肠出血性大肠杆菌O26∶H11鞭毛抗原的诱导及鉴定研究

目的对肠出血性大肠杆菌(EHEC)O26∶H11鞭毛抗原诱导及鉴定进行研究,为日常检测提供可借鉴的方法。方法对疑似EHEC O26∶H11菌株进行生化鉴定和血清分型,比较37℃及28℃2种温度Swarm琼脂诱导鞭毛抗原的效果,并采用荧光定量PCR检测8种致泻性大肠杆菌毒力基因eae、stx1、stx2、lt、st、bfp A、aggR和ipa H。结果 28℃诱导鞭毛抗原H11血清凝集为阳性,37℃诱导鞭毛抗原血清凝集为阴性;毒力基因检测结果显示stx1和eae基因阳性,其余均为阴性;菌株鉴定为EHEC O26∶H11。结论 28℃Swarm琼脂可诱导鞭毛H11,肠出血性大肠杆菌O26∶H11鉴定时应检测相关毒力基因。     

大肠埃希菌O157血清型分离株的脉冲场凝胶电泳分型

目的了解杭州地区分离的产志贺毒素和不产志贺毒素大肠埃希菌O157菌株的分子流行病学特征。方法对杭州地区于1997～2005年间自散发腹泻患者和动物中分离的10株大肠埃希菌O157血清型菌株,PCR检测毒力基因stx1、stx2、eae和ehxA,eae阳性者进行eae基因分型;按标准化的脉冲场凝胶电泳法(pulsefieldgelelectrophoresis,PFGE)进行分子分型,并与国内其他省份分离的产志贺毒素大肠埃希菌(Shigatoxin_producingEscherichiacoli,STEC)O157∶H7菌株进行比较。结果杭州分离菌株HZ1_11携带毒力基因stx2、γ型eae和ehxA,为浙江省检获的首株STECO157∶H7;3株杭州O157∶NM分离菌株(2_1、26_1和SR05株)仅携带β型eae基因;其他6株杭州O157∶H?分离菌株中,均未检出毒力基因。PFGE揭示,在杭州人源O157菌株中,STECO157∶H7HZ1_11株与近年江苏和安徽分离STECO157∶H7菌株密切近缘,且其图谱与江苏菌株几乎完全相同;3株eae阳性的O157∶NM分离菌株聚类成与STECO157∶H7菌株较远的一簇,杭州1株stx阴性的O157∶H?(1_68株)则处于另一独立的一簇。5株杭州O157∶H?动物分离菌株与其他人源菌株图谱差异极大。结论浙江省首株STECO157∶H7可能是源自近年在江苏流行的STECO157∶H7菌株。β型eae阳性的大肠埃希菌O157∶NM菌株可能具有引起散发腹泻的能力。     

浙江省首株产志贺毒素大肠埃希菌O157:H7的分子生物学特性研究

目的:了解自浙江省杭州市腹泻婴儿中分离的1株大肠埃希菌O157:H7(HZ1-11株)的分子生物学特性.方法:应用ATB1525细菌半动化生化鉴定系统鉴定菌种.应用O157特异性抗血清玻片凝集试验、H7特异性抗血清试管凝集试验、以及PCR检测O抗原特异性rfbE基因和H7特异性fliC基因,进行菌株血清型的鉴定.应用多重Real-timePCR和常规PCR检测stx1、stx2、hly和eae毒力基因.对菌株进行脉冲场凝胶电泳(PFGE)分型,并与国内代表菌株进行比较.ATB1525药敏检测仪和纸片法检测菌株的抗药性.结果:细菌生化鉴定为大肠埃希菌,山梨醇阴性.血清型为O157:H7.毒力基因stx2、hly和eae均阳性,stx1阴性.PFGE谱带同江苏分离O157:H7菌株几乎完全相同,带型的相似度为97%.结论:该菌株为浙江省首株产志贺毒素大肠埃希菌(STEC)O157:H7,与国内近年在江苏等地流行的STECO157:H7菌株密切相关.STEC O157:H7已开始对浙江地区的人民健康构成了威胁.     

