长春地区家禽(家畜)中E.coli O157∶H7调查

为了通过对宿主动物EHEC O157∶H7监测,了解长春地区肠出血性大肠埃希菌O157∶H7的污染状况,建立流行病学监测网,以便为疾病预防控制提供良好的科学依据。结果在采集的639份家禽、家畜粪便样品中共检出7株EHEC O157∶H7。     

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％.     

用含10%胎牛血清的DMEM培养肠出血性大肠杆菌O157∶H7可增强其对HeLa细胞的黏附/擦拭损伤

目的:研究生长在不同培养基中的肠出血性大肠杆菌(EHEC)O157∶H7对宿主细胞造成的黏附/擦拭损伤是否存在差异。方法:分别用LB、DMEM、DMEM(含10%胎牛血清)、DMEM(含终浓度为25mmol/L的HEPES)等4种培养基培养O157∶H7,然后感染HeLa细胞,利用Giemsa染色观察细菌黏附差异,进行肌动蛋白荧光染色实验并观察宿主细胞肌动蛋白聚集差异。结果:在含10%胎牛血清的DMEM培养基中培养EHECO157∶H7,其黏附力增加,聚集细胞骨架肌动蛋白的能力明显增强。结论:为进一步研究EHECO157∶H7的致病性,探索O157∶H7新的致病因子奠定了基础。     

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%。     

大肠杆菌O157:H7的sRNA伴侣蛋白Ufq的基因克隆、表达及纯化

目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7的sRNA伴侣蛋白Hfq.方法:利用PCR方法从EHEC O157:H7基因组中扩增出基因hfq,并插入含6xHis标签序列的原核表达载体pET28a(+)的多克隆位点中,构建重组表达质粒pET28a(+)-hfq,以重组质粒转化大肠杆菌BL21(DE3)...     

肠出血性大肠杆菌O157：H7监测及分析

为了了解长春地区动物和人感染肠出血性大肠杆菌O157H7状况,建立流行病学监测网.采集长春市动物养殖场动物粪便和腹泻病人便样进行监测.结果在牛粪和鸡粪中共检出2株O157H7大肠杆菌.可见,在长春地区存在肠出血性大肠杆菌O157H7菌潜在污染的威胁,需要加强监测力度.     

肠出血性大肠杆菌（EHEC）O157：H7紧密粘附素免疫保护性片段（Intimin-C）的克隆表达纯化及部分生物学活性研究

克隆表达并纯化肠出血性大肠杆菌(EHEC)O157:H7紧密粘附素免疫保护性片段(Intimin-C),并对其部分生物学活性进行研究.     

EHEC 的新型粘附因子研究进展*

摘要：肠出血性大肠杆菌（enterohemorrhage ，EHEC）是一种重要的人畜共患病，世界各地包括中国都有不同规模的暴发流 行。EHEC有多种血清型，其中毒力最强血清型是O157：H7。EHEC O157：H7 感染除可使人发生常规腹泻外，还可在5%-10%的病 例中引发严重并发症，甚至死亡。该菌是重要的食源性致病菌，危害严重，缺乏有效的防治手段，而抗生素治疗可能会加剧溶血性 尿毒症（haemolutic uraemic syndrome，HUS）。由于以上特点EHEC O157：H7 成为世界公共卫生问题，引起微生物学家和公共卫生 工作者的广泛关注。目前，临床针对EHEC 感染只是对症治疗和适当的抗菌治疗。粘附是EHEC感染宿主细胞的第一步，没有这 一步，细菌和宿主肠道细胞之间不会发生任何的相互作用，而且对于许多病原菌来说，粘附具有宿主特异性。本文概述了EHEC 的流行病学及粘附机理，并对近年在EHEC 研究中的发现一些新型粘附因子和一些假设的定植因子的研究背景及作用机理作一 综述。     

