江苏某奶牛场产志贺毒素大肠杆菌的分子流行病学与遗传相关性分析研究
发布时间：2018-10-26 16:38
【摘要】：产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC)又被称为产Vero细胞毒素大肠杆菌,是一类能够产志贺毒素的大肠杆菌,是近几十年来发现的一群最重要的能够引起人类潜在致命性疾病的食源性致病菌,而EHEC 0157:H7则是其中的典型代表。高危人群老人和儿童感染产志贺毒素大肠杆菌后表现为腹泻、出血性肠炎等症状,易继发感染溶血性尿毒综合征。本研究在2014年对江苏某奶牛场进行定期采样,分离鉴定产志贺毒素大肠杆菌并用Vero细胞和BALB/c小鼠做致病性研究,以脉冲场凝胶电泳作聚类分析绘制出系统进化树做遗传相关性分析,对于了解奶牛场STEC的流行情况等具有重要的意义。1江苏某奶牛场产志贺毒素大肠杆菌的分离鉴定对江苏某奶牛场于2014年3、6、9、12月份进行定期采样,应用本实验室已建立的方法分离产志贺毒素大肠杆菌,以初步了解STEC在该奶牛场的流行情况。用本实验室建立的多重PCR对病原菌的stx1、stx2两个基因进行初步检测,检测阳性的样品,通过挑取CT-SMAC上的菌落继续分离纯化再应用多重PCR检测stx1、stx2、eaeA、ehxA这四个毒力基因,经四重PCR检测阳性的样品则继续分离纯化。本次调查中江苏某奶牛场STEC总的阳性率为12.56%,总分离率为2.07%,犊牛的阳性率为17.50%,犊牛的分离率为4.44%。本次调查中共分离到12种不同的O血清型STEC,包括O13、O16、O25、O55、O68、O161等血清型,也分离到了O55等对人类具有严重危害的血清型,同时O55也是该牛场的优势血清型。本研究选用4株STEC制备细菌毒素接种于Vero细胞,24 h后观察发现细胞变圆、皱缩,之后逐渐死亡。选择3周龄的雌性BALB/c小鼠作为实验模型,将经萘啶酮酸诱导的STEC采用灌胃的方式接种于小鼠,给实验小鼠灌胃的同时采用腹腔注射的方式接种丝裂霉素C,在攻毒后的2-7d内,攻毒组小鼠出现不同程度的死亡,其中STEC 109攻毒组死亡率最高。从攻毒小鼠的病理切片可以观察到小鼠的结肠黏膜的上皮细胞发生坏死脱落,肝脏、脾脏均出现中性粒细胞浸润。2产志贺毒素大肠杆菌分离株的遗传相关性分析对江苏某奶牛场分离到的17株产志贺毒素大肠杆菌基因组用Xba Ⅰ酶切后进行脉冲场凝胶电泳,通过UPGMA进行聚类分析绘制出系统进化树,并分析其遗传相关性。这17株STEC通过Xba Ⅰ酶切得到了17种不同的酶切图谱,相似指数在41.8%-90.3%之间,STEC 13和STEC 65, STEC 41和STEC 78的Dice相似系数高于80%,其他的菌株之间的Dice相似系数均未达到80%,这反映了该奶牛场所分离到的STEC亲缘性较低,具有丰富的克隆群,存在感染人类的潜在危险性。
[Abstract]:Shiga toxin producing Escherichia coli (Shiga toxin-producing Escherichia coli,STEC), also known as Vero cytotoxin producing Escherichia coli, is a kind of Escherichia coli capable of producing Shigella toxin. EHEC 0157:H7 is one of the most important foodborne pathogens found in recent decades, which can cause potentially fatal diseases in humans. The elderly and children in high risk group showed diarrhea, hemorrhagic enteritis and secondary hemolytic uremic syndrome after infection with Shiga toxin producing Escherichia coli. In this study, a dairy farm in Jiangsu Province was sampled regularly in 2014 to isolate and identify Shiga toxin producing Escherichia coli and to do pathogenicity study on Vero cells and BALB/c mice. The phylogenetic tree was plotted by cluster analysis by pulsed field gel electrophoresis (PGE) for genetic correlation analysis. It is of great significance to understand the prevalence of STEC in dairy farms in Jiangsu Province. 1 the isolation and identification of Shiga toxin Escherichia coli from a dairy farm in Jiangsu Province were regularly sampled from a dairy farm in Jiangsu Province in December 2014. Shiga toxin Escherichia coli was isolated by using the method established in our laboratory to understand the prevalence of STEC in the dairy. The two stx1,stx2 genes of pathogenic bacteria were preliminarily detected by multiplex PCR established in our laboratory. The positive samples were detected, and the isolation and purification of the colonies from CT-SMAC were carried out, and then the multiple PCR was used to detect stx1,stx2,eaeA,. The four virulence genes of ehxA were isolated and purified from the positive samples tested by quadruple PCR. The total positive rate of STEC in a dairy farm in Jiangsu Province was 12.566.The total isolation rate was 2.070.The positive rate of calves was 17.50 and the separation rate of calves was 4.44. A total of 12 different serotypes of O STEC, were isolated, including O13, O16, O25, O55, O5, O68, O161, and O55 serotypes, which are harmful to human beings. O55 is also the dominant serotype of the cattle farm. In this study, four strains of STEC were used to prepare bacterial toxin and inoculated into Vero cells. After 24 hours, the cells became round, shrank, and then died gradually. Three week-old female BALB/c mice were selected as the experimental model. The STEC induced by Naphthyronic acid was inoculated into mice by gavage, and mitomycin C was injected intraperitoneally to the experimental mice. Within 2 to 7 