浙江省首株产志贺毒素大肠埃希菌0157：H7的分子生物学特性研究

目的：了解自浙江省杭州市腹泻婴儿中分离的1株大肠埃希菌O157：H7（HZI-11株）的分子生物学特性。方法：应用ATB1525细菌半动化生化鉴定系统鉴定菌种。应用0157特异性抗血清玻片凝集试验、H7特异性抗血清试管凝集试验、以及PCR检测O抗原特异性rfbE基因和H7特异性fliC基因，进行菌株血清型的鉴定。应用多重Real-time PCR和常规PCR检测stx1、stx2、hly和eae毒力基因。对菌株进行脉冲场凝胶电泳（PFGE）分型，并与国内代表菌株进行比较。ATB1525药敏检测仪和纸片法检测菌株的抗药性。结果：细菌生化鉴定为大肠埃希菌，山梨醇阴性。血清型为O157：H7。毒力基因stx2、hly和eae均阳性，stx1阴性。PFGE谱带同江苏分离O157：H7菌株几乎完全相同，带型的相似度为97％。结论：该菌株为浙江省首株产志贺毒素大肠埃希菌（STEC）O157：H7。与国内近年在江苏等地流行的STEC O157：H7菌株密切相关。STEC O157：H7已开始对浙江地区的人民健康构成了威胁。     

致病性大肠埃希菌毒素基因的Allglo探针技术鉴定

目的：基于Allglo探针技术结合多重荧光PCR，建立快速、简便鉴定致病性大肠埃希菌相关毒素基因的方法。方法选取致病性大肠埃希菌的紧密黏附素基因（ eae）、志贺样毒素Ⅰ基因（ stx I）、志贺样毒素II基因（ stx II）和转录激活因子基因（ aggR）作为靶点。通过设计引物和Allglo探针，建立多重荧光PCR反应体系。同时，构建了4种毒素基因标准质粒，进而评价方法的特异性、灵敏性和重复性。结果基于Allglo探针技术的多重荧光PCR方法可同时准确、特异地鉴定致病性大肠埃希菌所携带的4种毒素相关基因，eae基因和aggR基因的灵敏度为10 copies/μL，stx I基因和stx II基因的灵敏度为1 copies/μL；定量检测的批间和批内变异系数均小于5%。对356份腹泻粪便样本进行评价，结果显示检出39份基因阳性。39份阳性样本中，eae基因阳性为53.85%，stxⅠ基因和stx II基因阳性为23.08%，aggR基因阳性为46.15%。通过直接测序方法进行鉴定，符合率达到100.00%。结论本实验建立的基于Allglo探针技术结合多重荧光PCR方法具有操作简便、特异性好和灵敏度高等特点，为致病性大肠埃希菌鉴定提供了一种快速、可靠的检测方法。     

福建省致泻大肠埃希菌的实验室考核分析

目的分析福建省疾控中心微生物检验科实验室致泻大肠埃希菌的质控考核结果,总结经验,促进监测工作质量提高及防控疫情暴发中实验室检测的作用。方法福建省CDC微生物检验科实验室于2014—2016年参加4次国家相关部门组织的致泻大肠埃希菌考核,依据每次考核作业指导书及相关国家标准要求选择检测方法,对考核株进行分离培养、生化鉴定、血清分型及多重PCR或荧光多重PCR基因检测。结果各考核结果均获满意,考核株均为肠出血性大肠埃希菌,但血清型有些不同,包括O55:H7、O157:H7、O26:H11,检出毒力基因存在uidA、escV、eae、stx1、stx2的不同组合。结论福建省CDC微生物检验科实验室质控考核结果,具备致泻大肠埃希菌检测能力。随着病原的变异,今后应加强产志贺毒素大肠埃希菌亚型分型能力及其他类致泻大肠埃希菌的考核。     

大肠埃希菌所致儿童急性腹泻流行病学调查及耐药性分析

目的 分析大肠埃希菌在急性腹泻儿童中的流行病学和耐药性特征.方法 收集2019年1-12月期间于芜湖市第五人民医院就诊的564例急性腹泻儿童的粪便样本,培养后鉴定,用PCR法扩增大肠埃希菌的毒力基因uida、eae、aggR、bfpB、escV、stx1,用血清凝集法鉴定致病性大肠埃希菌,用琼脂稀释法进行常见抗生素的药...     

