屎肠球菌多位点序列分型及间接血凝诊断方法的建立

发布时间：2018-12-13 08:42

【摘要】：屎肠球菌(Enterococcus faecium)属于革兰氏阳性、过氧化氢酶阴性球菌,是人和动物肠道内的主要菌群。自上世纪90年代以来,屎肠球菌逐渐成为人和动物重要的条件性致病菌。在我国,对屎肠球菌的研究主要集中于人感染屎肠球菌的病例报道,及分离菌株的分类研究,在动物方面,则少有报道。本研究成功分离到18株肠球菌临床分离株,对进一步研究肠球菌诊断方法,治疗肠球菌感染以及耐药机制和多位点序列分型提供基础。首先通过细菌分离,染色鉴定,凝固酶试验,延伸因子tuf及16S rRNA序列鉴定,从采自甘肃、青海等地的羊及猪组织病料中,分离到18株屎肠球菌,分别命名为LVRI1101,LVRI1102,LVRI1103,LVRI1104,LVRI1301,LVRI1302,LVRI1303,LVRI1304,YYAN1401,YYAN1402,YYAN1403,YYAN1404,lby1,lby2,lby3,lbg1,lbg2,lbg3。药敏试验结果表明,所分离菌株均为多重耐药性菌株。小鼠致病性实验结果表明,所分离菌株均具有致病力。多位点序列分型(multilocus sequence type,MLST)结果发现,所分离菌株属于3个新的序列型(Sequence type),分别命名为Enterococcus faecium ST989,Enterococcus faecium ST1017,Enterococcus faecium ST1018,已添加到数据库中。由于目前在基层尚缺乏快速有效的屎肠球菌血清学诊断方法,从而可能会造成误诊及漏诊,因此,我们采用超声细胞破碎法提取屎肠球菌菌体抗原作为抗原,致敏醛化鞣酸化的绵阳红细胞,与屎肠球菌高免兔血清,屎肠球菌阳性血清进行间接血凝实验。结果显示该方法特异性好、敏感性高,建立了检测屎肠球菌抗体的间接血凝实验方法(IHA),可为屎肠球菌病的血清学快速诊断及流行病学调查提供依据。
[Abstract]:Enterococcus faecium (Enterococcus faecium) belongs to Gram-positive and catalase negative cocci. Since 1990 s, Enterococcus faecium has become an important conditional pathogen in human and animal. In China, the studies on Enterococcus faecium mainly focus on the case reports of Enterococcus faecium infection in human beings, and the taxonomic studies of isolated strains, but there are few reports on animals. In this study, 18 strains of Enterococcus were successfully isolated, which provided the basis for further study on the diagnosis of Enterococcus, the treatment of enterococcal infection, the mechanism of drug resistance and the classification of multilocus sequence. At first, 18 strains of Enterococcus faecium were isolated from sheep and pig tissue samples collected from Gansu, Qinghai and other places by bacterial isolation, staining, coagulase test, extension factor tuf and 16s rRNA sequence identification. They were named LVRI1101,LVRI1102,LVRI1103,LVRI1104, respectively. LVRI1301,LVRI1302,LVRI1303,LVRI1304,YYAN1401,YYAN1402,YYAN1403,YYAN1404,lby1,lby2,lby3,lbg1,lbg2,lbg3. The results of drug sensitivity test showed that the isolates were multidrug resistant. The pathogenicity test of mice showed that all the isolated strains had pathogenicity. The results of multi-locus sequence typing (multilocus sequence type,MLST) showed that the isolated strains belonged to three new sequence-type (Sequence type), named Enterococcus faecium ST989,Enterococcus faecium ST1017,Enterococcus faecium ST1018, which had been added to the database. Due to the lack of a rapid and effective serological diagnosis method for Enterococcus faecium at present, it may lead to misdiagnosis and missed diagnosis. Therefore, we use the method of ultrasonic cell fragmentation to extract Enterococcus faecium antigen as antigen. The indirect hemagglutination test was carried out between the sensitized mianyang erythrocytes and the high immune rabbit serum of Enterococcus faecium and the positive serum of Enterococcus faecium. The results showed that the method had good specificity and high sensitivity. The indirect hemagglutination assay (IHA),) for the detection of Enterococcus faecium antibody could provide the basis for rapid serological diagnosis and epidemiological investigation of Enterococcus faecium.
【学位授予单位】：西北师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446

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