用于Escherichia coli O157∶H7直接快速检测的倏逝波荧光核酸适配体传感器研究

通过融合倏逝波荧光光纤传感器和特异性核酸适配体的优势,提出了一种基于倏逝波荧光原理及其与病原菌尺寸效应的Escherichia coli O157∶H7(E.coli O157∶H7)直接快速检测方法。基本原理是当一定浓度荧光标记E.coli O157∶H7核酸适配体加入样品检测池时,倏逝波激发荧光分子发出荧光,利用倏逝波全光纤生物传感器即可实现荧光信号的定量检测;当荧光标记的核酸适配体与E.coli O157∶H7混合后加入样品检测池,因倏逝波渗入深度仅为100 nm,导致特异性结合E.coli O157∶H7的核酸适配体标记荧光分子不能被激发,从而使得检测荧光信号降低;利用荧光信号强度与E.coli O157∶H7浓度的比例关系即可实现其定量检测。结果表明:该方法检测E.coli O157∶H7的检测限可达610 CFU/mL,线性检测区间为1.1×10~3-1.4×10~7 CFU/mL。实际水样加标回收率在40%-180%之间,相对标准偏差在10%之内,水样基质对E.coli O157∶H7的检测没有明显影响。本研究建立基于倏逝波荧光原理及其与病原菌尺寸效应的生物传感分析方法具有普适性,仅需使用不同荧光标记的生物识别分子即可实现其他病原菌的直接快速检测。     

大肠杆菌O157:H7致病因子及其保护性免疫研究现状

大肠杆菌O157:H7是肠出血型大肠杆菌(EHEC)的主要病原血清型,它可产生特殊的粘附因子粘附靶细胞,产生Vero毒素和肠溶血素毒力因子.近几年对大肠杆菌O157:H7的毒力因子有了深入的了解,对致病机理作了一些探讨,用实验动物对保护性免疫进行了研究.本文对近几年来0157:H7大肠杆菌的致病因子及其主要的保护性免疫的研究作一简要综述.     

The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been considered as an agent responsible for outbreak of hemorrhagic colitis and the hemolytic uremic syndrome. We examined the effect of the probiotic agent Clostridium butyricum MIYAIRI strain 588 on EHEC O157:H7 infections in vitro and in vivo using gnotobiotic mice. The growth of EHEC O157:H7 and the production of Shiga-like toxins in broth cultures were inhibited by co-incubation with C. butyricum. The antibacterial effects of butyric and lactic acid were demonstrated in a dose-dependent manner. In addition, the inhibitory effect of butyric acid on the viability of EHEC was demonstrated not only at low pH, but also at neutral pH adjusted to 7.0. Flowcytometric analysis showed that pre-incubation of Caco-2 cells with C. butyricum and E. coli K12 inhibited the adhesion of EHEC O157:H7. However, the effect of C. butyricum on adhesion of EHEC to Caco-2 cells was more inhibitory than that of E. coli K12. Gnotobiotic mice mono-associated with EHEC O157:H7 died within 4-7 days after the infection. On the other hand, all gnotobiotic mice prophylactically pre-treated with C. butyricum survived exposure to EHEC O157:H7 and of the gnotobiotic mice therapeutically post-treated with C. butyricum, 50% survived. Both counts of EHEC O157:H7 and the amounts of shiga-like toxins (Stx1 and Stx2) in fecal contents of gnotobiotic mice di-associated with EHEC O157:H7 and C. butyricum were less than those of gnotobiotic mice mono-associated with EHEC O157:H7. These results indicated that the probiotic bacterium C. butyricum MIYAIRI strain 588 has preventive and therapeutic effects on EHEC O157:H7 infection in gnotobiotic mice.     