2005～2007年中国食品中疑似O157大肠埃希菌的鉴定及毒素基因的检测

目的对中国2005～2007年食源性疾病监测网分离疑似O157大肠埃希菌进行鉴定,了解各种不同类型监测食品中大肠埃希菌O157的分布及毒力基因的携带情况。方法运用API20E生化试剂条进行初步鉴定,使用血清分型和PCR方法确定菌株,并检测毒力基因。结果(1)通过API20E生化初步鉴定共获得154株疑似O157大肠埃希菌。(2)通过血清学方法和特异基因的检测,共确定89株大肠埃希菌O157,其中42株是O157:H7(47%),其余的菌株为O157:NM和O157:hund(未确定型)。(3)毒力基因的检测:42株O157:H7和6株O157:NM携带eaeA+hlyA基因;共29株菌携带stx基因,主要分布在不发酵山梨醇O157:H7菌株中。(4)监测的生羊肉、生牛肉、生猪肉、生鸡肉、蔬菜沙拉等食品中均检出了携带stx基因的O157:H7。结论鉴定结果显示中国部分监测食品中均分离到有一定程度致病力的大肠埃希菌O157。     

2005-2009年河南省肠出血性大肠埃希菌O157：H7监测分析

目的监测河南省肠出血性大肠埃希菌O157∶H7在腹泻患者和宿主动物中带菌及毒力基因情况,探索疾病发生的原因和相关因素。方法对2005-2009年河南省监测点所送可疑菌株进行血清学和PCR复核,对确认菌株进行stx1、stx2、eaeA、hlyA毒力基因测定。结果河南省2005-2009年共监测各类标本10 732份,检出O157∶H7菌株255株,检出率为2.38%;其中动物粪便标本的检出率为6.31%,检出率最高的为羊粪（8.04%）,其次为牛粪（7.20%）;各年份菌株检出率之间有一定差异;河南省肠出血性大肠埃希菌O157∶H7的产毒株主要来自于羊、牛、鸡粪便及蝇类和生肉标本;毒株类型主要为stx2、eaeA、hlyA组合型。结论河南省肠出血性大肠埃希菌O157∶H7在人群和各类动物中均存在,最重要的动物宿主是羊和牛;部分食品在加工环节有可能被污染,存在引起暴发的危险性。     

健康人群致泻性大肠埃希菌耐药性及多位点序列分型分析

目的 通过分析健康人群致泻性大肠埃希菌(Diarrheagenic Escherichia coli,DEC)携带率、耐药性及多位点序列分型(multilocus sequence typing, MLST),探究其病原学特征。方法 收集2018年北京市朝阳区健康人群粪便标本809份,进行细菌分离培养,采用聚合酶链式反应(Polymerase Chain Reaction, PCR)法检测六种DEC特异毒力基因(aggR、eae、bfpA、ipaH、lt、st、stx1、stx2、afaD)。应用微量肉汤稀释法对检测出的DEC进行26种常用药物的敏感试验。对所有DEC菌株进行MLST分析。结果 共检出38株DEC分布于各年龄组,检出率为4.70%。主要型别为肠道致病性大肠埃希菌(EPEC)、弥散粘附性大肠埃希菌(DAEC)、肠聚集性大肠埃希菌(EAEC)。药敏结果显示,38株DEC对卡那霉素、阿米卡星、亚胺培南、美罗培南全部敏感,对三代头孢、三代喹诺酮类抗生素耐药率较低。对四环素耐药率最高(76.32%),氨苄西林次之(65.79%),对复方新诺明、磺胺异恶唑、萘啶酸耐药率超过50.0...     

首次从病人中分离到产毒O157:H7菌株的多重PCR鉴定

目的 鉴定从病人粪便中分离到的大肠杆菌O157：H7菌株的毒素基因和rfbO157特异性基因。方法 多重PCR技术同时检测大肠杆菌O157：H7的四种毒素基因和O157特异性基因。结果 可疑菌株含有志贺氏毒素2（stx2)基因，溶血素基因（hlyA)，肠上皮细胞纤毛清除素基因（eaeA)和O157：H7特异性基因（rfbO157)，但不含有志贺氏毒素1（stx1)基因。结论 菌株为产志贺氏毒素大肠杆菌O157：H7血清型。     

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.     

