【作者】 王亚宾；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2009， 博士

【摘要】 肠球菌为球形或卵圆形、呈链状排列的革兰阳性细菌。自从1984年从链球菌属中分离出来单独成属以来,其成员已由原来的2个种增加到41个种。肠球菌不但可引起住院病人的腹膜炎、心内膜炎、尿道炎和脑膜炎等,也可引起猪、羊、鸡和鸭等动物的感染。感染动物的肠球菌可直接传染给人,而食品中污染的肠球菌可导致人的食物中毒,因此肠球菌在公共卫生学上具有重要的意义。2003～2008年期间,我们在河南省许昌、南阳、郑州、洛阳等地送检的病死猪分离出43株疑似肠球菌的病原性细菌,该些细菌主要引起患猪败血症、脑膜炎和关节炎。对其进行了常规的形态学观察、耐受性试验、Vitek-32生化鉴定和全细胞蛋白型分析,并做了16S rRNA和RAPD等分子水平的研究。在此基础上,调查了各分离株的毒力基因携带情况,并建立了小鼠腹膜炎模型,进一步比较了毒力基因、毒力表型和抗生素抗性与分离菌的致病性关系,以期为研究细菌致病机制、猪感染的流行病学以至最终的预防和控制提供参考依据。细菌学检验结果表明,所有的分离菌均呈G（?）,卵圆形或球形,并呈长短不一的链状排列,能够在6.5%NaCl肉汤、pH 9.6葡萄糖肉汤和10℃、45℃和60℃30 min条件下生长。Vitek-32全自动细菌鉴定系统仅成功鉴定出了43个分离株中的35株,分别为粪肠球菌（E.faecalis）11株、屎肠球菌（E. faecium）2株、铅黄肠球菌（E. casseliflavus）22株,另有8个分离菌株未能鉴定到种的水平。细菌全细胞蛋白型分析结果与Vitek-32有明显不同,43株分离菌全细胞蛋白条带的分子量差异主要在97 KDa、90 KDa、67 KDa和46 KDa处,其中具有蛋白分子量90 KDa条带的菌株有13个,与参考菌株ATCC29212和ATCC33186相似；而其余的菌株均具有46KDa、67 KDa和97 KDa 3个条带,故认为它们是另外一种不同于粪肠球菌的肠球菌种类。16S rRNA基因序列分析结果表明,43株肠球菌的16S rRNA序列与GenBank中登录的各种肠球菌同源性均较高。经利用DNAStar软件将感染猪的疑似肠球菌分离株与GenBank中32种肠球菌的16S rRNA序列进行序列同源性分析,所有43个分离株均被鉴定到种的水平,其中13株与GenBank中登录的4株粪肠球菌（E.faecalis）模式菌株AF515223、AJ301831、EU728747、NC₀04668的同源性在99.3%～99.9%之间；而另30个分离株除HE25之外均与GenBank中登录的3株屎肠球菌（E.faecium）模式菌株EU547780、DQ411813、Y18294的同源性在99.3%～100%之间；HN-25与其余29个屎肠球菌菌株的同源性在99%～99.4%之间。所测定的42株肠球菌（HN-N1～HN-N42）16S rRNA序列已被GenBank收录,其登录序列号依次为FJ378656～FJ378697。合成了19条随机引物对肠球菌基因组DNA多态性进行分析,以从基因组水平上进一步验证鉴定的正确性,并通过分析各菌株亲缘关系为肠球菌的流行病学积累资料。结果显示4条引物对所有菌株均扩增出了多态性较好的条带,通过SPSS11.5软件计算其相似性系数,发现HN-3和HN-6菌株间遗传距离指数为0.000,可能为同源菌株,其他菌株亲缘关系则较远。在聚类分析中,聚类重新标定距离（rescaled distance cluster combine）为21时,具有鉴定肠球菌至种水平的能力,试验的肠球菌菌株聚类成4人类群,分别为粪肠球菌聚类群、屎肠球菌聚类群、鸟肠球菌聚类群和仅有HN-25菌株聚类群。在粪肠球菌聚类分析中,聚类重新标定距离为19时,可将试验的粪肠球菌分成4个聚类群；而屎肠球菌在聚类重新标定距离为19时,则分成2个聚类群,除HN-25菌株为一聚类群外,其他屎肠球菌形成一个人的聚类群。为了研究肠球菌致病机制,通过调查猪临床菌株、肉品菌株和猪粪便菌株12种毒力基因携带情况,发现粪肠球菌较屎肠球菌携带更多的毒力基因（7.83：3.73）；而3种来源的肠球菌中以临床菌株毒力基因最多,其次是肉品菌株,粪便菌株携带毒力基因最少。临床粪肠球菌分离株中毒力基因gelE、efaA和sprE的检出率为100%,ace、cylA和esp的检出率分别达到84.6%、84.6%和69.2%,均明显高于其他来源的菌株；而在临床屎肠球菌菌株中,cylA和gelE的检出率分别为83.3%和66.7%,也明显比其他来源的高。在小鼠腹膜炎模型中,粪肠球菌均比屎肠球菌致病性强,而在试验的粪肠球菌中,各菌株的致病性也不相同,其中,以HN-41的致病性最强,其LD50为106.01/ml,而HN-1致病性最弱,其LD50为107.01。屎肠球菌HN-32也较HN-15的LD50高。通过分析各菌株的毒力基因、毒力表型和抗生素抗性与致病性关系时发现,致病性的大小与毒力基因携带多少成正相关,gelE、cylA、ace和esp等毒力基因对致病性影响较大；毒力表型与致病性大小关系与肠球菌种有关,同种肠球菌中,β溶血性表型比明胶溶解表型与致病性大小更密切：肠球菌耐药种类多少与其致病性关系不明显。更多还原

【Abstract】 ABSTRACT Enterococci are globular or orbicular-ovate, catenulate gram-positive bacteria. Since Enterococcus was separated from Streptoococcus as a new genus in 1984, its members have increased from the original two species to forty-one. Enterococci may not only cause peritonitis, endocarditis, urethritis and meningitis in hospital patients, but may lead to infection in animals such as swine, sheep, chicken, duck, and so on. Enterococcus that infects animals may directly infect humans, and Enterococcus-contaminated food may also cause food poisoning, therefore Enterococcus is of great significance in public health. During the years 2003 to 2008,43 Enterococcus-like isolates were obtained from dead pigs, respectively from farms in Xuchang, Nanyang, Zhengzhou and Luoyang of Henan province, which showed symptoms of septicaemia, meningitis and arthritis. Conventional morphological observation, tolerance experiments, Vitek-32 automatic biochemical bacteria identification and whole-cell protein profiles （WCPP） analysis were carried out, followed by the 16S rRNA and RAPD molecular approaches. Upon these bases, virulence factor genes carried by those strains were investigated and mice peritonitis models were set up with selected isolates, and further analysis was done on the relevance between pathogenicity of the isolates and virulence genes, virulence phenotypes, and antibiotic tolerance, so that information could be provided on epidemiology, pathogenicity, and eventually prevention and control of swine enterococcal infections.The conventional morphological observation and tolerance experiments showed the characteristics of the isolates were:gram-positive, globular or orbicular-ovate, catenulately arrayed, tolerant to 6.5% NaCl broth、pH 9.6 glucose broth, and temperatures of 10℃,45℃and 60℃in 30 minutes.35 among the 43 isolates were identified to