Influence of Plasmid pO157 on Escherichia coli O157:H7 Sakai Biofilm Formation

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.Enterohemorrhagic Escherichia coli serotype O157:H7 is a major food-borne pathogen causing hemorrhagic colitis and the hemolytic-uremic syndrome (17). Many E. coli O157:H7 outbreaks have been associated with contaminated undercooked ground beef, vegetables, fruits, and sprouts (20, 31). One of the largest disease outbreaks occurred in Sakai City, Japan, in 1996 with nearly 8,000 confirmed cases. The E. coli isolate responsible for this outbreak, referred to as “Sakai,” is one of the best-characterized isolates and one of only three O157 strains for which the genome has been fully sequenced (8, 16). Because of its importance as a human pathogen and its characterization, Sakai was the focus of this investigation.There is significant phenotypic diversity among E. coli O157:H7 strains, including the ability to form biofilm. Previous studies show that certain E. coli O157:H7 strains form biofilm on various surfaces, and biofilm on food or food-processing surfaces can serve as a source or vehicle of contamination that may result in human infection (6, 18, 25). Biofilm is an organized and structured community of microorganisms that attaches to solid surfaces and contains cells embedded in an extracellular polymer matrix (4, 26). Exopolysaccharide (EPS) is a major component of the biofilm matrix and is required for the development of characteristic biofilm architecture (5, 29). Bacteria gain a variety of advantages from biofilm formation that include attachment, colonization, and protection from adverse environments (4, 11).E. coli O157:H7 carries a 92-kb virulence plasmid (pO157) encoding a number of putative virulence determinants, including ehxA, etpC to etpO, espP, katP, toxB, ecf, and stcE (31). However, the biological role of pO157 is not fully understood, and only 19 genes among the 100 open reading frames (ORFs) in pO157 have been characterized (2, 15). Our previous work indicates that pO157 is a colonization factor in cattle and may regulate several chromosomal genes (14, 24, 31).To investigate the role of pO157 in biofilm formation, we characterized the biofilm of wild-type E. coli O157:H7 strain Sakai and an isogenic pO157-cured Sakai (Sakai-Cu). Both strains were kindly provided by C. Sasakawa (University of Tokyo). Sakai-Cu was generated using a plasmid incompatibility method (27). This method is not prone to secondary mutations and requires minimal passage in laboratory medium. The mini-R plasmid pK2368, harboring a chloramphenicol (CM) resistance gene and being in the same plasmid incompatibility group as pO157, was introduced into wild-type Sakai by transformation. Transformants were isolated on LB agar containing CM and selected for loss of pO157 by agarose gel electrophoresis analysis. CM-resistant transformants were cured of pKP2368 by subculturing in LB broth without CM. The absence of pO157 was confirmed by Southern blot hybridization with a pO157-specific gene probe (derived from ecf1), and chromosomal DNA integrity was confirmed by pulsed-field gel electrophoresis (data not shown).Because E. coli O157:H7 strains are generally not strong biofilm producers, the condition most conducive to biofilm production, a fluorometric flow cell method, was used to compare separately grown Sakai and Sakai-CU (3). The biofilm cultivation systems consisted of seven parts: (i) medium reservoir, (ii) multichannel pump (205S; Watson Marlow, United Kingdom), (iii) bubble trap (BioSurface Technologies Co., Bozeman, MT), (iv) flow cell, (v) outflow reservoir, (vi) air pump (DrsFosterSmith, Rhinelander, WI), and (vii) flow meter (Gilmont, BC Group, St. Louis, MO). The flow cell was constructed from two rectangular acrylic plates that were 104 by 48 mm. Sidewalls (62 by 26 by 5 mm) were glued to the top plate to form an elongated hexagonal growth chamber. There were 56- by 20-mm square openings in the top and bottom rectangular plates that were sealed with 60- by 24-mm glass slides (Fisher, Pittsburgh, PA). The upper and lower plates were assembled with screws and sealed using a microseal B film (MJ Research, Waltham, MA). The flow cell volume was about 10.4 ml, the medium flow rate was 10.5 ml/h, and the hydraulic retention time was 1 h. Under these conditions, the linear surface velocity was about 80 mm/h at the center of the flow cell. The biofilm was grown with BGM2 medium (21). To prepare the inoculums, Sakai and Sakai-Cu were grown at 37°C in BGM2 medium to mid-exponential phase, and cells were harvested by centrifugation and resuspended in 0.85% NaCl. One hundred μl of the resuspended cell solution was inoculated from the effluent side of flow cells through a long stainless steel needle (Fisher, Pittsburgh, PA). The cells were incubated for at least 3 h without supplying fresh medium, and then fresh medium was supplied to the biofilm cultivation system at 30°C.At various times, the resulting biofilms were stained with a green fluorescent dye, wheat germ agglutinin (WGA)- Alexa Fluor 488 (Invitrogen, Carlsbad, CA), and analyzed using the Olympus FluoView confocal laser scanning microscopy system (Olympus, Tokyo, Japan). Using