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。     

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。     

一株不产志贺毒素的肠出血性大肠杆菌O157:H7菌株的鉴定

目的 对一株不产志贺毒素的肠出血性大肠杆菌O157:H7菌株进行血清生化特征及毒力基因鉴定.方法 对154菌株进行常规血清生化鉴定,使用聚合酶链反应(PCR)方法检测该菌的志贺毒素基因及其他毒力因子,同时以southern杂交、Hela细胞毒实验进行证实.结果 菌株154的志贺毒素PCR检测、shouthern杂交为阴性,Hela细胞毒定量结果显示该菌株不产生志贺毒素,毒力因子eae,hly检测为阳性.结论 菌株154是不产志贺毒素的肠出血性大肠杆菌O157:H7菌株,且具有O157:H7菌株特异性的毒力因子.     

产志贺毒素大肠埃希菌的特殊类型

产志贺毒素大肠埃希菌（STEC）的菌种以O157：H7为代表，但现在已扩展至近70个血清型。STEC的重要生化反应特征是不发酵山梨醇，现已在德国，捷克发现能在24h内发酵山梨醇的菌株，且其H抗原缺失。少数不产生志贺毒素，stx基因为阴性的发酵山梨醇的O157：H菌株也有欧洲发现。以上新情况及STEC的检出更具挑战性。     

双重实时荧光定量PCR检测产志贺毒素大肠埃希菌stx1和stx2基因

摘要：目的：建立一种检测产志贺毒素大肠埃希菌(STEC)的双重实时荧光定量PCR法。 方法：根据stx1和stx2及其变种序列,设计PCR引物和TaqMan探针,建立双重实时荧光定量PCR检测体系,进行灵敏度和特异性评价,并对STEC模拟粪便标本及动物粪便标本进行检测分析。 结果：双重实时荧光定量PCR检测含志贺毒素基因重组质粒的灵敏度为1×10² copies/反应体系;该法对17种常见肠道病原菌均无特异性扩增,对47株不同志贺毒素类型及变种的已知STEC菌株的检测特异性为100%;对模拟粪便标本的检测下限为3.55×10³ CFU/g;对动物粪便标本的检测阳性率高于细菌分离培养。 结论: 建立的双重实时荧光定量PCR可作为不同志贺毒素类型及变种的STEC菌株的快速鉴定方法,亦可用于人感染性腹泻标本和动物粪便标本STEC感染的快速筛查。     

产志贺毒素大肠埃希菌的特殊类型

产志贺毒素大肠埃希菌 (STEC)的菌种以O15 7∶H7为代表 ,但现在已扩展至近 70个血清型。STEC的重要生化反应特征是不发酵山梨醇 ,现已在德国、捷克发现能在 2 4h内发酵山梨醇的菌株 ,且其H抗原缺失。少数不产生志贺毒素、stx基因为阴性的发酵山梨醇的O15 7∶H菌株也在欧洲发现。以上新情况使STEC的检出更具挑战性。     

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.     

产志贺毒素大肠埃希菌生物被膜形成能力与毒力基因表达的关系

目的探讨临床腹泻患者产志贺毒素大肠埃希菌(STEC)生物被膜形成能力与致病毒力基因表达的关系。方法收集从520例临床腹泻患者标本中检测分离到的STEC,进行体外药敏试验和生物被膜形成能力定量分析;应用多重聚合酶链反应(PCR)检测产志贺毒素相关基因stx1、stx2,实时定量逆转录PCR(RT-PCR)检测eae、ehx A基因表达。以健康体检人群粪便标本中分离的98株大肠埃希菌作为对照菌株。结果从520例腹泻患者标本中分离出328株大肠埃希菌,其中stx1、stx2基因阳性菌(即STEC)78株(23.8%)。78株STEC中产生物被膜菌57株(73.1%),eae和ehx A基因的相对表达量分别为6.57(2.82~27.8)、1.81(1.07~5.28);不产生物被膜菌21株(26.9%),eae和ehx A基因的相对表达量分别为3.41(0.96~12.1)、1.76(0.89~6.06);另分离出副溶血弧菌18株[其中产生物被膜菌10株(55.6%)]、奇异变形杆菌3株。98株对照菌中产生物被膜菌39株(39.7%),eae和ehx A基因的相对表达量分别为1.02(0.86~3.47)、1.03(0.88~3.03)。产生物被膜STEC对氨苄西林/舒巴坦、复方磺胺甲噁唑、环丙沙星的耐药率明显高于不产生物被膜STEC和对照菌株。结论STEC生物被膜形成率明显高于健康人群定植大肠埃希菌,产生物被膜STEC的eae基因表达明显提高;ehx A基因表达与不产生物被膜的STEC比较无明显变化。     