species level by the Vitek-32 method, with a result of 11 strains of E. faecalis,2 of E. faecium and 22 of E. casseliflavus, whereas 8 others remained unidentified. The whole-cell protein profiles （WCPP） analysis presented a different conclusion that the predominant difference among the 43 isolates dwelled at 97KDa, 90KDa, 67KDa and 46KDa bands of protein molecular weight in electrophoresis. Accordant to the reference strains ATCC29212 and ATCC33186, thirteen isolates presented the 97KDa band, whilst the others had 46KDa,67 KDa and 97 KDa ones, implying another group of Enterococcci.The results of 16S rRNA gene sequence analysis indicated a high homology between the trial strains and those registered in the GenBank database. By comparison through the software DNAStar, all the 43 clinical isolates were successfully identified at the species level, among which 13 trial strains showed homology of 99.3%～99.9% to 4 E. faecalis type strains （AF515223, AJ301831, EU728747, NC₀04668）, and the other 30 isolates except HE25 presented homology of 99.3%～100% to 3 E. faecium strains （EU547780, DQ411813, Y18294）, the HE25 strain showing 99%～99.4% homology the other 29 identified E.faecium strains. The 16S rRNA gene sequences of forty-two enterococci（HN-Nl to HN-N42） were registered to GenBank, with accession numbers FJ378656 to FJ378697 respectively.Nineteen random primers were synthesized and the genomic polymorphism of the isolates was analysed so as to clarify in genomic level the identification results, and then to collect reference information for epidemiological purpose. It showed that all the isolates performed good polymorphism straps when amplified with 4 of the random primers. The similarity coefficients and genetic distance index were calculated by SPSS11.5 software, and the phylogenetic tree was drawn with the method of nearest neighbor. The result indicated that the genetic distance index of HN-N3 and HN-N6 was 0.000, suggesting an identical origin of them. When the rescaled distance cluster combine was 21, the test strains could be identified to species level, and they dropped into four clustering groups, namely E.faecalis, E.faecium and E. avium groups, and the forth which had a single member of HN-25. Under a rescaled distance cluster combine of 19, the cluster of E. faecalis could be further divided into four subgroups, whereas the E. faecium members except HN-25 belonged to one large cluster.Genes of twelve virulence factors, which were predominant in E. faecalis and E. faecium, were detected on test strains of different origins -clinically infected pigs, fresh pork and 15 healthy-looking feces. Generally, E. faecalis strains carried more virulence factor genes than the E. faecium ones（7.83:3.73）; clinically originated strains carried the highest numbers of virulence genes, pork strains being the second and fecal ones the fewest. Among the clinical E.faecalis isolates, the virulence genes gelE, efaA and sprE were 100% detectable, and the percentage of ace、cylA and esp genes were 84.6%、84.6% and 69.2%, which were much higher than those of other origins; likewise, the percentages of virulence genes cylA and gelE （83.3% and 66.7%） in clinical E.faecium isolates were also higher than the other counterparts.In the mice peritonitis models, pathogenicity of E. faecalis strains were more potent than the E.faecium bacteria, and different E. faecalis isolates also varied in their capacity of infection, among which HN-41was the most potent strain （LD50 of 106.01/ml）, while HN-1 the weakest （LD50 of 107.01/ml）. Similarly, E. faecium HN-32presented a higher LD50 than HN-15 did. Analysis implied a positive correlation between the pathogenecity of an isolate and its virulence gene numbers; different kinds of genes contributed unequally to the bacterial pathogenicity; the influence of virulence phenotypes on pathogenicity depended on the species of Enterococcus, and within the species theβ-hemolysis phenotype was more predominant than glutin liquefaction; the antibiotic resistance of an isolate, by contrast, did not show clear correlation with its potential, which remains to further study.更多还原