the Olympus FluoView software program, version 1.7b, for analysis, the fluorescence intensities of Sakai and Sakai-Cu biofilm matrices were each analyzed from >20 three-dimensional-complexity images. Fluorescence was greater for Sakai than for Sakai-Cu, with average values of 2,448 ± 668 and 2,022 ± 619, respectively (Student''s t test; P < 0.05). Overhead images from the Sakai-Cu strain biofilm revealed more-compact cell clusters than images from wild-type Sakai (Fig. 1A and B). Comparisons of images taken sideways indicated that the Sakai-Cu biofilms were not as thick as those of wild-type Sakai (Fig. 1C and D), and typical ratios were consistently 9:11, respectively (P < 0.05). A previous study demonstrated that the biofilm of a wcaF::can mutant of E. coli K-12, which is deficient in EPS production, lacked depth and complex architecture (5). Sakai-Cu showed a similar but less dramatic phenomenon. These observations indicated that pO157 influenced biofilm formation and architecture.Open in a separate windowFIG. 1.Wild-type Sakai (A and C) or Sakai-Cu (B and D) biofilms after 3 days of incubation. Both strains were grown at 30°C in an individual flow cell apparatus. The biofilm was stained with WGA-Alexa Fluor 488 and examined by confocal microscopy. Representative overhead (A and B) or sagittal (C and D) images are shown and were generated using the deconvolution software. Bar, 50 μm.To quantitatively compare Sakai and Sakai-Cu biofilms, the contents of each flow cell apparatus were collected at various times and analyzed for bacterial cell number, viscosity, and EPS production. Biofilms were harvested by a standard technique that preserves cell numbers and minimizes viscosity changes (9). Briefly, floating cells in the biofilm were carefully collected with a pipet, and the remaining cells were scraped from the flow cell apparatus with sterilized applicator sticks. Biofilm samples were collected on days 1, 3, 5, 8, and 12, and measurements were means ± standard deviations (SD) of at least triplicate measurements from separately grown biofilms. There was no significant difference in bacterial number (CFU/ml) from Sakai and Sakai-Cu biofilms at any of the times measured (data not shown). A Cannon-Fenske routine viscometer (Size 100; Cannon Instrument Co., Pennsylvania) was used to determine biofilm viscosity. The conversion constant was 0.015 cSt/s (mm²/s²), and viscosities were measured according to the manufacturer''s instructions. Briefly, the viscometer was aligned vertically in the holder, and the sample was charged into the viscometer tube until the sample reached the “F” mark in the tube. A suction bulb was used to draw the sample slightly above mark “E.” The sample was allowed to flow freely, and the efflux time was measured as the time for the meniscus to pass from mark “E” to mark “F.” Measurements were repeated at least six times, and the kinematic viscosity in mm²/s (cSt) of the samples was calculated by multiplying the efflux time in seconds by the viscometer constant. The viscosity of Sakai biofilm was dramatically increased after 8 days (P < 0.001), while there was no significant change in the viscosities of Sakai-Cu biofilms through day 12 (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Comparison of Sakai and Sakai-Cu biofilm viscosity. Three or four separately grown biofilms were each harvested on the days indicated, and viscosity was measured using a Cannon-Fenske Routine viscometer.Bacterial EPS are associated with attachment to both inanimate surfaces and host cells (29). EPS can be categorized as extracellular carbohydrate complexes (ECC) that are loosely associated with cells and easily removed, referred to as slime (fraction I), or ECC that are closely associated with cells and removed only after heat treatment, referred to as capsule (fraction II) (22). No significant difference in ECC was observed until days eight and 12, when the level of total ECC produced from Sakai biofilms was significantly higher than that from the Sakai-Cu biofilms (P < 0.05) (Fig. ​(Fig.3).3). Also, by days eight and 12, levels of Sakai ECC fraction I, representing primarily secreted slime carbohydrates, were 5 and 10 times higher than Sakai-Cu ECC fraction I, respectively. These results correlated with the results of increased viscosity in Sakai biofilm samples that had aged for 8 or 12 days.Open in a separate windowFIG. 3.Comparison of Sakai and Sakai-Cu biofilm extracellular carbohydrate (ECC) production. ECC I was collected from cells by centrifugation, and ECC II was collected by centrifugation after heat treatment on each indicated day. Bar height represents total ECC production from each biofilm sample. The proportion of total ECC that was either ECC I (dark gray) or ECC II (light gray) is shown. Asterisks indicate significant differences between wild-type Sakai (Wt) and Sakai-Cu (Cu); day 8, P < 0.05; day 12, P < 0.001.Interestingly, during biofilm sampling, two colony morphology variants were isolated that are referred to here as sticky and mucoid. These variants were found only in wild-type Sakai biofilms that had aged for ≥8 days and were not found in Sakai-Cu biofilms even after screening of 10⁴ colonies and even among biofilms aged for 18 