sites of parC genes and 57~369 sites of gyrA genes. S80→I mutation of parC gene were occurred in 18strains of fluoroquinolone resistant S.suis and Ser81→Ior F or Y of gyrA gene in 11 highly resistance strains; no amino acid change in gyrA or parC was detected when strains MIC of ciprofloxacin were under 1μg/ml.A single substitution in parC was found in the strains whose MIC of ciprofloxacin was higher than 2μg/ml.Strains in which the mutation of S81 of gyrA were occurred at the same time had a high resistance level. Mutations in parC gene result in low level resistance against fluoroquinolones, and high level resistance is the outcome of the mutations in both genes.The numbers of mutations is proportional to the level of fluoroquinolone resistance.更多还原