多重实时PCR检测致泻性大肠埃希菌志贺毒素和紧密素基因

目的 建立多重实时PCR检测志贺毒素（stx1、stx2）基因和紧密素基因（eae）的方法。方法 优化多重实时PCR反应条件，检测系列稀释的阳性菌DNA提取物及纯化阳性质粒。检测42株携带已知毒力基因的大肠埃希菌株，并比较其阳性和阴性符合率。对比3种粪便样品处理和DNA提取方法，选出最适方法。同时用该方法对36份腹泻患者粪便标本进行直接检测且对阳性标本进行菌株分离和鉴定。结果 多重实时PCR方法最低可以检测到10^1拷贝／pJ的毒力基因和10。CFU／p．1的DEL933大肠埃希菌。检测42株携带已知毒力基因的大肠埃希菌的阳性和阴性符合率均为100％。粪便样品的DNA提取以BP肉汤增菌6h后煮沸提取效果最好。36份腹泻患者粪便中2份eae阳性，均鉴定为大肠埃希菌。结论 建立的同时检测stx1、stx2、eae基因的多重实时PCR方法，具有较高的敏感性，可用于产志贺毒素大肠埃希菌（STEC）和肠致病性大肠埃希菌（EPEC）毒力基因鉴定及临床腹泻粪便标本的快速筛检。     

江苏省东台地区家畜非O157产志贺毒素大肠埃希菌耐药性及多位点序列分型分析

  目的  了解江苏省东台地区家畜来源非O157产志贺毒素大肠埃希菌（STEC）抗生素耐药表型、耐药基因,以及多位点序列分型（MLST）情况。  方法  于2019年5月,江苏省东台市采集家畜粪便样本301份（羊粪便231份,牛粪便70份）,分离非O157 STEC菌株。 采用改良微量肉汤法测定菌株对21种抗生素最小抑菌浓度（MIC）;通过全基因组测序技术对O∶H血清型、耐药基因和多位点序列型别进行预测。  结果  在68株非O157 STEC中,有32株对至少1种药物耐药。 其中,STEC对四环素耐药率最高（42.6%）,其次分别对阿奇霉素（36.8%）、复方新诺明（35.3%）、链霉素（32.3%）、氯霉素（30.9%）、环丙沙星（29.4%）等9种抗生素耐药;共识别出25种耐药基因,其中,氨基糖苷类耐药基因和叶酸途径抑制剂类耐药基因携带率最高（44.1%）,其次为四环素类耐药基因tet(A)（42.6%）;MLST将分离株分为13种ST型,其中ST43（19.1%）和ST155（16.2%）比例较高。 最小生成树显示,具有相同血清型的STEC菌株多聚集在一起。 STEC中4种ST型别（ST25、ST40、ST43、ST675）分别与引起肠溶血性尿毒综合征相关的肠出血性大肠埃希菌（HUSEC）聚类成簇。  结论  江苏东台地区家畜中非O157 STEC耐药形势复杂,且存在多重耐药现象。 此外,该地区STEC菌株对人群存在潜在威胁,家畜作为非O157 STEC宿主应引起重视。     

A multiple protocol to improve diagnosis and isolation of Shiga toxin-producing Escherichia coli from human stool specimens

