安徽省114株EHECO157∶H7菌株毒力因子基因的初步分析

目的　对安徽省近几年分离的肠出血性大肠杆菌O157∶H7(EHECO157∶H7)菌株的毒力因子基因进行实验室检测 ,以分析和研究肠出血性大肠杆菌O157∶H7的毒力特征和致病特性。方法　采用聚合酶链反应(PCR)检测EHECO157∶H7的两种重要的毒力因子志贺样毒素 (SLT)和溶血素 (hly)的特异性基因片段 ,并辅以血清学试验、生化试验进行菌株的生物学特性鉴定。结果和结论　检测结果显示 ,有 63.2%的EHECO157∶H7菌株携带SLT、hly基因 ,动物源的菌株以家禽较高 ,家畜次之 ,3株源自腹泻病人的菌株毒力因子基因携带率为33.3% ,说明分离的EHECO157∶H7菌株具有较强的致病性 ;114株菌株中,有 14.0%的菌株在 24h内发酵山梨醇 ,发酵山梨醇菌株的毒力因子基因携带率 18.8% ,显著低于不发酵菌株的 70.4% ,显示出山梨醇发酵特性变异与毒力因子基因缺失之间具有一定的关联性。     

安徽省114株EHECO_(157)∶H_7菌株毒力因子基因的初步分析

目的　对安徽省近几年分离的肠出血性大肠杆菌O1 57∶H7(EHECO1 57∶H7)菌株的毒力因子基因进行实验室检测 ,以分析和研究肠出血性大肠杆菌O1 57∶H7的毒力特征和致病特性。方法　采用聚合酶链反应(PCR)检测EHECO1 57∶H7的两种重要的毒力因子志贺样毒素 (SLT)和溶血素 (hly)的特异性基因片段 ,并辅以血清学试验、生化试验进行菌株的生物学特性鉴定。结果和结论　检测结果显示 ,有 6 3 2 %的EHECO1 57∶H7菌株携带SLT、hly基因 ,动物源的菌株以家禽较高 ,家畜次之 ,3株源自腹泻病人的菌株毒力因子基因携带率为33 3% ,说明分离的EHECO1 57∶H7菌株具有较强的致病性 ;114株菌株中 ,有 14 0 %的菌株在 2 4h内发酵山梨醇 ,发酵山梨醇菌株的毒力因子基因携带率 18 8% ,显著低于不发酵菌株的 70 4 % ,显示出山梨醇发酵特性变异与毒力因子基因缺失之间具有一定的关联性     

不同来源STECO157:H7生物学特性研究

目的 探讨浙江省产志贺毒素大肠埃希菌O157:H7菌株PFGE分型及携带毒力基因以及耐药情况.方法 菌株生化、血清鉴定按API 20E鉴定系统;大肠杆菌O157:H7诊断血清凝集试验;采用聚合酶链反应(PCR)法检测O、H抗原及毒力基因SLT1、SLT2、Hly、eaeA;STEC O157:H7分型用脉冲场凝胶电泳(pulse field gel electrophoresis,PFGE)方法进行;14种抗生素进行药敏试验采用K-B法.结果 5株可疑菌株经生化和血清鉴定符合O157:H7特性;毒力基因检测所有分离株SLT2、Hly、eaeA三种毒力基因均阳性而SLT1均阴性;脉冲场凝胶电泳分型结果显示,有2株菌PFGE电泳条带完全相同,其余3株菌电泳条带不同,5株菌共分为4个带型;经耐药性分析,菌株对头孢噻吩、头孢噻肟、庆大霉素、环丙沙星100%敏感;对复方新诺明、氨苄西林100%耐药.结论 STEC O157:H7菌株在浙江省一些地区存在,并且携带SLT2毒力基因,PFGE型别较多,同时也有人间感染病例的出现,提示应加强O157:H7监测力度,防止该菌在人间的感染和流行.     