days. The percentages of sticky and mucoid variants in Sakai biofilms ranged from 5 to 30% and 0 to 5%, respectively. The differences in colony morphology were readily distinguished, as shown in Fig. ​Fig.4.4. The sticky variant was raised in elevation and shinier than the Sakai parent strain but was not difference in size. When single bacterial colonies grown on agar plates were touched with a sterilized toothpick and that toothpick was gently lifted up, the colonies had a hyperadherence phenotype and elongated to approximately 1 cm between the plate and the toothpick. This phenomenon was unique to the sticky colony variants and was not observed among colonies of the parent Sakai strain (Fig. ​(Fig.4D).4D). The mucoid colony variants were convex in elevation and shiny in texture, had irregular colony shapes, and were larger than the Sakai parent strain but were not hyperadherent. The motility of variants was determined using 0.3% soft agar, and both sticky and mucoid variants exhibited 30- to 90%-reduced motility compared to the parent Sakai strain (data not shown). The characteristics of both sticky and mucoid variants were inherited, and the variant characteristics were maintained in laboratory subculture through 15 generations.Open in a separate windowFIG. 4.Colony morphologies of wild-type, mucoid, and sticky variants. The wild-type E. coli O157:H7 Sakai strain formed small, flat, and nonsticky colonies on LB agar (A). The mucoid variant formed irregular, large, shiny, mucoid, convex, and nonsticky colonies (B). The sticky variant formed small, slightly raised, and sticky colonies (C). The sticky variant adheres to a toothpick touched to the colony surface (D). Bar, 1 cm.It is known that mutation is a powerful mechanism of adaptation when bacteria are faced with environmental change (1). Like other bacterial variants, the sticky and mucoid phenotypic biofilm variants may provide a survival advantage in specific niches (10, 19). Pseudomonas aeruginosa is a well-known biofilm model, and colony morphology variants are a common biofilm-related phenomenon. Both reduced-motility and hyperadherence variants have been described (10) and have characteristics similar to those of the E. coli O157:H7 biofilm variants described here. However, unlike the P. aeruginosa biofilm variants, the sticky and mucoid Sakai variants were not smaller, rougher, or more wrinkled than the parent colony.Although it is possible that the changes measured in biofilm formation and the generation of hyperadherent variants were not due to the plasmid, it is highly unlikely. The method of plasmid curing by incompatibility is gentle and is not prone to secondary mutation. A powerful and common approach to address possible secondary mutations is complementation; however, it was not used here because reintroduction of the plasmid requires the manipulation of a very large piece of DNA (92 kb) and the procedure itself is likely to introduce mutation. Also, reintroduction of the large 92-kb pO157 plasmid would require antibiotic resistance for efficient selection, and this may influence biofilm formation.Many regulatory mechanisms are involved in biofilm formation (7, 12, 13, 28, 30, 32). Among those mechanisms, the relationship between biofilm formation and acid resistance is well known. Biofilm formation is upregulated after the deletion of the gad or hde gene, which allows bacteria to survive under acidic conditions (12). Previously we showed that an isogenic pO157-cured strain of E. coli O157:H7, ATCC 43894, enhanced acid resistance through increased expression of Gad (14). Similarly, Sakai-Cu has enhanced acid resistance compared to wild-type Sakai (data not shown and J. Y. Lim, B. Hong, H. Sheng, S. Shringi, R. Kaul, and C. J. Hovde, submitted for publication). The link between increased acid resistance and reduced biofilm formation, reduced ESP production, reduced viscosity, and lack of colony morphology variants was not explored here. Comparisons of biofilm formation were not made between these two strains because neither wild-type E. coli O157:H7 ATCC 43894 nor its plasmid-cured strain form significant biofilm under the laboratory conditions tested (data not shown).Two pO157-cured E. coli O157 strains (ATCC 43894 and Sakai) do not colonize cattle as well as their wild-type counterpart (14, 24). The mechanism for this difference may be related to pO157 encoding a set of putative type II secretion genes, etpC to etpM, etpO, and etpS, and these etp genes may be associated with protein secretion required for efficient adherence (23). Tatsuno et al. reported that the toxB gene encoded on pO157 is required for the full epithelial cell adherence phenotype (27). These results may relate to the defect of Sakai-Cu in biofilm formation.In conclusion, this is the first report that pO157 affects biofilm formation of E. coli O157:H7 Sakai through increased EPS production and generation of hyperadherent variants. Further study of biofilm formation under a variety of conditions and comparisons of Sakai with other E. coli O157:H7 strains will be important for understanding the relationship between biofilm formation and E. coli O157:H7 virulence and survival on foods and in the farm environment.     