2010年福建南平及永安监测点致泻大肠杆菌的监测报告

目的:为了解福建省腹泻病人中致泻大肠杆菌的感染情况。方法:应用多重PCR或单重PCR方法检测致泻大肠杆菌的EPEC/EHEC eaeA基因、EHEC stx基因、EAEC aggR基因、EIEC ipaH、EIEC virA、ETEC ST、ETEC LT、EPEC bfp基因。API 20E生化鉴定条进行生化试验。血清学鉴定。结果:2010年度共分离得19株致泻大肠杆菌,检出率为10.9%。1株EAEC;1株tEPEC;4株aEPEC;13株ETEC。19株分离的致泻大肠杆菌经API 20E鉴定均为大肠埃希菌。1株tEPEC与EPEC三种多价诊断血清型均不凝集,而1株aEPEC血清型为O111:K58(B4)。13株ETEC菌株血清型:多数为O6:K15,1株O25:K19(L),3株未能分型。结论:监测结果对于我们了解福建省致泻大肠杆菌感染情况,预警潜在发生疫情的可能性有其重要性。这些菌种的获得可以为下一步研究其毒力特征、分子分型及耐药性积累原始材料。     

衢州市2000-2009年肠出血性大肠埃希菌O157∶H7监测

目的了解浙江省衢州市肠出血性大肠埃希菌O157∶H7人的感染、食品污染以及动物和媒介昆虫带菌情况。方法在流行季节采集腹泻患者、动物粪便以及各类食品、苍蝇等样本,MEC肉汤增菌后,用特异性免疫磁珠集菌,以科玛嘉显色平板分离,纯化的菌株进行生化鉴定、血清分型及毒力基因检测。结果 2000-2009年,共检测样本12 292份,检出EHEC O157∶H7 19株,总检出率为0.15%。其中4种宿主动物粪便样本中检出18株,检出率分别为羊2.14%、牛1.29%、猪0.45%、鸭0.19%;食品中从生猪肉中检出1株,检出率0.17%;腹泻患者和苍蝇样本中均未检出。经毒力基因检测,其中16株stx2+hly+eaeA阳性,1株hly+eaeA阳性,其余2株不带毒力基因,带毒率89.47%。结论浙江省衢州市部分动物中产毒的EHEC O157∶H7带菌率较高,表明该地区存在暴发或流行的潜在危险,应加强综合监测。     

Occurrence of shigatoxinogenic Escherichia coli O157 in Norwegian cattle herds.

To investigate if there is a reservoir of Escherichia coli O157 in Norwegian cattle, faecal samples from 197 cattle herds were screened for E. coli O157 by the use of immunomagnetic separation (IMS) and PCR during the 1995 grazing season. Six E. coli O157:H-isolates were detected in two herds, one isolate in one and five in the other. The isolates carried the stx1, stx2, and eae genes, and a 90 MDa virulence plasmid. They were toxinogenic in a Vero cell assay. From 57 other herds, 137 faecal samples were positive for stx1 and/or stx2 genes detected by PCR run directly on IMS-isolated material. Among these samples, stx2 were the most widely distributed toxin encoding genes. No difference was found among milking cows and heifers in the rate of stx1 and/or stx2 in positive samples.     

Genetic characterization of a diverse Escherichia coli O157:H7 population from a variety of farm environments