江苏省建湖地区大肠埃希菌O157:H7毒力基因分析

目的对检测出的17份肠出血性大肠埃希菌0157：H7阳性菌株进行毒力基因分析。方法收集江苏省建湖地区2008年5月～9月监测出的17株肠出血性大肠埃希菌0157：H7阳性菌株,利用单一和多重PCR法检测不同来源菌株0157抗原编码（rfbE）、H7鞭毛抗原编码（fliC）、志贺样毒素（stx1和stx2）、溶血素（hly）和黏附抹平因子（eaeA）。结果通过血清学方法确定的17株肠出血性大肠埃希菌0157：H7阳性菌株,再通过PCR法检测,其中15株大肠埃希菌rfbE和fliC基因检测为阳性,确认为EHEC O157：H7,其中9株菌株扩增出全部毒力基因,5株菌株扩增出除stxl外其它全部毒力基因,1株仅检出stx2,hly毒力基因;2株大肠埃希菌rfbE基因检测阳性、fliC基因检测为阴性,确认为O157：H7-大肠埃希菌,无毒力基因检出。结论该地区存在O157：H7大肠埃希菌强毒株污染,从流行病学角度看具潜在的流行性危险,当地疾控部门应加强O157：H7的综合监测。     

产志贺毒素大肠埃希菌的鉴定及分布特征

目的 探讨吉林地区产志贺毒素大肠埃希菌（STEC）分离株的分布和特征。方法 采集牛羊粪便及其肉类食品、蔬菜，分离大肠埃希菌，PCR鉴定毒力因子及血清学分型。结果 380份标本中鉴定出12株STEC,3株为产志贺毒素O157：H7型，9株为产志贺毒素非O157：H17型。结论 STEC存在于不同来源的标本中，菌株血清型与毒力因子存在一定差异。     

浙江省检出带毒力基因的人源性肠出血性大肠杆菌O157:H7

目的了解肠出血性大肠杆菌O157:H7在浙江省人群中的感染情况和菌型分布特征,制定相应的防治对策。方法5～10月份肠道传染病高发季节,在浙江省各地(市)肠道门诊采集腹泻病人粪便,对O157:H7菌株进行分离培养,并用PCR方法检测其毒力基因。结果1998年开始至今的人群监测中,共检出2株无毒力的肠出血性大肠杆菌O157:H7。PCR毒力基因检测,SLT2和Hly阳性,SLT1阴性。结论与以往不同,此次分离到带有毒力基因的人源性肠出血性大肠杆菌O157:H7,说明浙江省O157:H7在菌型特征方面发生了变化,对人群健康构成了更大的威胁。     

O157大肠杆菌生物学特性及分离鉴定技术研究

目的　研究肠出血性大肠杆菌 (EnterohemorrhagicEscherichiacoli)O15 7:H7和其他O15 7大肠杆菌 (以下简称O15 7:非H7)的生物学特性 ,制定高效、实用的O15 7:H7分离程序及鉴定指标。方法　生化试验和玻片凝集试验分别检测菌株的生化特性和菌体抗原 ;穿刺法接种抗 -H7抗血清琼脂检测H7鞭毛抗原 ,试管凝集试验检测其它鞭毛抗原。聚合酶链反应 (PCR)分别检测特异性基因和毒力基因。结果　 194株O15 7:H7与10 1株O15 7:非H7在山梨醇、蔗糖、赖氨酸和鸟氨酸等生化反应中有差异。 194株O15 7:H7中 192株(98 97% )携带O15 7特异性基因 ,189株 (97 4 2 % )菌体抗原表型阳性 ;10 1株O15 7:非H7均携带O15 7特异性基因同时表型亦为阳性。 194株O15 7:H7均携带H7特异性基因 ,186株 (95 88% )鞭毛抗原表型阳性。 194株O15 7:H7中除 1株 (0 5 2 % )四种毒力基因检测均为阴性外 ,其余 193株 (99 4 8% )均携带 2种及其以上毒力基因 ;10 1株O157:非H7四种毒力基因检测均为阴性。结论 O157:H7特征性生化反应、O157和H7特异性基因检测及其携带毒力基因的特性,在区分不同类型的0157方面有重要意义,据此可以制定针对性强的O157:H7分离鉴定程序。     

非O157产志贺毒素大肠埃希菌分离株血清型鉴定及主要毒力基因分析

目的 了解中国不同来源非O157产志贺毒素大肠埃希菌分离株血清型及主要毒力基因的流行情况。方法 采用O抗原基因簇特异性聚合酶链反应（PCR）结合全套O抗血清凝聚法确定O血清群,以PCR扩增和测序fliC基因确定H型;采用PCR方法对所有菌株进行stx1、stx2、eae及ehxA基因检测。结果 434株非O157 STEC分离株中,除不可分型菌株外,共检测出82种O血清群和28种H型,其中O20:H30、O2:H32和O2:H45为优势血清型。stx1、stx2和stx1+stx2 3种志贺毒素基因型的检出率分别为25.35%、64.98%和9.68%,而eae和ehxA基因的阳性率分别为3.92%和32.95%。仅15株菌同时携带3种毒力基因,且主要为血清型O26:H11的腹泻患者分离株。结论 我国非O157 STEC分离株血清型复杂多样,同时检测eae和ehxA基因对于发现高致病潜力的菌株具有重要参考价值。     