Characterization of the StcE protease activity of Escherichia coli O157:H7

The StcE zinc metalloprotease is secreted by enterohemorrhagic Escherichia coli (EHEC) O157:H7 and contributes to intimate adherence of this bacterium to host cells, a process essential for mammalian colonization. StcE has also been shown to localize the inflammatory regulator C1 esterase inhibitor (C1-INH) to cell membranes. We tried to more fully characterize StcE activity to better understand its role in EHEC pathogenesis. StcE was active at pH 6.1 to 9.0, in the presence of NaCl concentrations ranging from 0 to 600 mM, and at 4 degrees C to 55 degrees C. Interestingly, antisera against StcE or C1-INH did not eliminate StcE cleavage of C1-INH. Treatment of StcE with the proteases trypsin, chymotrypsin, human neutrophil elastase, and Pseudomonas aeruginosa elastase did not eliminate StcE activity against C1-INH. After StcE was kept at 23 degrees C for 65 days, it exhibited full proteolytic activity, and it retained 30% of its original activity after incubation for 8 days at 37 degrees C. Together, these results show the StcE protease is a stable enzyme that is probably active in the environment of the colon. Additionally, k(cat)/K(m) data showed that StcE proteolytic activity was 2.5-fold more efficient with the secreted mucin MUC7 than with the complement regulator C1-INH. This evidence supports a model which includes two roles for StcE during infection, in which StcE acts first as a mucinase and then as an anti-inflammatory agent by localizing C1-INH to cell membranes.     

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.     

A genome rearrangement has orphaned the Escherichia coli K-12 AcpT phosphopantetheinyl transferase from its cognate Escherichia coli O157:H7 substrates

