Interaction of enterohemorrhagic Escherichia coli O157:H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.     

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.     

Molecular subtyping for Escherichia coli O157: H7 isolated in Taiwan

Enterohaemorrhagic Escherichia coli O157: H7 is an important pathogen these days. Outbreaks of its infection have been reported all over the world, in Australia, Canada, Japan, the United States, south Africa, and various countries in Europe. In the summer of 2001, the first clinical infection by E. coli O157: H7 was identified in Taiwan. In this study, the standard procedures for molecular subtyping were applied to several strains collected in Taiwan as well as from elsewhere. The two molecular subtyping methods we used are pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The isolates from the U.S.A., Canada, Japan, and Taiwan each showed a unique molecular fingerprinting pattern. The environmental strains isolated in Taiwan showed closer relationships with each other, and their similarity was in the range of 75-85%. The first clinical strain isolated in Taiwan in 2001 was similar to the strains from North America but not closely related to the Taiwanese environmental strains. Our surveys showed that some local E. coli O157: H7 strains did exist in Taiwan, but there had been only one official case report of the infection by local E. coli O157: H7. The eating habits of the people and the geographic distribution of the pathogen are considered crucial risk factors in Taiwan. The establishment of a database of our own and joining the global network database are important tasks if we want to control such agricultural and food-borne pathogens, and reduce the number of victims and amount sufferings, as well as the economic losses due to the infection.     

Highly diverse variable number tandem repeat loci in the E. coli O157:H7 and O55:H7 genomes for high-resolution molecular typing

AIM: Evaluation of the Escherichia coli genome for variable number tandem repeat (VNTR) loci in order to provide a subtyping tool with greater discrimination and more efficient capacity. METHODS AND RESULTS: Twenty-nine putative VNTR loci were identified from the E. coli genomic sequence. Their variability was validated by characterizing the number of repeats at each locus in a set of 56 E. coli O157:H7/HN and O55:H7 isolates. An optimized multiplex assay system was developed to facility high capacity analysis. Locus diversity values ranged from 0.23 to 0.95 while the number of alleles ranged from two to 29. This multiple-locus VNTR analysis (MLVA) data was used to describe genetic relationships among these isolates and was compared with PFGE (pulse field gel electrophoresis) data from a subset of the same strains. Genetic similarity values were highly correlated between the two approaches, through MLVA was capable of discrimination amongst closely related isolates when PFGE similar values were equal to 1.0. CONCLUSIONS: Highly variable VNTR loci exist in the E. coli O157:H7 genome and are excellent estimators of genetic relationships, in particular for closely related isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 MLVA offers a complimentary analysis to the more traditional PFGE approach. Application of MLVA to an outbreak cluster could generate superior molecular epidemiology and result in a more effective public health response.     

Clonal turnover of enterohemorrhagic Escherichia coli O157:H7 in experimentally infected cattle

A total of 401 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates from two experimentally infected calves were analyzed using molecular biological methods. Genetic differences detected by pulsed-field gel electrophoresis were observed between the inoculated and recovered strains as early as 1 day post inoculation. The loss of the inoculated clone was observed in one calf. Replication and dissemination of the EHEC O157:H7 strains that mutated in cattle may result in the diversification of this organism among cattle populations.     

Design and activity of novel lactoferrampin analogues against O157:H7 enterohemorrhagic escherichia coli

Lactoferrampin 265–284 (LFampin 265–284) is a peptide consisting of residues 265–284 of N1‐domain of bovine Lactoferrin (LF). This peptide has several cationic groups in the C‐terminal lobe, exhibiting an antibacterial activity against a wide range of microorganisms. However, LFampin 265–284 exhibits low antimicrobial activity against the O157:H7 enterohaemorrhagic Escherichia coli (EHEC O157:H7) when compared with Lactoferrin chimera and Lactoferricin. Here, we have designed three analogues of LFampin 265–284 based on the distribution of cationic groups, hydrophobicity, size, and sequence. Analogues were synthesized by solid phase chemistry using Fmoc methodology obtaining peptides with 95% purity. All peptides maintain the ability to adopt helical conformations (checked by circular dichroism spectra and molecular simulations). Some of these analogues exhibited a significant increase in antimicrobial activity by counting colony forming units against EHEC O157:H7 compared to native LFampin 265–284, with MIC of 10 and 40 µM for 264G‐D265K and 264G‐D265K/S272R, respectively. The incorporation of a GKLI sequence in the N‐terminal lobe increased dramatically its antibacterial activity, an effect which has been attributed to the addition of cationic groups in the N‐terminal side that may stabilize the helical conformation of the new designed peptides. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 319–328, 2014.     