多重PCR检测肠出血性大肠埃希菌O157：H7

目的 建立一种快速、特异的检测肠出血性大肠埃希菌O157:H7菌株的多重PCR方法。 方法 针对O157:H7菌株的O157、H7抗原特异基因rfbE O157、fliC H7以及stx1、stx2、eaeA和hlyA四种毒力基因设计相应引物,在同一扩增体系中进行PCR反应,通过优化多重PCR 反应条件和循环参数,建立检测O157:H7菌株的多重PCR方法,并测定其特异性和灵敏度。 结果 6 对特异性引物各自扩增相应的基因片段,检测结果与常规PCR 获得的结果一致。细菌纯培养物的检测灵敏度为1.33×10⁴ CFU。 结论 该多重PCR方法能在一次检测中同时反映待测菌株是否为肠出血性大肠埃希菌O157:H7及其携带毒力基因的情况,可为O157:H7大肠埃希菌感染的诊断及流行病学调查提供一种简便、快速的检测手段。     

非O157产志贺毒素大肠杆菌分离株的志贺毒素基因变种及黏附相关基因分析

目的 了解我国部分地区非O157产志贺毒素大肠杆菌分离株的志贺毒素基因变种及其黏附相关基因,为进一步研究致病机制提供依据。 方法 采用聚合酶链反应(polymerase chain reaction,PCR)方法对29株分离菌株的stx1、stx2基因全长扩增并测序,通过与GenBank中已公布的变种序列比对确定菌株stx1、stx2基因的变种类型。对位于LEE毒力岛上的eaeA、escF、escC、tir、espA、espB、espD基因及LEE以外其他黏附相关基因iha、toxB、efa1、sfpA、lpfAO157/OI-141、 lpfAO157/OI-154、saa、lpfAO113、eibG进行PCR检测。 结果 25株stx1阳性菌株中有13株携带stx1a原毒素,12株携带stx1c变种;10株stx2阳性菌株中,7株携带stx2d变种,1株为stx2a原毒素,1株携带stx2g变种,1株携带与stx2e A亚单位、stx2d B亚单位最接近的stx2变种。LEE岛上的7个基因检测结果均为阴性,黏附相关基因iha阳性率为89.7%(26/29),saa阳性菌株3株、eibG阳性菌株1株,其余6个黏附相关基因均为阴性。 结论 我国部分非O157 STEC菌株的志贺毒素基因以stx1c、stx2d变种为主,LEE毒力岛不存在,而黏附相关基因iha广泛存在于不同血清型的产志贺毒素大肠杆菌菌株中。     

Optimization of a 3'-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR

Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA (gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5' nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non-E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.     

Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR

Enterohemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, but more serious complications such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) can result. A real-time PCR method to detect the presence of Shiga toxin producing E. coli (STEC) and E. coli O157:H7 was investigated using SYBR Green I (SG). Primers were designed to target the Shiga toxin genes (stx1 and stx2) and a highly conserved base substitution at +93 of the beta-glucuronidase gene (uidA) unique to E. coli O157:H7. An initial test panel of five E. coli and non-E. coli isolates was tested with individual primer sets (simplex assay) and all primer sets including stx1, stx2, and uidA (multiplex assay). All strains were correctly identified in both assays. Average melt temperatures (Tm's, degrees C) for PCR products were 85.42--stx1, 81.93--stx2, and 88.25--uidA in simplex assays and 85.20--stx1, 81.20--stx2, and 88.16--uidA when multiplexed. Each of the three gene targets in one multiplex reaction could be distinguished by melt curve data with significantly different Tm's. The assay was expanded to a panel of 138 isolates consisting of STEC, E. coli O157:H7, non-toxigenic E. coli, and non-E. coli isolates with melt peaks consistent with those stated above.     

