Tir细胞骨架偶联蛋白:大肠杆菌O157：H7的一种新的毒力因子

肠出血性大肠杆菌O157:H7是一种重要的传染性病原菌,可引起多种致死性疾病的爆发流行。Tir细胞骨架偶联蛋白(TccP)是近年来发现的O157:H7的一种新的重要的毒力因子,在O157:H7黏附宿主细胞造成黏附擦拭(A/E)损伤过程中起重要作用,TccP相关研究对阐明O157:H7致病机制具有重要意义。     

Contribution of dps to Acid Stress Tolerance and Oxidative Stress Tolerance in Escherichia coli O157:H7

An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log₁₀-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log₁₀-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log₁₀ CFU/ml while the parent strain decreased by approximately 2 log₁₀ CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.     

Contribution of dps to acid stress tolerance and oxidative stress tolerance in Escherichia coli O157:H7

An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log(10)-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log(10)-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log(10) CFU/ml while the parent strain decreased by approximately 2 log(10) CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.     

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.     

Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant.

Virulence and pathogenicity of an Escherichia coli O157:H7 sorbitol-positive mutant were investigated with an infant rabbit animal model as well as a battery of in vitro assays. Total cell lysate protein profiles, outer membrane protein profiles, plasmid profiles, and levels of cytotoxic activity against Vero cells were similar in the wild-type and mutant strains. Both adhered to intestinal epithelial cells in culture and reacted with fluorescein isothiocyanate-labeled antiserum against E. coli O157:H7. The mutant appeared to be similar to the wild type in all respects except in its ability to ferment sorbitol. [14C]sorbitol uptake and sorbitol-6-phosphate dehydrogenase activities were notably increased in the mutant strain. Diarrhea developed in rabbits administered the wild-type strain and in those fed the sorbitol-positive mutant. There was greater bacterial attachment and mucosal damage in the cecum and large intestine than in the small intestine. Scanning electron microscopy revealed bacteria adhering as single cells and as aggregates closely associated with mucus. Mucosal lesions consisted of areas of tissue necrosis with sloughing of epithelial cells. By transmission electron microscopy, electron-dense necrotic epithelial cells were visible in areas where bacteria were present, and epithelial cell debris containing bacteria was observed between the villar luminal surfaces. Light microscopy of epithelial cells of intestinal sections of infected rabbits revealed noticeable vacuolation and spherical, pyknotic nuclei. These data indicate that the sorbitol-negative phenotype is not associated with the pathogenicity of E. coli O157:H7.     

TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.     

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.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

Reduction of Escherichia coli O157:H7 by stimulated Pediococcus acidilactici

This study was conducted to determine if stimulating the growth of meat starter culture (Pediococcus acidilactici) in a laboratory medium (Brain Heart Infusion broth +2.3% NaCl + 1.5% sucrose; LBHI) and during meat fermentation would control Escherichia coli O157:H7. In LBHI medium without P. acidilactici, the numbers of E. coli O157:H7 increased from 4.00 to 8.34 log10 cfu ml-1, whereas in the presence of P. acidilactici (approximately 6.0 log10 cfu ml-1) in LBHI, LBHIM (LBHI + 0.005% MnSO4), LBHIO (LBHI + 0.3 unit ml-1 Oxyrase), and LBHIMO (LBHI + M + O), the numbers of E. coli O157:H7 increased from 4.00 to 8.05, 7.50, 7.99, and 6.50 log10 cfu ml-1, respectively, after incubation at 40 degrees C for 15 h. During salami fermentation, the numbers of E. coli O157:H7 changed from 7.00 to 6.40 and 5.10 log10 cfu g-1 without and with P. acidilactici (approximately 7.0 log10 cfu g-1), respectively. Stimulated P. acidilactici by M, O, and MO further reduced the number of E. coli O157:H7 from 7.00 to 4.00, 4.80, and 3.65 log10 cfu g-1, respectively. The combination of MO was a better growth stimulator for P. acidilactici, which controlled E. coli O157:H7 in both systems (P < 0.05).     

Cardiovascular disease after Escherichia coli O157:H7 gastroenteritis

Background:

Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, which can be devastating in outbreak situations. We studied the risk of cardiovascular disease following such an outbreak in Walkerton, Ontario, in May 2000.