Many infections caused by Shiga toxin-producing Escherichia coli (STEC) are undiagnosed, particularly non-O157 STEC. We evaluated the use of a multiple protocol approach to improve diagnosis, isolation, and characterization of STEC strains. Among 18 presumptive STEC-positive stool samples received by the INOVA Fairfax Hospital, Falls Church, VA, in 2006, 16 were Shiga toxin positive. From these 16 stool samples, 8 O157:H7 and 5 non-O157 STEC were isolated by plating onto sorbitol MacConkey (SMAC) agar. The remaining 5 stool samples that did not yield colonies on SMAC agar plates were enriched. All enriched samples were Shiga toxin positive, and 2 O157:H7 and 1 non-O157 STEC were subsequently isolated. The 2 remaining enriched samples did not yield isolates; however, based on polymerase chain reaction (PCR) analysis, both samples contained STEC genes. Based on PCR analysis of non-O157 strains, 3 strain types were identified. Samples from 3 patients, received within 2 days of one another, had a similar gene profile-eae and stx(1) negative and stx(2) positive-suggesting that these patients were likely infected with the same strain. Our results indicate that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of outbreaks.     

Semi-automated fluorogenic PCR assays (TaqMan) forrapid detection of Escherichia coli O157:H7 and other shiga toxigenic E. coli.

Semi-automated detection of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC) was achieved using fluorogenic polymerase chain reaction (PCR). These PCR assays were designed to amplify 80, 120 and 150 bp regions of virulence genes stx1, stx2 and eaeA, respectively, using specific primers. The fluorogenic probes were used for specific detection of amplified products of the stx1 and stx2 genes of STEC, and the eaeA gene of EHEC O157:H7. For multiplex PCR assay, the three sets of primers and fluorogenic probes were included in one reaction to simultaneously amplify and detect any of the three targeted virulence genes. In non-multiplex PCR assay, each of the three virulence genes was amplified and detected in independent reactions. The specificity of these assays was evaluated using suspensions of STEC and other bacterial species lacking stx1, stx2 and eaeA. The multiplex assay detected all STEC harbouring any combination of three virulence genes. Three non-multiplex PCR reactions identified types of Shiga toxin genes carried by a STEC and identified STEC as either EHEC O157:H7 or non-O157:H7 STEC. Sensitivity limits of these assays in beef and faeces inoculated with EHEC O157:H7 were 5.8 to 580 cfu and 1.2 to 1200 cfu, respectively. These assays can be completed within 8-10 h when performed simultaneously or within 13 h if the multiplex assay is used as an initial screen for detecting STEC and the non-multiplex assay is used for subsequent detection of stx1 and stx2 of STEC and eaeA of EHEC O157:H7 Copyright 1999 Academic Press.     

Detection by 5'-nuclease PCR of Shiga-toxin producing Escherichia coli O26, O55, O91, O103, O111, O113, O145 and O157:H7, associated with the world's most frequent clinical cases

This paper describes 5'-nuclease PCR assays for detecting eight O-serogroups, H7 flagellar antigen and stx genes from the Shiga toxin-producing Escherichia coli (STEC) associated with the world's most frequent clinical cases. A single set of primers was used to detect the genes stx1 and stx2 in the same reaction by 5'-nuclease PCR. Serotyping by 5'-nuclease PCR of STEC was based on the selection of primers and probes targeting the O-antigen gene clusters of E. coli O26, O55, O91, O111, O113, O157, the eae gene of E. coli O103, the O-island 29 of E. coli O145, and the flagellar H7 antigen gene. Results obtained on a collection of 190 strains indicate that the 5'-nuclease PCR assays used here could serve as a basis for rapid specific stx, O and H7 typing of these major pathogenic serogroups of E. coli. This work provides sensitive and specific tests for the rapid, reliable detection of the main pathogenic E. coli O-serogroups of major public health concern.     

Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes

A multiplex PCR assay was developed which allowed the simultaneous detection of five trait genes or virulence markers in enterohemorrhagic Escherichia coli (EHEC) serotypes. A primer pair, designed to detect a single base-pair mutation in the uidA gene, is specific only for the prototypic EHEC of O157:H7 serotype and its toxigenic, non-motile variants. In a similar way, primers to the eaeA gene of the gamma-intimin derivative specifically detects strains in the EHEC 1 clonal group, which consists mostly of O157:H7 and some O55:H7 serotypes. The other three primer pairs, specific for stx1, stx2 and both variants of ehxA genes, will detect the presence of these virulence genes in all EHEC serotypes. Analysis of 34 strains, including various serotypes of EHEC, Shiga toxin-producing E. coli and enteropathogenic E. coli, confirmed that the multiplex PCR assay detected the presence of these genes in a manner consistent with the known genotype of each respective strains.     

Comparison between ImmunoCard STAT! and real-time PCR as screening tools for both O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in Southern Alberta,Canada

An increasing number of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections and outbreaks have been reported. In this study, we evaluated the performance of ImmunoCard STAT!^® (Meridian Bioscience, Inc., Cincinnati, OH, USA) as a method to screen stool specimens for STEC (O157 and non-O157). An in-house real-time PCR method was used as the “gold standard”. We also evaluated the prevalence and clinical characteristics of STEC infections in the Alberta South West Zone. From July to November 2011, 819 stool specimens submitted for routine stool culture were tested. With our in-house real-time PCR, 7 O157:H7 and 10 non-O157 STEC isolates were identified for a total of 17 STECs. In comparison, ImmunoCard STAT!^® identified a total of 6, resulting in a sensitivity and specificity of 35% and 99%, respectively (P < 0.05). Because of the low sensitivity, ImmunoCard STAT!^® cannot be recommended as a routine screening test for STEC from enriched stool specimens. The rate of STEC positivity as detected by PCR was 2.08%, of which 0.86% was O157:H7 and 1.22% non-O157 STEC. Five of the 7 cases of STEC O157 infection experienced bloody diarrhea, and 1 developed hemolytic uremic syndrome.     

产志贺毒素大肠埃希菌分离株多位点可变数目串联重复序列分析

目的 探讨多位点可变数目串联重复序列分析[multiple-locus VNTR (variable-number tandem repeats) analysis,MLVA]分型方法对产志贺毒素大肠埃希菌分离株的分型效果,初步了解菌株分子流行病学特征。 方法 采用7个VNTR位点对70株产志贺毒素大肠埃希菌分离株进行分析,并根据多态性位点的重复数目利用BioNumerics软件进行聚类分析。 结果 70株菌株被分为46种MLVA型别,O157与非O157菌株可分为A、B两个明显不同的群。除少部分菌株外,不同宿主来源、志贺毒素型别及血清型的非O157菌株与MLVA型别存在较好的相关性。 结论 7个VNTR位点的MLVA分型方案对于产志贺毒素大肠埃希菌的菌株分型、暴发溯源等具有一定的参考价值,但仍需进一步完善。     

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.     

Shiga toxin- and enterotoxin-producing Escherichia coli isolated from subjects with bloody and nonbloody diarrhea in Bangkok,Thailand

A total of 314 stool samples collected from 92 subjects with bloody diarrhea, 119 subjects with non-bloody diarrhea and 103 normal subjects in Bangkok, Thailand, were investigated for the presence of Shiga toxin-producing Escherichia coli (STEC) and enterotoxin-producing E. coli (ETEC) by multiplex PCR assay. Virulence genes and cytotoxic effect to Vero cells of STEC were also determined. STEC (5 isolates) and ETEC (18 isolates) were detected in 3 and 14 subjects, respectively. Among subjects containing ETEC, only one person belonged to normal control group. The detected STEC included two isolates (serotypes O26:H(-) and O111:H(-)) of Shiga toxin type 1 (Stx1-only) STEC from a child with non-bloody diarrhea, two isolates (Stx1-Stx2 STEC and Stx1-only STEC) from an adult with bloody diarrhea, and one isolate of Stx1-Stx2v STEC (O157:H7) from normal child. Only Stx1-Stx2 STEC isolate was found to exhibit toxicity to Vero cells and carry hlyA gene. The intimin encoding gene locus eaeA was not detected in any isolate. These results indicate that most of STEC isolates in Thailand were low virulent.