大肠埃希菌O157及其检验

以往,对食品中的大肠埃希菌检验主要是从食品卫生指标角度进行。通过对大肠埃希菌的检验,以确定食品受粪便污染的程度,而O157：H7大肠埃希菌系临床腹泻病原菌,是致病性大肠埃希菌的重要成员。近年,由该菌引起的食物中毒来势颇为凶猛,深受世界各国政府所重视。本文试对该菌的有关情况做一简述。1腹泻原性大肠埃希菌的分类和引起的疾病（见表1）引起腹泻的大肠埃希菌,已知有肠致病性大肠埃希菌（EPEC）、肠侵袭性大肠埃希菌（EIEC）、产肠毒素大肠埃希菌（EIEC与肠出血性大肠埃希菌（EHEC）或称产Vero毒素大肠埃希菌（VTEC）…     

分型检测致泻性大肠埃希氏菌PCR技术研究进展

致泻性大肠埃希氏菌是一类能够引起人类和动物腹泻的食源性致病菌,迅速确定致泻性大肠埃希氏菌的污染来源可有效缩小疫情影响范围,建立简便高效的致泻性大肠埃希氏菌检测与分型技术是保障食品安全和控制疫情的关键。为适应对时间敏感度较高要求的现场或在线检测,基于PCR技术的检测分型方法不断地被标准化和规范化。对近年来国内外的致泻性大肠埃希氏菌分子检测与分型的PCR技术研究进展进行了综述,并详细地介绍了多重聚合酶链反应、荧光实时定量聚合酶链反应和核酸等温扩增技术的原理及其优缺点。为致病菌溯源方法的选择提供参考,对防御并控制致病菌引起流行病传播具有重要的意义。     

新腹泻原性大肠杆菌的致病机理

<正> 以往的研究将肠道病原性大肠杆菌分成三类。一类是主要引起婴幼儿流行性腹泻的致病性大肠杆菌(BPEC);另一类是引起旅游者腹泻和发展中国家婴幼儿腹泻的产毒性大肠杆菌(ETEC);再一类是导致痢疾等疾病的侵袭性大肠杆菌(EIEC)。关于ETEC和EIEC的致病机理研究的比较清楚,ETEC中要产生LT和ST肠毒素,EIEC象志贺氏菌一样能侵入肠上皮细胞并繁殖。EPEC的致病性可能与某种粘附因子和细胞毒素产生有关。近来人们又提出两类新的腹泻性大肠杆菌,即肠出血性大肠杆菌(En-     

多重PCR检测无公害畜禽肉和水产品中4种致病菌

建立无公害畜禽肉和水产品中肠出血性大肠杆菌(EHEC)、沙门氏菌、副溶血性弧菌(VP)和单核细胞增生性李斯特氏菌(LM)的多重PCR检测方法,为这些致病菌的快速诊断提供实验依据。选择分别针对EHEC溶血素基因hlyAB、副溶血性弧菌属保守序列toxR基因、沙门氏菌侵袭基因invA和LM的iap基因特异的4对引物,先分别进行单重PCR扩增,再同时加入4对引物进行多重PCR扩增,扩增产物经测序验证。建立的多重PCR方法可简便、快速、灵敏地实现对EHEC、LM、沙门氏菌和VP的同时检测,在畜禽肉和水产品中的检测灵敏度达到10^3cfu/mL。     

整合子介导的肠致病性大肠埃希菌多重耐药研究

目的了解肠致病性大肠埃希菌（EPEC）多重耐药菌株中整合酶基因的携带情况,研究整合子与抗生素多重耐药的相关性。方法使用血清学的方法对EPEC进行初筛,用PCR扩增EPEC毒力基因（eae,EAF,bfpA）进行确证。对确证为EPEC的细菌DNA进行提取,使用PCR方法对整合酶基因及在整合子中插入的基因盒进行扩增。EPEC药敏试验采用K-B琼脂扩散法。结果在34株EPEC中,ESBL为14株,其中在lI株ESBL阳性细菌中扩增出整合子I整合酶片段,在20株ESBL阴性细菌中,有7株扩增出相应的片段。在这所有的34株细菌中未检出整合子Ⅱ和Ⅲ。结论I类整合子在肠致病性大肠埃希菌多重耐药菌株中最常见,是导致细菌多重耐药的一个重要因素,合理用药,控制耐药基因的传播是当前医学面临的一个重要问题     