Methods:

In this community-based cohort study, we linked data from the Walkerton Health Study (2002–2008) to Ontario’s large healthcare databases. We included 4 groups of adults: 3 groups of Walkerton participants (153 with severe gastroenteritis, 414 with mild gastroenteritis, 331 with no gastroenteritis) and a group of 11 263 residents from the surrounding communities that were unaffected by the outbreak. The primary outcome was a composite of death or first major cardiovascular event (admission to hospital for acute myocardial infarction, stroke or congestive heart failure, or evidence of associated procedures). The secondary outcome was first major cardiovascular event censored for death. Adults were followed for an average of 7.4 years.

Results:

During the study period, 1174 adults (9.7%) died or experienced a major cardiovascular event. Compared with residents of the surrounding communities, the risk of death or cardiovascular event was not elevated among Walkerton participants with severe or mild gastroenteritis (hazard ratio [HR] for severe gastroenteritis 0.74, 95% confidence interval [CI] 0.38–1.43, mild gastroenteritis HR 0.64, 95% CI 0.42–0.98). Compared with Walkerton participants who had no gastroenteritis, risk of death or cardiovascular event was not elevated among participants with severe or mild gastroenteritis.

Interpretation:

There was no increase in the risk of cardiovascular disease in the decade following acute infection during a major E. coli O157:H7 outbreak.Escherichia coli O157:H7 is one cause of acute bacterial gastroenteritis, causing 63 000 infections each year and 12 major outbreaks since 2006 in the United States alone.^1,2 This strain was most recently implicated in the outbreak involving beef from XL Foods (September 2012), with 17 confirmed cases across Canada.³ A similar enterohemorrhagic strain E. coli O104:H4 was responsible for an outbreak in Germany in May 2011, causing 3792 cases of gastroenteritis and 43 deaths.^4,5Most patients fully recover from acute gastroenteritis caused by E. coli. However, such an illness may predispose patients to long-term disease. Shiga toxin is produced by E. coli O157:H7; this toxin damages the microvasculature of the kidneys leading to hypertension^6–13 and directly damages the systemic vasculature.^14–16 Infected people may progress from a state of acute inflammation of the vasculature to subclinical chronic inflammation, which could promote atherosclerosis.^17–20In Walkerton, Ontario, in May 2000, heavy rains transported bovine fecal matter into the town’s well, contaminating the inadequately chlorinated municipal water supply with E. coli O157:H7.²¹ Over 2300 people developed acute gastroenteritis, and 7 people died.²² The unique circumstances of this outbreak provided a rare opportunity to study the natural history following exposure to this pathogen in a single cohort.²³ Other outbreaks have been geographically dispersed, making it difficult to track cases.^24,25In Walkerton, affected individuals were followed annually in a clinic to assess their long-term outcomes (Walkerton Health Study, 2002–2008). We previously reported that adults who experienced acute gastroenteritis during the outbreak had a higher than expected incidence of hypertension, chronic kidney disease and self-reported cardiovascular disease in follow-up.²³ However, 46% of participants were lost to follow-up by the end of the study, and there were limitations associated with the assessment of cardiovascular disease by participant recall. Thus, we conducted an expanded and extended follow-up study, linking the Walkerton study data to Ontario’s health care databases. Our objective was to more accurately determine the 10-year risk of major cardiovascular events after exposure to E. coli O157:H7.     

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.     

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.     

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.     

Production of an autoinducer of growth by norepinephrine cultured Escherichia coli O157:H7

Abstract Escherichia coli O157:H7 were cultured in the presence or absence of norepinephrine to generate conditioned media. The presence of a growth-inducing factoKs) in the conditioned media was examined by measurement of the ability of conditioned media to support the growth of fresh cultures of E. coli O157:H7. Supplementation of fresh cultures with as little as 0.024% (v/v) norepinephrine conditioned medium resulted in increased growth as compared to controls, thereby indicating the presence of an autoinducer of growth. Analysis of the production kinetics for the autoinducer during the generation of conditioned media indicates that it differs from other more well characterized autoinducers. It is proposed that the neurohumoral environment of the host may contribute to the production of bacterial growth factors.     

Fate of Escherichia coli O157:H7 in Manure-Amended Soil

Escherichia coli O157:H7 cells survived for up to 77, >226, and 231 days in manure-amended autoclaved soil held at 5, 15, and 21°C, respectively. Pathogen populations declined more rapidly in manure-amended unautoclaved soil under the same conditions, likely due to antagonistic interactions with indigenous soil microorganisms. E. coli O157:H7 cells were inactivated more rapidly in both autoclaved and unautoclaved soils amended with manure at a ratio of 1 part manure to 10 parts soil at 15 and 21°C than in soil samples containing dilute amounts of manure. The manure-to-soil ratio, soil temperature, and indigenous microorganisms of the soil appear to be contributory factors to the pathogen's survival in manure-amended soil.