非致病性大肠埃希菌能增多大肠埃希菌O157:H7志贺毒素的产生

大肠埃希菌(简称大肠杆菌)O157：H7毒力因子为志贺毒素2(stx2),其基因由温和噬菌体编码,由晚期基因启动子调控表达。stx2的合成与释放需要诱导噬菌体溶菌周期,而且正常肠道大肠杆菌感染了毒素编码的噬菌体就能制造毒素和噬菌体,使毒素水平远远超过病原性菌株本身的产量,作者在体外以及鼠肠道验证了这一假设。     

大肠埃希菌O157：H7毒力的分子生物学研究进展

肠出血性大肠埃希菌是一种致病性大肠埃希菌,与人类的出血性肠类和溶血性尿毒症有关,本文介绍了肠出血性大肠埃希菌O157：H7毒力基因研究的新进展,包括研究较多的噬菌体携带的slt基因,引起上皮细胞粘附/消除损害的eae基因,特异质粒上的hly基因和一些新的毒力基因,即tir,toxB,iha和hokw基因。     

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%。     

从蜣螂肠道中分离大肠埃希菌O157:H7

作为一种新出现的细菌性病原体,大肠埃希菌(简称大肠杆菌)0157：H7曾引起出血性结肠炎(HC)和溶血性尿毒综合征(HUS)的几起大规模暴发和无数散在病例。它通常与动物源性食物联系在一起。流行病学调查发现,大肠杆菌O157：H7株普遍存在于家畜和野生动物的胃肠道中。在携带病原体     

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％.     

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）…     

一种新的大肠埃希菌O157:H7 vero细胞毒素的纯化及特性分析

目的研究出血性大肠埃希菌的vero细胞毒素。方法通过超声破碎,硫酸铵沉淀及两次离子交换层析,再经过凝胶过滤,最终从大肠埃希菌O157：H7 882364菌株获得了一种新的vero细胞毒素。对此毒素进行细胞毒性测定和分子量及等电点的测定,并对毒素的N端15个氨基酸序列进行了检测。结果用凝较过滤层析测定纯化的vero细胞毒素分子量为43KD,等电点为3．8,蛋白N端15个氨基酸序列为SFELPALPYAKDALA,其对vero细胞的CD50为4bg。结论这种毒素与VT1毒素和VT2毒素明显不同,因此初步确定为新毒素,称之为VT3。通过实验将推动我国对出血性大肠埃希菌O157：H7的致病性研究和对VT类毒素和抗毒素的进一步研制和应用。     

肠侵袭性大肠埃希菌噬菌体DK-13的生物学特性及应用

【目的】筛选并分离出能裂解肠侵袭性大肠埃希菌噬菌体,分析其生物学特性,并探究其对污染猪肉的杀菌作用。【方法】采用双层平板法分离鉴定噬菌体,并测定其最佳感染复数,一步生长曲线等,对其基因组进行测序分析以及裂解功效的测定。【结果】从医院污水中分离鉴定能特异裂解肠侵袭性大肠埃希菌（Enteroinvasive Escherichia coli,EIEC）的噬菌体并命名为DK-13,其呈典型的蝌蚪状外形,包含一个正二十面体的头部和一个可收缩的螺旋对称的尾部,属于肌尾噬菌体科（Myoviridae）噬菌体;生物学特性表明：最佳感染复数为0.01,潜伏期约为10 min,裂解期约为70 min,噬菌体DK-13能在50℃,pH 5.0-10.0条件下存活。全基因组测序表明,噬菌体基因组长约172 275 bp,GC含量为40.18%,预测其共有293个开放阅读框（open reading frames,ORF）,未发现已知耐药基因和毒力基因。应用试验表明：被污染的猪肉中表现的杀菌效果良好,宿主菌的数量明显减少。【结论】分离并鉴定一株新的烈性噬菌体DK-13,DK-13具有潜伏期短、裂解效率高等优势...     