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。     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

B-cell epitope KT-12 of enterohemorrhagic Escherichia coli O157:H7: a novel peptide vaccine candidate

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is associated with hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic-uremic syndrome in humans. B-cell epitopes of intimin γ from EHEC O157:H7 were predicted and synthesized for evaluating their immunogenicity and protective effect and for screening a novel synthetic peptide vaccine. In the present study, five B-cell epitopes of IntC300 were predicted by Hopp-Woods, Chou-Fasman, Karplus-Schulz, Emini, Jameson-Wolf and Kolaskar-Tongaonakar analysis. One of them, KT-12 (KASITEIKADKT) was coupled with keyhole limpet hemocyanin, and used to immunize BALB/c mice three times by subcutaneous and intranasal injection. Mouse serum titers of IgG and IgA were assessed by indirect ELISA. Oral inoculation of EHEC O157:H7 resulted in infection and death of the mice. It was found that B-cell epitopes are located within or near the peptide segments 658-669, 711-723, 824-833, 897-914, 919-931. Both subcutaneous and intranasal immunization induced higher concentrations of IgG antibodies, as detected by indirect ELISA, and nasal-mucosal immunization induced the production of high concentrations of IgA antibodies. After infection with a lethal dose of EHEC O157:H7, the survival rate of mice that had received subcutaneous immunization was not significantly different from that of the control group (P > 0.05). On the other hand, mice that received intranasal immunization showed a better survival rate than the group that received subcutaneous immunization (P < 0.05). The synthesized antigenic peptide KT-12 induced mice to produce higher concentrations of IgG and IgA after immunization, but only intranasal immunization of KT-12 succeeded in protecting most mice from infection with EHEC O157:H7. This study suggests that the synthesized antigenic peptide KT-12 is be a potential vaccine candidate against EHEC O157:H7.     

Isolation of Escherichia coli O157:H7 from dung beetles Catharsius molossus

In an epidemiological survey, Escherichia coli O157:H7 was isolated from the intestine 4 of 113 dung beetle Catharsius molossus captured below ground at Tongshan County, Jiangsu Province of China. In parallel, 10 strains of E. coli O157:H7 were isolated from fecal samples of 383 diarrhea patients from the same region. Most importantly, using pulsed field gel electrophoresis (PFGE) of chromosomal DNA restriction fragments and PCR method, we found that the PFGE pattern and virulence genes of beetle isolates were identical to those of the human isolates, such as Shiga-toxins (stx) and enterohemorrhagic Escherichia coli hemolysin A (EHEC-hlyA). Therefore, dung beetle might acquire pathogenic E. coli O157:H7 through contact with feces of domestic animals.     

Combined Use of Bacteriophage Typing and Pulsed-Field Gel Electrophoresis in the Epidemiological Analysis of Japanese Isolates of Enterohemorrhagic Escherichia coli O157:H7

A total of 236 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates in Japan were investigated by bacteriophage typing, and the results were compared with those of pulsed-field gel electrophoresis (PFGE). Seven phage types (PTs) were observed in 71 isolates which were derived from 22 outbreaks. All of the isolates from ten outbreaks in the Kinki region (midwestern part of Japan) in July-August 1996 were grouped into the same PFGE type (IIa) and PT 32, while among total isolates, there were such varieties as PFGE type IIa containing five phage types and PT32 containing two PFGE types. These results suggest that the ten outbreaks should be considered to be a single outbreak, and show that the combined use of bacteriophage typing and PFGE enhances reliability in epidemiological surveys.     

Proteomic characterization of enterohemorrhagic escherichia coli O157:H7 in the oxidation-induced viable but non-culturable state

Enterohemorrhagic Escherichia coli (EHEC) O157 strain F2, a food isolate of an outbreak, is resistant to oxidative stress, but has increased stress-sensitivity after passage through mice. The stress-sensitive variant of F2 (designated MP37) has decreased culturability, but retains membrane integrity under stress conditions, indicating that the cells enter a viable but non-culturable (VBNC) state. Proteomic analyses revealed that MP37 in the VBNC state had decreased levels of some oxidation-responsive factors (AhpCF, AceF), but it markedly increased levels of outer membrane protein W (OmpW). Because F2 expressed higher levels of some ribosome-associated proteins (RaiA, S6, Bcp) than MP37, the effect of animal passage on the induction of the VBNC state in the EHEC O157 cells might be due to ribosomal activity.     

Rumen contents as a reservoir of enterohemorrhagic Escherichia coli

Abstract We investigatedthe role of the rumen fermentation as a barries to the foodborne pathogen, Escherichia coli O157:H7. Strains of E. coli , including several isolates of O157:H7, grew poorly in media which simulated the ruminal environment of a well-fed animal. Strains of E. coli O157:H7 did not display a superior tolerance to ruminal conditions which may facilitate their colonization of the bovine digestive tract. Unrestricted growth of E. coli was observed in rumen fluid collected from fasted cattle. Growth was inhibited by rumen fluid collected from well-fed animals. Well-fed animals appear less likely to become reservoirs for pathogenic E. coli . These results have implications for cattle slaughter practices and epidemiological studies of E. coli O157:H7.     

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.     

Escherichia coli O157:H7 colonization in small domestic ruminants

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

Release of membrane vesicles containing endotoxic lipopolysaccharide in Escherichia coli O157:H7 clinical isolates

Membrane vesicles released by E. coli O157:H7 strains were investigated by immuno-electron microscopy using anti-O157 antibody. Anti-O157 antibody enhanced the negative-staining of vesicles and we found numerous small vesicles clearly formed around bacterial cells. An immunogold-electron microscopic examination confirmed that lipopolysaccharide (LPS) including the O-side chain is present on the surface of the vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified vesicles showed that the vesicles contained LPS consisting of a lipid-A and an O polysaccharide. In addition, the endotoxic activity of the vesicle was confirmed by a limulus test. These results suggest that the vesicles may play an important role in the pathogenesis of Escherichia coli O157:H7.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.