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.     

Identification and Characterization of Spontaneous Deletions within the Sp11-Sp12 Prophage Region of Escherichia coli O157:H7 Sakai

Prophages make up 12% of the enterohemorrhagic Escherichia coli genome and play prominent roles in the evolution and virulence of this food-borne pathogen. Acquisition and loss of and rearrangements within prophage regions are the primary causes of differences in pulsed-field gel electrophoresis (PFGE) patterns among strains of E. coli O157:H7. Sp11 and Sp12 are two tandemly integrated and putatively defective prophages carried by E. coli O157:H7 strain Sakai. In this study, we identified 3 classes of deletions that occur within the Sp11-Sp12 region, at a frequency of ca. 7.74 × 10⁻⁴. One deletion resulted in a precise excision of Sp11, and the other two spanned the junction of Sp11 and Sp12. All deletions resulted in shifts in the XbaI fragment pattern observed by PFGE. We sequenced the inducible prophage pool of Sakai but did not identify any mature phage particles corresponding to either Sp11 or Sp12. Deletions containing pchB and psrC, which are Sp11-carried genes encoding proteins known or suspected to regulate type III secretion, did not affect the secretion levels of the EspA or EspB effector. Alignment of the Sp11-Sp12 DNA sequence with its corresponding regions in other E. coli O157:H7 and O55:H7 strains suggested that homologous recombination rather than integrase-mediated excision is the mechanism behind these deletions. Therefore, this study provides a mechanism behind the previously observed genetic instability of this genomic region of E. coli O157:H7.     

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Characterization of Escherichia coli O157:H7 Strains Isolated from Supershedding Cattle

Previous reports have indicated that a small proportion of cattle shedding high levels of Escherichia coli O157:H7 is the main source for transmission of this organism between animals. Cattle achieving a fecal shedding status of 10⁴ CFU of E. coli O157:H7/gram or greater are now referred to as supershedders. The aim of this study was to investigate the contribution of E. coli O157:H7 strain type to supershedding and to determine if supershedding was restricted to a specific set of E. coli O157:H7 strains. Fecal swabs (n = 5,086) were collected from cattle at feedlots or during harvest. Supershedders constituted 2.0% of the bovine population tested. Supershedder isolates were characterized by pulsed-field gel electrophoresis (PFGE), phage typing, lineage-specific polymorphism assay (LSPA), Stx-associated bacteriophage insertion (SBI) site determination, and variant analysis of Shiga toxin, tir, and antiterminator Q genes. Isolates representing 52 unique PFGE patterns, 19 phage types, and 12 SBI clusters were obtained from supershedding cattle, indicating that there is no clustering to E. coli O157:H7 genotypes responsible for supershedding. While being isolated directly from cattle, this strain set tended to have higher frequencies of traits associated with human clinical isolates than previously collected bovine isolates with respect to lineage and tir allele, but not for SBI cluster and Q type. We conclude that no exclusive genotype was identified that was common to all supershedder isolates.     

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.     

Characterization of a novel two-partner secretion system in Escherichia coli O157:H7

Gram-negative bacteria contain multiple secretion pathways that facilitate the translocation of proteins across the outer membrane. The two-partner secretion (TPS) system is composed of two essential components, a secreted exoprotein and a pore-forming beta barrel protein that is thought to transport the exoprotein across the outer membrane. A putative TPS system was previously described in the annotation of the genome of Escherichia coli O157:H7 strain EDL933. We found that the two components of this system, which we designate OtpA and OtpB, are not predicted to belong to either of the two major subtypes of TPS systems (hemolysins and adhesins) based on their sequences. Nevertheless, we obtained direct evidence that OtpA and OtpB constitute a bona fide TPS system. We found that secretion of OtpA into the extracellular environment in E. coli O157:H7 requires OtpB and that when OtpA was produced in an E. coli K-12 strain, its secretion was strictly dependent on the production of OtpB. Furthermore, using OtpA/OtpB as a model system, we show that protein secretion via the TPS pathway is extremely rapid.     

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.     

Survival of Escherichia coli O157:H7 on cattle hides

The objective of this study was to determine the time period that Escherichia coli O157:H7 survives on the hides of cattle. Extensive research has been conducted and is ongoing to identify and develop novel preharvest intervention strategies to reduce the presence of E. coli O157:H7 on live cattle and subsequent transfer to processed carcasses. If a reduction of E. coli O157:H7 levels in feces can be achieved through preharvest intervention, it is not known how long it would take for such reductions to be seen on the hide. In the study presented herein, three trials were conducted to follow E. coli O157:H7 hide prevalence over time. For each trial, 36 animals were housed in individual stanchions to minimize or prevent hide contamination events. Through prevalence determination and isolate genotyping with pulsed-field gel electrophoresis, survival of E. coli O157:H7 on the hides of live cattle was determined to be short lived, with an approximate duration of 9 days or less. The results of this study suggest that any preharvest interventions that are to be administered at the end of the finishing period will achieve maximum effect in reducing E. coli O157:H7 levels on cattle hides if given 9 days before the cattle are presented for processing. However, it should be noted that interventions reducing pathogen shedding would also contribute to decreasing hide contamination through lowering the contamination load of the processing plant lairage environment, regardless of the time of application.     

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.     

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 degrees 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 degrees 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.     

Factors affecting the heat resistance of Escherichia coli O157 : H7

Escherichia coli O157 : H7 has been reported as being not particularly heat resistant. However, several factors which might increase its heat resistance have been investigated in this study using five strains. Increase in growth temperature to 40 °C, as found in the cow gut, heat-shock at sub-lethal temperatures of 42, 45, 48 and 50 °C, and variable heating rate (1 °C min⁻¹ to 23 °C min⁻¹) had no dramatic effect on heat resistance. Growth phase had a marked impact on heat resistance ; late stationary phase cells were more heat-resistant than were log phase cells. The difference in heat resistance between the two phases of growth became more pronounced when cells were resuspended in fresh nutrient broth ; heat resistance of late stationary phase cells increased dramatically whereas no such effect was observed with log phase cells. The addition of polyphosphates to the heating medium did not increase heat resistance. A reduction in water activity of the heating medium from 0·995 to levels between 0·980 and 0·960 also resulted in a marked increase in heat resistance. This effect was more pronounced under conditions of extremely low water activity created by resuspending late stationary phase cells in sunflower oil. Survivors were detected even after a heat treatment at 60 °C for 1 h or 70 °C for 5 min. It can be confirmed that this serotype has no unusual heat resistance and that the heating environment markedly affects resistance.     

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.