Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.     

Screening for Escherichia coli O157:H7 in Illinois

Objective: To determine the incidence of infection with Escherichia coli O157:H7 in a tertiary referral center in Chicago, where a similar study had been performed in 1984, to evaluate cases of disease reported to the Illinois Department of Public Health (IDPH) in 1993, and to determine laboratory practices used to detect this infection throughout the state.
Methods: During a 6-month period in 1993, all stool specimens at Rush-Presbyterian-St Luke's Medical Center (RPSLMC) were tested for E. coli O157:H7. Reports of diagnosed E. coli O157:H7 cases investigated by IDPH were also reviewed. A survey of 73 hospitals in the Chicago area was performed to determine routine culturing practices, specifically, the selection of stool specimens for evaluation for this pathogen.
Results: In the RPSLMC survey, two cases were identified among 1985 samples (incidence 0.1%), similar to the 0.08% incidence detected in a similar study conducted at the same institution in 1984. Through passive surveillance, the IDPH received 44 reports of E. coli O157:H7 in 1993. The hospital survey revealed that, in the seven labs testing all stool specimens for E. coli O157:H7, an incidence of 16/8137 specimens (0.2%) was determined.
Conclusions: These data suggest that sporadic E. coli O157:H7 remains uncommon in Illinois and that the incidence may not have changed over a 9-year period. The low yield and substantial cost of culturing all stools suggest that only specimens from patients with bloody diarrhea should be evaluated routinely in areas of low endemicity.     

由苍蝇传播引起E．coli O157：H7感染爆发的评估模型的建立

目的 建立苍蝇对E．coli O157：H7传播作用的定量危险性评估模型。方法 在Excel工作表中模拟E．coli O157：H7的粪口传播过程，利用@RISK软件对模型进行Monte Carlo模拟。结果 由此交叉污染途径引起人群感染的概率为10^-5～10^-3／餐。模型预测的结果与爆发调查数据基本吻合。通过模型分析可知，苍蝇排泄物中的含菌量及苍蝇的数量是影响人群感染危险性的重要因素。结论 以苍蝇污染途径模拟E．coli O157：H7感染危险性的定量评估模型，该模型是一个非常有用的评估危害与危险性关系的评估工具。     

江苏省113株大肠杆菌O157:H7的扩增片段长度多态性分析

目的:分析江苏省不同地区和年代分离的Escherichia coli O157:H7(E. coli O157:H7)之间的同源性?方法:用扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分析法对113株 E. coli O157:H7菌株进行分子分型,采用软件BioNumerics Version 4.0对分型数据进行处理和分析?结果:113株E. coli O157:H7共分37个型别,不同来源的菌株存在相同的基因型别,不同的地区和物种之间存在着相互传播?结论:AFLP可以用于对不同来源的E. coli O157:H7进行分子分型?     

espP基因在O157∶H7大肠埃希菌分离株中的分布

目的了解espP基因在中国部分地区产志贺毒素O157大肠埃希菌分离株中的分布情况及特点。方法根据O157∶H7的溶血素基因hlyA和细胞外分泌蛋白基因espP设计特异性引物,对113株O157大肠埃希菌分离株进行PCR检测。结果 hlyA的阳性率为99.1%,espP的阳性率为72.5%,且只在有大质粒pO157的菌株中检测到espP。结论 espP存在于携带pO157大质粒的O157∶H7大肠埃希菌中,其分布与菌株来源、志贺毒素携带情况等没有相关性。     

大肠杆菌O157∶H7 CVa9株O抗原变异的研究

目的　研究本实验室保藏的大肠杆菌O15 7:H7(E coliO15 7:H7)日本分离株CVa9O抗原的变异。方法　血清凝集试验检测CVa9菌株O和H抗原 ,区分O抗原变异株与非变异株。聚合酶链反应 (PCR)法分别检测O15 7和H7特异性基因。生化试验检测生化特性。观察菌株在麦康凯、山梨醇麦康凯、棉子糖麦康凯及ChromagarO15 7显色培养基上菌落形态 ,菌落计数法计算变异率。结果　抗 -O15 7抗血清与CVa9菌株进行玻片凝集试验出现强凝集 (CVa9- 8)和不凝集 (CVa9- 1、CVa9- 15 )两种菌落 ,试管凝集试验结果相同 ,证实不凝集菌落O抗原发生变异。抗 -HT 抗血清分别与变异株和非变异株H抗原进行试管凝集试验 ,均为强凝集。两种菌株O15 7和H7特异性基因均为阳性。变异株与非变异株生化特性基本相同 ,但变异株不发酵棉子糖。两种菌株在麦康凯及山梨醇麦康凯培养基上菌落形态特征没有区别。在ChromagarO15 7显色培养基上培养 4 8h ,变异株为浅紫红色 ,菌落周边呈毛边状 ,非变异株为紫红色 ,菌落边缘整齐。变异株在用棉子糖代替乳糖制备的棉子糖麦康凯培养基上菌落呈粉红色 ,非变异株呈红色。菌落计数显示变异率为 99 80 %。结论　CVa9菌株在保存过程中O抗原发生变异 ,菌落形态及部分生化特征亦有所改变。变异株和非变异株均携带O15     

肠出血性大肠埃希菌O157∶H7环介导恒温扩增快速检测方法的建立与应用

目的肠出血性大肠埃希菌(Enterohemorrhagic Escherichia coli,EHEC)O157∶H7是一种重要的人畜共患病致病菌,主要通过被污染的食物传播,引起出血性结肠炎,建立其快速检测方法具有重要意义。方法基于EHEC O157∶H7保守的rfbE基因序列,设计4条引物,利用环介导等温扩增技术(loop-mediatedisothermal amplification,LAMP),成功建立了EHEC O157∶H7LAMP快速检测方法 ,60min内即可完成致病菌检测。结果利用该LAMP方法对30种共38株细菌进行检测,所试EHEC O157∶H7均为阳性结果 ,说明该方法具有高度特异性。本方法对纯培养的EHEC O157∶H7检测限为12CFU/mL,污染食品中EHEC O157∶H7的检测限为18CFU/g。实践应用表明,对1121份进出口肉类、奶类制品及人工污染样品等进行检测,检出57份LAMP阳性,与采用AOAC标准检测结果的符合率为100%。结论该LAMP方法操作简便、特异性强、灵敏度高,具有良好的实用性。     

表达肠出血性大肠杆菌 O157:H7 EspA 抗原的减毒沙门氏菌株的构建

目的 利用平衡致死系统构建表达O157:H7 EspA 的减毒鼠伤寒沙门氏菌.方法 构建表达EspA的重组质粒,再将其转入终宿主菌减毒鼠伤寒沙门氏菌x4550 株中构建成口服活疫苗株,用IPTG进行诱导表达,经聚丙烯酰胺凝胶电泳、Westen-blot检测EspA蛋白的表达情况.并观察重组菌体外培养的稳定性.结果 利用宿主-载体平衡致死系统构建了表达O157:H7 EspA的重组减毒沙门氏菌,经Tricine-SDS-PAGE电泳出现了1条Mr约21 000的蛋白条带,Westen-blot检测能与抗EspA的单克隆抗体发生特异性反应.且在没有选择压力的条件下体外能稳定地繁殖、生长和传代.结论 表达O157:H7 EspA的重组减毒沙门氏菌构建成功,为发展口服抗EHEC O157:H7的疫苗奠定了初步基础.