江蘇某奶牛場產(chǎn)志賀毒素大腸桿菌的分子流行病學(xué)與遺傳相關(guān)性分析研究
發(fā)布時間：2018-10-26 16:38
【摘要】：產(chǎn)志賀毒素大腸桿菌(Shiga toxin-producing Escherichia coli,STEC)又被稱為產(chǎn)Vero細胞毒素大腸桿菌,是一類能夠產(chǎn)志賀毒素的大腸桿菌,是近幾十年來發(fā)現(xiàn)的一群最重要的能夠引起人類潛在致命性疾病的食源性致病菌,而EHEC 0157:H7則是其中的典型代表。高危人群老人和兒童感染產(chǎn)志賀毒素大腸桿菌后表現(xiàn)為腹瀉、出血性腸炎等癥狀,易繼發(fā)感染溶血性尿毒綜合征。本研究在2014年對江蘇某奶牛場進行定期采樣,分離鑒定產(chǎn)志賀毒素大腸桿菌并用Vero細胞和BALB/c小鼠做致病性研究,以脈沖場凝膠電泳作聚類分析繪制出系統(tǒng)進化樹做遺傳相關(guān)性分析,對于了解奶牛場STEC的流行情況等具有重要的意義。1江蘇某奶牛場產(chǎn)志賀毒素大腸桿菌的分離鑒定對江蘇某奶牛場于2014年3、6、9、12月份進行定期采樣,應(yīng)用本實驗室已建立的方法分離產(chǎn)志賀毒素大腸桿菌,以初步了解STEC在該奶牛場的流行情況。用本實驗室建立的多重PCR對病原菌的stx1、stx2兩個基因進行初步檢測,檢測陽性的樣品,通過挑取CT-SMAC上的菌落繼續(xù)分離純化再應(yīng)用多重PCR檢測stx1、stx2、eaeA、ehxA這四個毒力基因,經(jīng)四重PCR檢測陽性的樣品則繼續(xù)分離純化。本次調(diào)查中江蘇某奶牛場STEC總的陽性率為12.56%,總分離率為2.07%,犢牛的陽性率為17.50%,犢牛的分離率為4.44%。本次調(diào)查中共分離到12種不同的O血清型STEC,包括O13、O16、O25、O55、O68、O161等血清型,也分離到了O55等對人類具有嚴(yán)重危害的血清型,同時O55也是該牛場的優(yōu)勢血清型。本研究選用4株STEC制備細菌毒素接種于Vero細胞,24 h后觀察發(fā)現(xiàn)細胞變圓、皺縮,之后逐漸死亡。選擇3周齡的雌性BALB/c小鼠作為實驗?zāi)Ｐ?將經(jīng)萘啶酮酸誘導(dǎo)的STEC采用灌胃的方式接種于小鼠,給實驗小鼠灌胃的同時采用腹腔注射的方式接種絲裂霉素C,在攻毒后的2-7d內(nèi),攻毒組小鼠出現(xiàn)不同程度的死亡,其中STEC 109攻毒組死亡率最高。從攻毒小鼠的病理切片可以觀察到小鼠的結(jié)腸黏膜的上皮細胞發(fā)生壞死脫落,肝臟、脾臟均出現(xiàn)中性粒細胞浸潤。2產(chǎn)志賀毒素大腸桿菌分離株的遺傳相關(guān)性分析對江蘇某奶牛場分離到的17株產(chǎn)志賀毒素大腸桿菌基因組用Xba Ⅰ酶切后進行脈沖場凝膠電泳,通過UPGMA進行聚類分析繪制出系統(tǒng)進化樹,并分析其遺傳相關(guān)性。這17株STEC通過Xba Ⅰ酶切得到了17種不同的酶切圖譜,相似指數(shù)在41.8%-90.3%之間,STEC 13和STEC 65, STEC 41和STEC 78的Dice相似系數(shù)高于80%,其他的菌株之間的Dice相似系數(shù)均未達到80%,這反映了該奶牛場所分離到的STEC親緣性較低,具有豐富的克隆群,存在感染人類的潛在危險性。
[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，
本文編號：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，
本文編號：2296346
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