Phosphopantetheinyl transferases (PPTases) are enzymes that catalyse the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of a carrier protein. These carrier proteins use the 4'-phosphopantetheine thiol to shuttle intermediates between the active sites of biosynthetic enzymes involved in fatty acid, non-ribosomal peptide and polyketide synthesis. Three PPTases have been previously been identified in Escherichia coli K-12 and other E. coli strains by homology searches and are encoded by the genes acpS, entD and acpT. Both AcpS and EntD have been well studied whereas the function of AcpT has been an enigma because no carrier protein substrate could be found. We report genetic and biochemical evidence that AcpT modifies two carrier proteins encoded in O-island 138, a cluster of fatty acid biosynthesis-like genes located adjacent to acpT in the genome of the pathogenic E. coli strain O157:H7 (E. coli K-12 and several other sequenced E. coli and Shigella strains lack O-island 138). The two carrier proteins of O-island 138 of strain O157:H7 are not modified (or only very poorly modified) by AcpS, the PPTase responsible for 4'-phosphopantetheine attachment to the acyl carrier protein (AcpP) of fatty acid synthesis. We demonstrate that AcpT cannot functionally replace AcpS in E. coli K-12 either in its native chromosomal location or upon insertion of acpT into the acpS chromosomal location. However, in the absence of AcpS activity AcpT does allow very slow growth thus providing a rationale for its retention in the absence of its cognate substrates. These results together with phylogenetic analyses and comparisons of the E. coli and Shigella strains of known genome sequence strongly argue that AcpT has been orphaned from its cognate substrates by a deletion event that occurred in a common ancestor of these organisms. This seems one of the few cases where a chromosomal rearrangement has been functionally demonstrated to be a deletion event rather than an insertion event in the reference organism. We also show that the previously reported suppression of an acpS mutation by the deletion of Lon protease is an artifact of the increased capsular polysaccharide production of lon strains.     

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.     

Heat adaptation alters Escherichia coli O157:H7 membrane lipid composition and verotoxin production

The influence of heat adaptation (growth at 42 and 45 degrees C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57 degrees C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1omega7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.     

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.     

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.     

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.     

Antimicrobial activity of a 14-residue peptide against Escherichia coli O157:H7

An amphiphilic, cationic peptide composed of eight leucines and six lysines was synthesized by solid phase peptide synthesis (SPPS). The synthetic peptide was bactericidal within 10 min at concentrations as low as 3 microg ml - 1 against mid-exponential Escherichia coli O157:H7 suspended in buffer. Concentrations of 25 microg ml - 1 caused up to 7 log10 cfu ml - 1 reductions. When tested against E. coli O157:H7 grown in TSB, the peptide was bactericidal and bacteriostatic at concentrations of 50 and 25 microg ml - 1, respectively. An inhibitory effect was also observed against stationary phase cells. The synthetic peptide caused the release of u.v.-absorbing materials from the E. coli O157:H7 as well as an increase in its O.D.600 nm. Intracellular K+ and ATP depletion were also observed. These results suggest that the peptide increased the cell membrane permeability but it did not lyse the cells.     

Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7

AIMS: To quantify the antibacterial properties of five essential oils (EO) on a non-toxigenic strain of Escherichia coli O157:H7 in the presence and absence of a stabilizer and an emulsifier and at three different temperatures. METHODS AND RESULTS: Five EOs known to exhibit antibacterial properties were screened by disc diffusion assay and the most active were selected for further study in microdilution colorimetric assays. Oregano (Origanum vulgare) and thyme (Thymus vulgaris; light and red varieties) EO had the strongest bacteriostatic and bactericidal properties, followed by bay (Pimenta racemosa) and clove bud (Eugenia caryophyllata synonym: Syzygium aromaticum) EO. Oregano oil was colicidal at 625 microl l(-1) at 10, 20 and 37 degrees C. The addition of 0.05% (w/v) agar as stabilizer reinforced the antibacterial properties, particularly at 10 degrees C, whereas 0.25% (w/v) lecithin reduced antibacterial activity. Scanning electron micrographs showed extensive morphological changes to treated cells. CONCLUSIONS: Oregano and thyme EO possess significant in vitro colicidal and colistatic properties, which are exhibited in a broad temperature range and substantially improved by the addition of agar as stabilizer. Bay and clove bud EO are less active. Lecithin diminished antibacterial properties. The bactericidal concentration of oregano EO irreversibly damaged E. coli O157:H7 cells within 1 min. SIGNIFICANCE AND IMPACT OF THE STUDY: Oregano and light thyme EO, particularly when enhanced by agar stabilizer, may be effective in reducing the number or preventing the growth of E. coli O157:H7 in foods.     

