Gastrointestinal Tract Location of Escherichia coli O157:H7 in Ruminants

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of Escherichia coli O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with E. coli O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of E. coli O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum. E. coli O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of E. coli O157:H7 persistence was the lower GIT. E. coli O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for E. coli O157:H7 persistence and proliferation in mature ruminant animals.     

Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Rectal Administration of Escherichia coli O157:H7: Novel Model for Colonization of Ruminants

Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening complications. Because healthy cattle are reservoirs for the bacterium, ruminant infection models have applications in analyzing the relationship between cattle and this human pathogen and in testing interventions to reduce or prevent bovine colonization with this bacterium. Current approaches often do not reliably mimic natural, long-term bovine colonization with E. coli O157:H7 in older calves and adult animals (ages that enter our food chain). Based on the recent identification of the bovine rectoanal junction mucosa as a site of E. coli O157:H7 colonization, we developed a novel rectal swab administration colonization model. We compared this method with oral dosing and direct contact transmission (Trojan) methods. E. coli O157:H7 carriage status was determined by fecal or rectoanal mucosa swab culture. High (~10¹⁰ CFU) and low (~10⁷ CFU) oral doses of E. coli O157:H7 in sheep and cattle resulted in variable infection with the bacterium. Some animals became colonized with the bacteria and remained culture positive for several weeks, and some animals did not become colonized and rapidly cleared the bacteria in a few days. Pen mates of E. coli O157:H7 culture-positive Trojan cattle had a low infection rate and variable colonization status. However, rectal swab administration of E. coli O157:H7 to cattle resulted in consistent long-term colonization in all animals. The surprising ease with which long-term infections resulted from a single application of bacteria to the rectoanal mucosa also strongly supported this location as a site of E. coli O157:H7 colonization in cattle.     

Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10⁻⁶ CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10⁻⁴ CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10⁻⁶ CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.     

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.     

Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages

Escherichia coli O157:H7 is a food-borne bacterium that causes hemorrhagic diarrhea and hemolytic uremic syndrome in humans. While cattle are a known source of E. coli O157:H7 exposure resulting in human infection, environmental reservoirs may also be important sources of infection for both cattle and humans. Bacteriophage-encoded Shiga toxins (Stx) carried by E. coli O157:H7 may provide a selective advantage for survival of these bacteria in the environment, possibly through their toxic effects on grazing protozoa. To determine Stx effects on protozoan grazing, we co-cultured Paramecium caudatum, a common ciliate protozoon in cattle water sources, with multiple strains of Shiga-toxigenic E. coli O157:H7 and non-Shiga toxigenic cattle commensal E. coli. Over three days at ambient laboratory temperature, P. caudatum consistently reduced both E. coli O157:H7 and non-Shiga toxigenic E. coli populations by 1–3 log cfu. Furthermore, a wild-type strain of Shiga-toxigenic E. coli O157:H7 (EDL933) and isogenic mutants lacking the A subunit of Stx 2a, the entire Stx 2a-encoding bacteriophage, and/or the entire Stx 1-encoding bacteriophage were grazed with similar efficacy by both P. caudatum and Tetrahymena pyriformis (another ciliate protozoon). Therefore, our data provided no evidence of a protective effect of either Stx or the products of other bacteriophage genes on protozoan predation of E. coli. Further research is necessary to determine if the grazing activity of naturally-occurring protozoa in cattle water troughs can serve to decrease cattle exposure to E. coli O157:H7 and other Shiga-toxigenic E. coli.     

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.     

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°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.     

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.     

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.     

Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli

Applied Microbiology and Biotechnology - Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of...     

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.     

Experimental Escherichia coli O157:H7 carriage in calves.

Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding.     

Escherichia coli O157:H7 colonization in small domestic ruminants

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

Enterohemorrhagic Escherichia coli O157:H7 Present in Radish Sprouts

Using cultivation, immunofluorescence microscopy, and scanning electron microscopy, we demonstrated the presence of viable enterohemorrhagic Escherichia coli O157:H7 not only on the outer surfaces but also in the inner tissues and stomata of cotyledons of radish sprouts grown from seeds experimentally contaminated with the bacterium. HgCl₂ treatment of the outer surface of the hypocotyl did not kill the contaminating bacteria, which emphasized the importance of either using seeds free from E. coli O157:H7 in the production of radish sprouts or heating the sprouts before they are eaten.     

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

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