TaqMan探针荧光PCR检测肠出血性大肠埃希菌O157:H7方法的建立

肠出血性大肠埃希菌(Enterohemorrhagie Escherichia coli,EHEC)O157:H7是一种重要的传染病病原菌,以EHEC O157:H7标准菌株rfbE保守区设计一对特异引物和一条探针,建立了检测EHEC O157:H7核酸的荧光定量PCR检测方法。实验结果表明荧光定量PCR检测方法特异性好,最低检测限为20 cfu/mL,线性范围是102～108cfu/mL。稳定性试验表明批内变异系数和批间变异系数分别为2.06%和2.45%。     

TaqMan探针荧光PCR检测肠出血性大肠埃希菌O157∶H7方法的建立

肠出血性大肠埃希菌(Enterohemorrhagie Escherichia coli,EHEC)O157∶H7是一种重要的传染病病原菌,以EHEC O157∶H7标准菌株rfbE保守区设计一对特异引物和一条探针,建立了检测EHEC O157∶H7核酸的荧光定量PCR检测方法.实验结果表明荧光定量PCR检测方法特异性好,最低检测限为20 cfu/mL,线性范围是102～108 cfu/mL.稳定性试验表明批内变异系数和批间变异系数分别为2.06％和2.45％.     

多重PCR检测无公害畜禽肉和水产品中4种致病菌*

建立无公害畜禽肉和水产品中肠出血性大肠杆菌(EHEC)、沙门氏菌、副溶血性弧菌(VP)和单核细胞增生性李斯特氏菌(LM)的多重PCR检测方法,为这些致病菌的快速诊断提供实验依据。选择分别针对EHEC溶血素基因hlyAB、副溶血性弧菌属保守序列toxR基因、沙门氏菌侵袭基因invA和LM的iap基因特异的4对引物,先分别进行单重PCR扩增,再同时加入4对引物进行多重PCR扩增,扩增产物经测序验证。建立的多重PCR方法可简便、快速、灵敏地实现对EHEC、     

2007年上海市儿童社区获得性腹泻致病菌谱分析

为了解上海地区社区获得性腹泻儿童致病菌流行病学情况,对2007年度就诊于我院的2871例社区获得性腹泻患儿进行粪便弯曲菌、志贺菌、沙门菌,霍乱弧菌、副溶血性弧菌、致腹泻大肠埃希菌、气单胞菌、耶尔森菌进行培养分离、鉴定及部分菌株的血清分型。结果发现,弯曲菌在腹泻患儿粪便中的分离率为11.1%(172/1556),4月份感染率最高(P<0.001),多见于1～4岁儿童(P<0.05);其次是志贺菌,分离率为3.2%(91/2871),其中福氏志贺菌占63.7%;第3位是致腹泻大肠埃希菌,分离率为1.0%(30/2871),其中肠致病性大肠埃希菌占70.0%,以O55/K59血清型为主。沙门菌、气单胞菌分离率不超过0.5%,未检测到霍乱弧菌、副溶血性弧菌和耶尔森菌。本次调查显示,弯曲菌、志贺菌、致腹泻大肠埃希菌是目前上海地区儿童社区获得性腹泻的3种主要致病菌。弯曲菌感染率明显高于志贺菌及致腹泻大肠埃希菌,提示临床微生物室应将弯曲菌、致腹泻大肠埃希菌作为儿童社区获得性腹泻常规监测的病原微生物。     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hybridization with the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O:H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.     

Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli

Diarrheagenic E. coli comprise a diverse group of microorganisms responsible for gastrointestinal diseases in humans. On the basis of their virulence traits they are distinguished from the non-pathogenic E. coli and classified in several categories. Molecular methods represent the most reliable techniques for distinguishing pathogenic from non-pathogenic E. coli and characterising their pathogenic features. In this paper we report the development of a set of three multiplex PCR assays for the simultaneous and rapid identification of diarrheagenic E. coli belonging to ETEC, EPEC, EHEC and EIEC groups. Assay 1 utilizes primer pairs specific for genes coding for ST and LT toxins of ETEC, and for the E. coli beta-glucuronidase (uidA); assay 2 detects the presence of the eae and bfpA genes of EPEC, and assay 3 recognizes stx1 and stx2 of EHEC, and ial of EIEC. This technique has been validated on 190 E. coli isolated in Angola, Italy and Mozambique from feces of children with diarrhea. Results obtained with the set of multiplex PCR demonstrated 100% accordance with those obtained for the same isolates by PCR on single target genes. The proposed set of multiplex PCRs is the first reported assay that allows the simultaneous characterization of the four categories of diarrheagenic E. coli.     

Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children

A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

Rapid categorization of pathogenic Escherichia coli by multiplex PCR

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.     

变性高效液相色谱检测食品中致泻性大肠杆菌

[目的]应用多聚酶链式反应(polymerase chain reaction,PCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中致泻性大肠杆菌的快速检测方法.[方法]分别根据4种致泻性大肠杆菌的特异性毒力因子基因序列设计引物,PCR扩增产物经变性高效液相色谱进行快速检测.以肠产毒性大肠杆菌等32株试验菌株做特异性检测;4种致泻性大肠杆菌标准菌株稀释成不同梯度,做灵敏度检测.[结果]试验结果表明该方法有很好的特异性,且灵敏度高,检测限可达到:肠产毒性大肠杆菌27 CFU/mL、肠致病性大肠杆菌33 CFU/mL、肠出血性大肠杆菌25 CFU/mL、肠侵袭性大肠杆菌42 CFU/mL.[结论]该方法可以快速、准确地检测食品中的致泻性大肠杆菌,是食品中病原菌检测的新技术和新方法.     

Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique

A study was conducted on the circulation of potentially diarrheagenic Escherichia coli in two groups of children, both under the age of seven. The first group (548 children) suffered from mild diarrhea and attended the Xipamanine Health Center of Maputo, in Mozambique. The second group (380 children) included randomly chosen, asymptomatic, children from the same population. A total of 503 E. coli strains were isolated from the two groups of children (n=375 and 128, respectively). All E. coli strains were genotypically and phenotypically screened. The presence of virulence-associated genes was assessed by a set of multiplex PCR specific for st and lt genes of enterotoxic Escherichia coli (ETEC), eae and bfpA genes of enteropathogenic E. coli (EPEC), stx(1) and stx(2) of enterohemorrhagic E. coli (EHEC), ial of enteroinvasive E. coli (EIEC) and the species-specific gene uidA. Adhesion and citotoxicity of isolated E. coli were evaluated in vitro on different cell cultures. A total of 37 isolates harbored virulence-associated genes: 18 were classified as ETEC, (15 from symptomatic, and three from asymptomatic children), 16 as EPEC (respectively, 13 and 3) and three EIEC in the symptomatic group. No stx(1) or stx(2) genes, associated with enterohemorrhagic E. coli were found. On the basis of the adhesion pattern on HeLa cells, 167 E. coli were classified as diffusely adhering, (125 in patients and 42 in controls) and 67 as enteroaggregative, (50 and 17, respectively). To the best of our knowledge, this is the first report in the literature on the circulation of potentially diarrheagenic E. coli in Mozambique.     

Rapid detection of diarrheagenic E. coli by real-time PCR

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.     

Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.