Many of the current studies on the genetic diversity of Escherichia coli O157:H7 have focused on pathogenic clinical, veterinary, or food isolates. These studies did not explore the diversity of the larger population in the farm environment. Research on selected farm isolates address this wider diversity but have typically been limited to a specific geographic locale or farm type, thus giving limited insight into the greater diversity across geographic regions and varied environments. The objective of this study was to evaluate a diverse population of E. coli O157:H7 collected from a variety of locations and farm environments. Eighty-eight isolates were collected from four farm types (swine, dairy, beef, and poultry) across the southeastern and western United States. Eighteen farms were sampled every 3 months over a period of 24 months. Isolates were analyzed by ribotyping and pulsed field gel electrophoresis (PFGE). Real-time PCR was used to determine the presence or absence of key pathogenic genes (stx1, stx2, and eae). The data indicate a significant amount of genetic diversity, however, ribotype analysis revealed meaningful clusters within the larger population. These groupings were consistent with PFGE analysis. Most of these isolates were clustered by location (i.e. from the same state or region) or farm type. Of the isolates in these clusters, most did not contain pathogenic genes. Of notable interest is a single group in which the majority of isolates, collected from four of the five states sampled, contained at least one stx gene and the eae gene suggesting the existence of a specific pathogenic cluster. These data suggest that, while there is notable diversity within the broader E. coli O157:H7 population, pathogenic isolates may be limited to a subset of strains within the population.     

A long-term study on the prevalence of shiga toxin-producing Escherichia coli (STEC) on four German cattle farms

The occurrence of Shiga toxin-producing Escherichia coli (STEC) was studied on four cattle farms. STEC were detected in 29-82% of the cattle. STEC with additional EHEC markers were detected on all farms. The occurrence of the complete virulence marker pattern (stx1 and/or stx2, eae, EHEC(hlyA), katP, espP) was correlated with the presence of known STEC serotypes. STEC O26:H11 and O165:H25 with the complete pattern of virulence markers were the most prevalent. STEC O157 (H7/H-) STEC O103:H2 and STEC O145:H- were found sporadically. Five clonal subgroups of the STEC O26:H11 isolates were identified by pulsed-field gel electrophoresis. STEC O26:H11 were present in three groups of cattle. This serotype was detected in a single group over the entire fattening period. Most STEC O26:H11 with the complete pattern of potential virulence markers were found in clinically healthy cattle. These animals may represent a risk factor for farmers and consumers.     

PCR versus hybridization for detecting virulence genes of enterohemorrhagic Escherichia coli

We compared PCR amplification of 9 enterohemorrhagic Escherichia coli virulence factors among 40 isolates (21 O/H antigenicity classes) with DNA hybridization. Both methods showed 100% of the chromosomal and phage genes: eae, stx, and stx2. PCR did not detect 4%-20% of hybridizable plasmid genes: hlyA, katP, espP, toxB, open reading frame (ORF) 1, and ORF2.     

Characterization of Salmonella enterica and detection of the virulence genes specific to diarrheagenic Escherichia coli from poultry carcasses in Ouagadougou, Burkina Faso

One hundred chicken carcasses purchased from three markets selling poultry in Ouagadougou, Burkina Faso, between June 2010 and October 2010 were examined for their microbiological quality. The presence of Salmonella was investigated using standard bacteriological procedures, and the isolates obtained were serotyped and tested for antimicrobial susceptibility. The presence of virulence-associated genes of the five main pathogroups of diarrheagenic Escherichia coli-Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli, and enteroinvasive E. coli-was investigated using 16-plex polymerase chain reaction (PCR) on the mixed bacterial cultures from the poultry samples. Of the 100 chicken carcasses studied, 57 were contaminated by Salmonella; 16 different serotypes were identified, the most frequent being Salmonella Derby, found in 28 samples. Four Salmonella strains were resistant to tetracycline, and two were resistant to streptomycin. Based on the PCR detection of the virulence genes, in total, 45 carcasses were contaminated by three pathogroups of E. coli: STEC, EPEC, or EAEC. The STEC and EPEC virulence genes were detected on six and 39 carcasses, respectively. EAEC virulence genes were only detected in combination with those of EPEC (on 11 carcasses) or STEC (on two carcasses). The STEC-positive carcasses contained the genes stx(1), stx(2), eaeA, escV, and ent in different combinations. None of the EPEC-positive carcasses contained the bfp gene, indicating that only atypical EPEC was present. EAEC virulence genes detected were aggR and/or pic. The high proportion of chicken carcasses contaminated by Salmonella and diarrheagenic E. coli indicates a potential food safety risk for consumers and highlights the necessity of public awareness of these pathogens.