Lettuce cultivar mediates both phyllosphere and rhizosphere activity of Escherichia coli O157:H7

Plant roots and leaves can be colonized by human pathogenic bacteria, and accordingly some of the largest outbreaks of foodborne illness have been associated with salad leaves contaminated by E. coli O157. Integrated disease management strategies often exploit cultivar resistance to provide a level of protection from economically important plant pathogens; however, there is limited evidence of whether the genotype of the plant can also influence the extent of E. coli O157 colonization. To determine cultivar-specific effects on colonization by E. coli O157, we used 12 different cultivars of lettuce inoculated with a chromosomally lux-marked strain of E. coli O157:H7. Lettuce seedlings grown gnotobiotically in vitro did exhibit a differential cultivar-specific response to E. coli O157 colonization, although importantly there was no relationship between metabolic activity (measured as bioluminescence) and cell numbers. Metabolic activity was highest and lowest on the cultivars Vaila-winter gem and Dazzle respectively, and much higher in endophytic and tightly bound cells than in epiphytic and loosely bound cells. The cultivar effect was also evident in the rhizosphere of plants grown in compost, which suggests that cultivar-specific root exudate influences E. coli O157 activity. However, the influence of cultivar in the rhizosphere was the opposite to that in the phyllosphere, and the higher number and activity of E. coli O157 cells in the rhizosphere may be a consequence of them not being able to gain entry to the plant as effectively. If metabolic activity in the phyllosphere corresponds to a more prepared state of infectivity during human consumption, leaf internalization of E. coli O157 may pose more of a public health risk than leaf surface contamination alone.     

Persistence and metabolic activity of Escherichia coli O157:H7 in farm animal faeces

Ruminants, and to a lesser extent monogastric farm animals, are known to be natural reservoirs of Escherichia coli O157:H7, and contact with contaminated faeces has been linked to human infection. This study used a nontoxigenic, chromosomally marked, lux reporter strain to compare the persistence and activity (bioluminescence) of E. coli O157:H7 over 21 days in the faecal liquor of five farm animals: horse, sheep, cow, pig and piglet. Samples were inoculated with the lux E. coli O157:H7 (7.82 log CFU mL(-1)) and stored at 20 +/- 1 degrees C. The organism was recovered from all samples throughout the experimental period, although lower numbers were recovered from horse faecal liquor relative to all other types (P<0.001). The organisms' activity declined in all samples over time and no luminescence could be detected in any sample 21 days postinoculation. However, activity did increase greatly within pig and piglet faeces during initial stages of monitoring and overall luminescence was greater in piglet samples compared with all other samples (P<0.001). This is the first study to demonstrate how both the persistence and metabolic activity of E. coli O157:H7 notably varies within a range of ruminant and nonruminant animal faeces. Further research is needed to elucidate the factors that govern differential persistence and metabolic activity of E. coli O157:H7 within such matrices.     

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.     

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.     

Escherichia coli O157:H7 in Environments of Culture-Positive Cattle

Outbreaks of Escherichia coli O157:H7 disease associated with animal exhibits have been reported with increasing frequency. Transmission can occur through contact with contaminated haircoats, bedding, farm structures, or water. We investigated the distribution and survival of E. coli O157:H7 in the immediate environments of individually housed, experimentally inoculated cattle by systematically culturing feed, bedding, water, haircoat, and feed bunk walls for E. coli O157:H7 for 3 months. Cedar chip bedding was the most frequently culture-positive environmental sample tested (27/96 or 28.15%). Among these, 12 (44.0%) of positive bedding samples were collected when the penned animal was fecal culture negative. Survival of E. coli O157:H7 in experimentally inoculated cedar chip bedding and in grass hay feed was determined at different temperatures. Survival was longest in feed at room temperature (60 days), but bacterial counts decreased over time. The possibility that urine plays a role in the environmental survival of E. coli O157:H7 was investigated. Cedar chip bedding moistened with sterile water or bovine urine was inoculated with E. coli O157:H7. Bedding moistened with urine supported growth of E. coli O157:H7, whereas inoculated bedding moistened with only water yielded decreasing numbers of bacteria over time. The findings that environmental samples were frequently positive for E. coli O157:H7 at times when animals were culture negative and that urine provided a substrate for E. coli O157:H7 growth have implications for understanding the on-farm ecology of this pathogen and for the safety of ruminant animal exhibits, particularly petting zoos and farms where children may enter animal pens.