Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7

The eae (Escherichia coli attaching and effacing) gene from enteropathogenic Escherichia coli (EPEC) was previously shown to be essential for production of the 'attaching and effacing' histopathology characteristic of EPEC infections (Jerse et al., 1990). We have now cloned the eae gene from enterohaemorrhagic E. coli (EHEC) which, in addition to producing Shiga-like cytotoxins, also produces the attaching and effacing effect. The sequence homology between the EPEC and EHEC sequences was 86% and 83% at the nucleotide and amino acid levels, respectively. The predicted amino acid sequence of the EHEC eae gene shared 31% identity and 51% similarity with invasin of Yersinia pseudotuberculosis. Alignment of the EPEC and EHEC Eae proteins and the Y. pseudotuberculosis and Y. enterocolitica invasins shows striking regions of identity with the greatest divergence at the C-terminal end, the putative receptor-binding portion of invasin.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.     

肠出血性大肠埃希菌O157:H7噬菌体FEC14和FEC19特性及污染牛肉杀菌潜在应用探究

【背景】肠出血性大肠埃希菌O157:H7是重要的食源性致病菌之一,并且其耐药性越来越严重,寻找裂解性强噬菌体用于防治大肠埃希菌感染具有广阔的应用前景。【目的】从环境中分离鉴定能特异裂解大肠埃希菌O157:H7的噬菌体,通过对生物学特性及裂解细菌功效的探究,为其在食品安全防控中提供理论依据和研究基础。【方法】通过双层平板法分离并纯化噬菌体,透射电镜观察噬菌体形态,测定最佳感染复数、一步生长曲线、pH稳定性、温度稳定性,对噬菌体进行全基因组测序及噬菌体裂解细菌功效。【结果】2株大肠埃希菌O157:H7噬菌体FEC14和FEC19的头部皆呈二十面体立体对称,FEC14头部直径约80nm,尾丝呈星形,FEC19头部直径约58 nm,尾丝呈针形;噬菌体FEC14的最佳感染复数为0.001,潜伏期为15 min,裂解期为65 min,平均暴发量为156 PFU/cell,FEC19的最佳感染复数为0.1,潜伏期为10 min,裂解期为80 min,平均暴发量为800 PFU/cell;噬菌体FEC14能在60℃、pH 4.0-11.0条件下存活,噬菌体FEC19在70℃、pH5.0-9.0条件下存...     

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.     

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice

An immunoglobulin enriched bovine colostrum preparation, IMMULAC (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin-resistant EHEC O157:H7 strain (O157-SM(R)). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3-week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim-milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin-pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim-milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food-borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.     

Electrophoretic Mobilities of Escherichia coli O157:H7 and Wild-Type Escherichia coli Strains

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.     

Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells

Adherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phase-contrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.     

Role of colanic acid exopolysaccharide in the survival of enterohaemorrhagic Escherichia coli O157:H7 in simulated gastrointestinal fluids

AIM: This study evaluated the production of colanic acid (CA) exopolysaccharide (EPS) by Escherichia coli O157:H7 in relation to the pathogen's ability to survive under acidic conditions simulating the environment in the human gastrointestinal tract. METHODS AND RESULTS: Escherichia coli O157:H7 W6-13 and its CA-deficient mutant M4020 were examined for their resistance to bile salts, and their ability to survive in simulated gastric fluid containing pepsin (pH 2.0) and simulated intestinal fluid containing pancreatin (pH 8.0). The effect of acid adaptation at pH 5.5 on the survival of E. coli O157:H7 in simulated gastric fluid was also determined. The results indicated that the survivability of M4020, under conditions simulating the environment in the human gastrointestinal tract, reduced more drastically than the viability of W6-13. The presence of bile salts had a slight effect on both types of E. coli O157:H7 cells. The loss of CA did not change the ability of M4020 to respond to acid adaptation. CONCLUSION: The EPS CA may serve as a protective barrier to E. coli O157:H7 for its survival in the human gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: The study contributes to a better understanding of the EPS affecting the ability of E. coli O157:H7 to combat acid stress.     

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.     

Inhibitory effect of creosote and its main components on production of verotoxin of enterohaemorrhagic Escherichia coli O157:H7

The inhibitory effects of creosote and its main components, alpha-methoxyphenol and o-ethylphenol, on production of verotoxin by enterohaemorrhagic Escherichia coli O157 (VTEC E. coli) were investigated. The production of verotoxin by VTEC E. coli was inhibited by the administration of 0.001-0.1% of creosote or o-ethylphenol (final concentration). On the other hand, weak inhibition of production of verotoxin was observed with 0.1% alpha-methoxyphenol administration. As the inhibitory effects were obtained below Minimal Inhibitory Concentration (MIC) values, these test compounds exerted their effects at the active site of VTEC E. coli cells prior to their production of verotoxin. These findings suggest that pre-administration of creosote and its main components might prevent human intestinal food poisoning by VTEC E. coli and contribute to maintenance of public health.     

Derivation of Escherichia coli O157:H7 from Its O55:H7 Precursor

There are 29 E. coli genome sequences available, mostly related to studies of species diversity or mode of pathogenicity, including two genomes of the well-known O157:H7 clone. However, there have been no genome studies of closely related clones aimed at exposing the details of evolutionary change. Here we sequenced the genome of an O55:H7 strain, closely related to the major pathogenic O157:H7 clone, with published genome sequences, and undertook comparative genomic and proteomic analysis. We were able to allocate most differences between the genomes to individual mutations, recombination events, or lateral gene transfer events, in specific lineages. Major differences include a type II secretion system present only in the O55:H7 chromosome, fewer type III secretion system effectors in O55:H7, and 19 phage genomes or phagelike elements in O55:H7 compared to 23 in O157:H7, with only three common to both. Many other changes were found in both O55:H7 and O157:H7 lineages, but in general there has been more change in the O157:H7 lineages. For example, we found 50% more synonymous mutational substitutions in O157:H7 compared to O55:H7. The two strains also diverged at the proteomic level. Mutational synonymous SNPs were used to estimate a divergence time of 400 years using a new clock rate, in contrast to 14,000 to 70,000 years using the traditional clock rates. The same approaches were applied to three closely related extraintestinal pathogenic E. coli genomes, and similar levels of mutation and recombination were found. This study revealed for the first time the full range of events involved in the evolution of the O157:H7 clone from its O55:H7 ancestor, and suggested that O157:H7 arose quite recently. Our findings also suggest that E. coli has a much lower frequency of recombination relative to mutation than was observed in a comparable study of a Vibrio cholerae lineage.