酶解法制备姜汁及其抑制大肠埃希菌O157：H7的研究

目的探究酶解对生姜出汁率及姜汁悬浮稳定性的影响,并测定经过酶解处理及未经酶解处理的生姜姜汁对大肠埃希菌O157：H7的抑菌活性。方法新鲜的生姜清水洗净后切块,放人组织捣碎机中打浆,所得浆液利用果胶酶和淀粉酶进行酶解。采用共培养法测定了制得的姜汁对大肠埃希菌O157：H7的抑菌活性。结果最适酶解条件为,加入3nag淀粉酶60℃酶解50min,灭酶后将pH调至4,再加入3mg果胶酶于45℃酶解50min。结果显示经酶解处理的姜汁对大肠埃希菌O157：H7的抑菌率远大于未经酶解处理的姜汁,酶解处理的姜汁其抑菌率最高达到86％。结论该方法能够有效提高生姜的出汁率及姜汁对大肠埃希菌O157：H7的抑菌效果。     

一种新的大肠埃希菌O157∶H7 vero细胞毒素的纯化及特性分析

目的研究出血性大肠埃希菌的vero细胞毒素。方法通过超声破碎,硫酸铵沉淀及两次离子交换层析,再经过凝胶过滤,最终从大肠埃希菌O157∶H7 882364菌株获得了一种新的vero细胞毒素。对此毒素进行细胞毒性测定和分子量及等电点的测定,并对毒素的N端15个氨基酸序列进行了检测。结果用凝较过滤层析测定纯化的vero细胞毒素分子量为43KD,等电点为3.8,蛋白N端15个氨基酸序列为SFELPALPYAKDALA,其对vero细胞的CD50为4bg。结论这种毒素与VT1毒素和VT2毒素明显不同,因此初步确定为新毒素,称之为VT3。通过实验将推动我国对出血性大肠埃希菌O157∶H7的致病性研究和对VT类毒素和抗毒素的进一步研制和应用。     

Dose-Response Envelope for Escherichia coli O157:H7

Escherichia coli O157:H7 is an emerging food and waterborne pathogen in the U.S. and internationally. The objective of this work was to develop a dose-response model for illness by this organism that bounds the uncertainty in the dose-response relationship. No human clinical trial data are available for E. coli O157:H7, but such data are available for two surrogate pathogens: enteropathogenic E. coli (EPEC) and Shigella dysenteriae. E. coli O157:H7 outbreak data provide an initial estimate of the most likely value of the dose-response relationship within the bounds of an envelope defined by beta-Poisson dose-response models fit to the EPEC and S. dysenteriae data. The most likely value of the median effective dose for E. coli O157:H7 is estimated to be approximately 190[emsp4 ]000 colony forming units (cfu). At a dose level of 100[emsp4 ]cfu, the median response predicted by the model is six percent.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Sequence analysis of Escherichia coli O157:H7 bacteriophage ΦV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen

Bacteriophage ΦV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the ΦV10 genome is 39 104 bp long and contains 55 predicted genes. ΦV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage ɛ15 and a prophage from E. coli APEC O1. The attachment site of ΦV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. ΦV10 encodes an O -acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block ΦV10 superinfection, indicating that the O157 antigen is most likely the ΦV10 receptor.     

Competition for proline between indigenous Escherichia coli and E. coli O157:H7 in gnotobiotic mice associated with infant intestinal microbiota and its contribution to the colonization resistance against E. coli O157:H7

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 mice became carriers. It has been reported that the lag time of E. coli O157:H7 growth in 50% GB-3 caecal suspension was extended when compared to GB-4 caecal suspension. In this study, competition for nutrients between intestinal microbiota of GB-3 and GB-4 mice and E. coli O157:H7 was examined. Amino acid concentrations in the caecal contents of GB-3 and GB-4 differed, especially the concentration of proline. The supplementation of proline into GB-3 caecal suspension decreased the lag time of E. coli O157:H7 growth in vitro. When E. coli O157:H7 was cultured with each of the strains used to produce GB-3 mice in vitro, 2 strains of E. coli (proline consumers) out of 5 enterobacteriaceae strains strongly suppressed E. coli O157:H7 growth and the suppression was attenuated by the addition of proline into the medium. These results indicate that competition for proline with indigenous E. coli affected the growth of E. coli O157:H7 in vivo and may contribute to E. coli O157:H7 elimination from the intestine.     

Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.     