days after the attack, the mice in the STEC 109 group had different degrees of death, and the death rate in the STEC 109 group was the highest. From the pathological sections of mice attacked with poison, the epithelial cells of the colonic mucosa of mice were observed to be necrotic and shedding, and the liver. (2) genetic correlation analysis of 17 Shigella toxin producing Escherichia coli isolates isolated from a dairy farm in Jiangsu Province was carried out by pulsed field gel electrophoresis (PGE) of 17 Shiga toxin producing Escherichia coli genomes digested with Xba 鈪，
本文编号：2296346
[Abstract]:Shiga toxin producing Escherichia coli (Shiga toxin-producing Escherichia coli,STEC), also known as Vero cytotoxin producing Escherichia coli, is a kind of Escherichia coli capable of producing Shigella toxin. EHEC 0157:H7 is one of the most important foodborne pathogens found in recent decades, which can cause potentially fatal diseases in humans. The elderly and children in high risk group showed diarrhea, hemorrhagic enteritis and secondary hemolytic uremic syndrome after infection with Shiga toxin producing Escherichia coli. In this study, a dairy farm in Jiangsu Province was sampled regularly in 2014 to isolate and identify Shiga toxin producing Escherichia coli and to do pathogenicity study on Vero cells and BALB/c mice. The phylogenetic tree was plotted by cluster analysis by pulsed field gel electrophoresis (PGE) for genetic correlation analysis. It is of great significance to understand the prevalence of STEC in dairy farms in Jiangsu Province. 1 the isolation and identification of Shiga toxin Escherichia coli from a dairy farm in Jiangsu Province were regularly sampled from a dairy farm in Jiangsu Province in December 2014. Shiga toxin Escherichia coli was isolated by using the method established in our laboratory to understand the prevalence of STEC in the dairy. The two stx1,stx2 genes of pathogenic bacteria were preliminarily detected by multiplex PCR established in our laboratory. The positive samples were detected, and the isolation and purification of the colonies from CT-SMAC were carried out, and then the multiple PCR was used to detect stx1,stx2,eaeA,. The four virulence genes of ehxA were isolated and purified from the positive samples tested by quadruple PCR. The total positive rate of STEC in a dairy farm in Jiangsu Province was 12.566.The total isolation rate was 2.070.The positive rate of calves was 17.50 and the separation rate of calves was 4.44. A total of 12 different serotypes of O STEC, were isolated, including O13, O16, O25, O55, O5, O68, O161, and O55 serotypes, which are harmful to human beings. O55 is also the dominant serotype of the cattle farm. In this study, four strains of STEC were used to prepare bacterial toxin and inoculated into Vero cells. After 24 hours, the cells became round, shrank, and then died gradually. Three week-old female BALB/c mice were selected as the experimental model. The STEC induced by Naphthyronic acid was inoculated into mice by gavage, and mitomycin C was injected intraperitoneally to the experimental mice. Within 2 to 7 days after the attack, the mice in the STEC 109 group had different degrees of death, and the death rate in the STEC 109 group was the highest. From the pathological sections of mice attacked with poison, the epithelial cells of the colonic mucosa of mice were observed to be necrotic and shedding, and the liver. (2) genetic correlation analysis of 17 Shigella toxin producing Escherichia coli isolates isolated from a dairy farm in Jiangsu Province was carried out by pulsed field gel electrophoresis (PGE) of 17 Shiga toxin producing Escherichia coli genomes digested with Xba 鈪，
本文编号：2296346
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