江苏省徐州市出血性大肠埃希菌O157∶H7 感染性腹泻的监测研究

目的 系统监测肠出血性大肠埃希菌O157∶H7(O157∶H7)感染并发肾衰(HUS)、腹泻患者、宿主动物、蝇类、食品携带O157∶H7的状况,为预警监测体系建立提供参考依据。 方法 采集徐州市疫情暴发点和监测点腹泻患者粪便、蝇类和食品标本分离菌株。应用PCR技术检测志贺毒素基因stx。 结果 1999-2011年徐州市累计报告O157∶H7引起的感染性腹泻并发HUS 132例。腹泻患者带菌率1.2%、蝇类带菌率0.5%、食品带菌率1.0%、宿主动物带菌率3.1%。菌株带毒由stx1和stx2共存发展为以不带毒为主。 结论 江苏省徐州市O157∶H7感染自1999年、2000年暴发流行后,该市疫情一直稳定。腹泻患者带菌率、宿主动物带菌率、蝇类带菌率、食品带菌率及菌株带毒情况发生了改变。     

肠出血性大肠埃希菌O157 ∶ H7质粒 pO157 上毒力因子的研究进展

肠出血性大肠埃希菌O157 ∶ H7是一种重要的致病菌,可以引起不同程度人类疾病。目前已知的O157 ∶ H7的致病因子有3个,志贺毒素、粘附抹平效应毒力岛和特异性质粒pO157。其中pO157在致病过程中的作用并不十分清楚。本文结合近年来的研究进展,介绍了pO157上与细菌致病性有关的毒力因子。     

Detection of Escherichia coli O157 using equal-length double-stranded fluorescence probe in a real-time polymerase chain reaction assay

BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) O157 is a dangerous pathogen, which causes bloody diarrhea and severe hemolytic uremic syndrome (HUS). Although several assay systems based on real-time polymerase chain reaction (PCR) have been integrated to detect this pathogen, most of them are not specific. We report a real-time quantitative PCR method targeting rfbE, a gene specifically expressed in E. coli O157. This method can therefore be used to diagnose enterohemorrhagic Escherichia coli (E. coli) O157. METHODS: A nucleic acid based diagnostic assay system, combining equal-length double-stranded fluorescence probe technique and real-time PCR, was developed to detect E. coli O157. This assay system take advantage of the highly conserved rfbE O-antigen synthesis gene, and a pair of fluorescence-quenching probes complementary to rfbE gene were used in a real-time PCR to quantify the presence of the pathogen. RESULTS: The specificity of the diagnostic method was assessed by comparing test results on 14 different related pathogens including common E. coli, enteroinvasive Escherichia coli (EIEC), Salmonella, Shigella and E. coli O157. The detection limit of the method was determined using 10-fold serial dilutions of an E. coli O157 standard sample, and as few as 1.49 x 10(3) CFU/ml could be detected. All E. coli with serotype O157, which expresses rfbE gene, were positive in this assay, while all other species without rfbE gene expression were negative. CONCLUSIONS: By combining equal-length double-stranded fluorescence probe technique and real-time PCR, we have developed a simple, rapid, specific and sensitive method to detect E. coli O157.     

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。     

Rapid PCR detection of enterohemorrhagic Escherichia coli (EHEC) in bovine food products and feces

Although Escherichia coli (E. coli) O157:H7 is a major cause of foodborne illness, other types of E. coli can also cause illness. E. coli that possess the eae gene for attachment and effacing have the potential to cause disease. Many real-time, molecular-based assays have been developed to detect Enterohemorrhagic E. coli (EHEC) including E. coli O157:H7. However, no assay currently exists to detect the eae gene present in E. coli O157:H7 and other EHEC strains with a confirmed positive or negative result in less than 12 h. Raw beef food products (raw ground beef and raw boneless beef) at 25 and 375 g samples and bovine fecal samples at 2 g were inoculated with 10(1), 10(3), 10(4), and 10(5) organisms of E. coli O157:H7 to test the sensitivity of this assay. Fourteen different foodborne bacteria (including E. coli O157:H7) and 19 various E. coli strains, obtained from the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) were tested for specificity. E. coli O157:H7 was detected at the level of 10(1) organisms in both 25 and 375 g samples of raw ground and raw boneless beef products as well as 2 g samples of bovine feces after pre-enrichment and concentration. None of the 14 foodborne bacteria screened for cross-reactivity was detected. All USDA E. coli strains confirmed to contain the eae gene were detected.     

Genetic diversity of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli(EHEC) causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Genomic comparison of an EHEC O157: H7 strain isolated from the Sakai outbreak and a benign laboratory strain K-12 revealed that acquisition of a large amount of foreign DNA has promoted the genetic diversification of E. coli strains. In the emergence of O157: H7, bacteriophages, in particular, played an important role. EHEC are a group of strains with several serotypes, each belonging to different E. coli lineages. Even in an O157 lineage, significant phenotypic and genetic heterogeneities are observed. Recent knowledge on the significance and the generating mechanism of such heterogeneity in EHEC strains are summarized.