Methods available for the detection of Escherichia coli O157 in clinical,food and environmental samples

Because of the potential severity of infections caused by Escherichia coli O157 it is important that the most sensitive laboratory methods are used both for outbreak investigation and surveillance. Selective culture of E. coli O157 remains the detection method of choice, particularly in investigation of outbreaks where strains isolated from various sources may need to be compared by various typing methods. Strains of E. coli O157 do not normally ferment sorbitol, whereas many other serogroups of E. coli do, and sorbitol MacConkey agar, or modified forms of this medium, have become widely used for their isolation. Detection of small numbers of E. coli O157 may be facilitated by enrichment culture which may include a recovery period during which selective agents are not added to the medium. Immunomagnetic separation of E. coli O157 after enrichment culture enhances sensitivity still further and has the potential to be fully automated. Alternatives to culture include immunoassays and PCR, both of which are available as commercial detection kits. The last 15 years has seen many advances in detection of E. coli O157 and has been accompanied by a plethora of reports in the scientific literature. However, it is an area which is continually developing and we are still far away from a universally accepted method for this purpose. This revised version was published online in November 2006 with corrections to the Cover Date.     

Impact of indigenous microorganisms on Escherichia coli O157:H7 growth in cured compost

Both autoclaving and dry-heat treatments were applied to dairy manure-based compost to achieve target populations of indigenous microorganisms. A 3 strain-mixture of Escherichia coli O157:H7 of ca. 2 log CFU/g was inoculated into acclimated autoclaved compost (AAC) and dry heat-treated compost (DHTC) with different moistures, and stored at 8, 22, or 30 °C. Only selected groups of microorganisms grew in AAC during acclimation, whereas the relative ratio of each group of microorganisms was maintained in DHTC after heat treatment. E. coli O157:H7 grew more in AAC than DHTC in the presence of same level of indigenous mesophiles. However, control compost (no heat treatment) did not support E. coli O157:H7 growth. Our results revealed that both the type and population of indigenous microorganisms is critical for suppressing E. coli O157:H7 growth in compost, and dry-heat treatment can result in a compost product which resembles cured compost with different levels of indigenous microorganisms.     

Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

Effect of organic acids on inhibition of Escherichia coli O157:H7 colonization in gnotobiotic mice associated with infant intestinal microbiota

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 became carriers. In this study, we analysed the mechanisms of E. coli O157:H7 elimination using GB-3 and GB-4. When GB-3 and GB-4 mice were challenged with E. coli O157:H7, the E. coli O157:H7 population was reduced in the caecum of GB-3 when compared to that in the GB-4 caecum, although the numbers of E. coli O157:H7 in the small intestine were not significantly different between these two groups of gnotobiotic mice. The lag time of E. coli O157:H7 growth in a 50% GB-3 caecal suspension increased when compared to that in a GB-4 caecal suspension. Acetate and lactate were detected in the GB-3 caecal contents, and acetate and propionate in those from GB-4. Although E. coli O157:H7 growth was not suppressed when it was cultured in anaerobic broth supplemented with these organic acids, the motility of E. coli O157:H7 was suppressed when it was cultured on semi-solid agar supplemented with the combination of acetate and lactate. These results indicate that the organic acid profile in the caecum is an important factor related to the elimination of E. coli O157:H7 from the intestine.     

The history and evolution of Escherichia coli O157 and other Shiga toxin-producing E. coli

Shiga toxin-producing Escherichia coli (STEC) O157 is a formidable human pathogen with the capacity to cause large outbreaks of gastrointestinal illness. The known virulence factors of this organism are encoded on phage, plasmid and chromosomal genes. There are also likely to be novel, as yet unknown virulence factors in this organism. Many of these virulence factors have been acquired by E. coli O157 by transfer from other organisms, both E. coli and non-E. coli species. By examination of biochemical and genetic characteristics of various E. coli O157 strains and the relationships with other organisms, an evolutionary pathway for development of E. coli O157 as a pathogen has been proposed. E. coli O157 evolved from an enteropathogenic E. coli ancestor of serotype O55:H7, which contained the locus of enterocyte effacement containing the adhesin intimin. During the evolutionary process, Shiga toxins, the pO157 plasmid and other characteristics which enhanced virulence were acquired and other functions such as motility, sorbitol fermentation and β-glucuronidase activity were lost by some strains. It is likely that E. coli O157 is constantly evolving, and changes can be detected in genetic patterns during the course of infection. A variety of mechanisms may be responsible for the development of the virulent phenotype that we see today. Such changes include uptake of as yet uncharacterised virulence factors, possibly enhanced by a mutator phenotype, recombination within virulence genes to produce variant genes with different properties, loss of large segments of DNA (black holes) to enhance virulence and possible adaptation to different hosts. Although little is known about the evolution of non-O157 STEC it is likely that the most virulent clones evolved in a similar manner to E. coli O157. This revised version was published online in November 2006 with corrections to the Cover Date.