Production of slime polysaccharide by EHEC and STEC as well as the influence of culture conditions on slime production in Escherichia coli O157:H7

AIMS: To quantify the slime polysaccharide, composed of colanic acid (CA), produced by enterohaemorrhagic and Shiga toxin-producing Escherichia coli (EHEC and STEC) and to determine the influence of culture conditions on CA production in E. coli O157:H7. METHODS AND RESULTS: The study examined the amounts of CA produced by EHEC and STEC, and evaluated the production of CA in E. coli O157:H7 as influenced by medium pH and incubation temperatures. The results indicated that the amounts of CA produced by EHEC and STEC vary to a great extent and CA production in E. coli O157:H7 is influenced by the tested culture conditions. CONCLUSIONS: The abilities of EHEC and STEC to produce CA differ. Medium pH and incubation temperature are among the important factors affecting CA production in E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Slime polysaccharide can affect the abilities of E. coli O157:H7 cells to combat environmental stress. This study contributes to a better understanding of the physiological factors influencing slime polysaccharide production in EHEC and STEC.     

Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and β-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).     

Adhesion and colonization of enterohemorrhagic Escherichia coli O157:H7 in cecum of mice

Infectious diseases due to enterohemorrhagic Escherichia coli (EHEC) are characterized by diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The adherence of EHEC on intestinal epithelial cells is a first step for developing these diseases. In the present study, we examined whether EHEC O157:H7 adhere to intestinal epithelial cells of mice and cause F-actin accumulation in the epithelial cells following the intragastric inoculation of the pathogen. Fecal shedding of the EHEC O157:H7 strain was observed in ICR mice up to 3 weeks. Fecal shedding periods of the type III secretion system-related gene (espA and sepL) deletion mutants were clearly shorter than that of the wild-type EHEC O157:H7 strain. The EHEC O157:H7 colonies were found on the epithelial surfaces of the ceca in association with F-actin accumulation beneath the attached bacteria.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

Fim operon variation in the emergence of Enterohemorrhagic Escherichia coli: an evolutionary and functional analysis

Fim operons were examined to illuminate the emergence of Escherichia coli O157:H7 from the less-virulent E. coli O55:H7. A fim invertible element deletion occurred only after O157:H7 descended from O55:H7, and after sorbitol nonfermenting O157 diverged. Type 1 pili nonexpression correlates with this deletion in all enterohemorrhagic E. coli (EHEC) tested. An N135K FimH mutation in the two most evolved O157:H7 clusters is not found in other EHEC. These data refine the evolutionary history of an emerging pathogen.     

Presence and characterization of a mosaic genomic island which distinguishes sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H(-) from EHEC O157:H7.     

Enterohemorrhagic Escherichia coli Specific Enterohemolysin Induced IL-1β in Human Macrophages and EHEC-Induced IL-1β Required Activation of NLRP3 Inflammasome

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major foodborne pathogen causing hemorrhagic colitis and hemolytic-uremic syndrome. The role of EHEC O157:H7-enterohemolysin (Ehx) in the pathogenesis of infections remains poorly defined. In this study, we used gene deletion and complement methods to confirm its putative functions. Results demonstrated that, in THP-1 cells, EHEC O157:H7-Ehx is associated with greater production of extracellular interleukin (IL)-1β than other cytokines. The data also showed that EHEC O157:H7-Ehx contributed to cytotoxicity in THP-1 cells, causing the release of lactate dehydrogenase (LDH). Although we observed a positive correlation between IL-1β production and cytotoxicity in THP-1 cells infected with different EHEC O157:H7 strains, our immunoblot results showed that the majority of IL-1β in the supernatant was mature IL-1β and not the pro-IL-1β that can be released after cell death. However, EHEC O157:H7-Ehx had no detectable effect on biologically inactive pro-IL-1β at the mRNA or protein synthesis levels. Neither did it affect the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, or NOD-like receptor family pyrin domain containing 3 (NLRP3). RNA interference experiments showed that EHEC O157:H7-induced IL-1β production required the involvement of ASC, caspase-1, and NLRP3 expression in THP-1 cells. Our results demonstrate that Ehx plays a crucial role in EHEC O157:H7-induced IL-1β production and its cytotoxicity to THP-1 cells. NLRP3 inflammasome activation is also involved in EHEC O157:H7-stimulated IL-1β release.