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.     

Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 x 10(3) CFU g(-1) or <1 x 10(3) CFU ml(-1) and animals with E. coli O157 levels of > or =1 x 10(3) CFU g(-1) or > or =1 x 10(3) CFU ml(-1) in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (> or =1 x 10(3) CFU g of feces(-1)) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Cattle Water Troughs as Reservoirs of Escherichia coli O157

Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of E. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.     

Chromosomal Dynamism in Progeny of Outbreak-Related Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx₂) in the outbreak strain or with the loss of this gene. The two stx₂ alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx₂ genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

Prevalence of Escherichia coli O157 in Cattle Feeds in Midwestern Feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Cattle water troughs as reservoirs of Escherichia coli O157.

Environmental survival of Escherichia coli O157 may play an important role in the persistence and dissemination of this organism on farms. The survival of culturable and infectious E. coli O157 was studied using microcosms simulating cattle water troughs. Culturable E. coli O157 survived for at least 245 days in the microcosm sediments. Furthermore, E. coli O157 strains surviving more than 6 months in contaminated microcosms were infectious to a group of 10-week-old calves. Fecal excretion of E. coli O157 by these calves persisted for 87 days after challenge. Water trough sediments contaminated with feces from cattle excreting E. coli O157 may serve as a long-term reservoir of this organism on farms and a source of infection for cattle.     

Longitudinal Study of Escherichia coli O157 in a Cattle Finishing Unit

In a longitudinal study in a Finnish cattle finishing unit we investigated excretion and sources of Escherichia coli O157 in bulls from postweaning until slaughter. Three groups of 31 to 42 calves were sampled in a calf transporter before they entered the farm and four to seven times at approximately monthly intervals at the farm. All calves sampled in the livestock transporter were negative for E. coli O157 on arrival, whereas positive animals were detected 1 day later. During the fattening period the E. coli O157 infection rate varied between 0 and 38.5%. The animals were also found to be shedding during the cold months. E. coli O157 was isolated from samples taken from water cups, floors, and feed passages. E. coli O157 was detected in 9.7 to 38.9% of the fecal samples taken at slaughter, while only two rumen samples and one carcass surface sample were found to be positive. E. coli O157 was isolated from barn surface samples more often when the enrichment time was 6 h than when the enrichment time was 24 h (P < 0.0001). Fecal samples taken at the abattoir had lower counts (≤0.4 MPN/g) than fecal samples at the farm (P < 0.05). E. coli O157 was isolated more often from 10-g fecal samples than from 1-g fecal samples (P < 0.0001). Most farm isolates belonged to one pulsed-field gel electrophoresis (PFGE) genotype (79.6%), and the rest belonged to closely related PFGE genotypes. In conclusion, this study indicated that the finishing unit rather than introduction of new cattle was the source of E. coli O157 at the farm and that E. coli O157 seemed to persist well on barn surfaces.     

Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Using colony blot hybridization with stx₂ and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx₂-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx₂-positive isolates fliC_H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx₂-positive strains by loss of stx₂ during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx₂-positive and stx-negative isolates demonstrated that yecE was occupied in stx₂-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx₂-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx₂-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx₂-harboring phages. The ability to recycle stx₂, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.     

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.     

Pulsed-Field Gel Electrophoresis Profile Changes Resulting from Spontaneous Chromosomal Deletions in Enterohemorrhagic Escherichia coli O157:H7 during Passage in Cattle

A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans worldwide (18). Cattle are considered the primary reservoir for this pathogen and play a central role in transmission to humans (6). Healthy cattle transiently carry EHEC O157:H7 and shed the bacteria in their feces (5, 7). Human infections have been associated with the consumption of contaminated meat and milk, direct contact with cattle, and the consumption of vegetables, fruits, and water contaminated with cattle manure (6).Because of its high discriminatory power, pulsed-field gel electrophoresis (PFGE) has been widely employed as a molecular typing method in many epidemiological investigations to identify various outbreaks and routes of transmission of EHEC O157:H7 (1, 12, 15, 17). Simpson''s index of diversity (9) was reported to be >0.985 in previous studies (1, 15), supporting the identification of richness (the number of types among isolates) and evenness (the relative distribution of individual strains among the different types) of molecular typing using PFGE.Instability of the PFGE patterns of EHEC O157:H7 isolates has been reported. Changes in PFGE patterns were observed among strains after repeated subculturing and prolonged storage at room temperature (11). Loss of Shiga toxin genes and a large-scale inversion within the genome were identified as genetic events generating changes in PFGE patterns in vitro (10, 13). Shifts in the genotypes of EHEC O157:H7 clinical isolates from patients and cattle have been reported (3, 14). This phenomenon was also observed in EHEC O157:H7 experimental infections of cattle. Spontaneous curing of a 90-kb plasmid resulted in the loss of two restricted fragments from the PFGE profiles of EHEC O157:H7 isolates obtained from experimentally infected cattle (2). The purpose of the present study was to identify the genetic events affecting the PFGE patterns of EHEC O157:H7 after passage through the intestinal tract of cattle, especially for restriction fragments that are >90 kb long.Four 5-month-old Holstein steers were housed individually in climate-controlled biosafety level 2 containment barns in accordance with the guidelines for animal experimentation defined by the National Institute of Animal Health of Japan. The pens had individual floor drains and were cleaned twice daily with water and disinfectant. All animals were healthy and culture negative for EHEC O157:H7 strains, as determined by a previously described technique (2), prior to inoculation.EHEC O157:H7 strain Sakai-215 (12, 23), which was isolated from an outbreak in Sakai, Osaka Prefecture, in 1996 was used for inoculation. This strain harbors the genes encoding Stx1 and Stx2. A spontaneous resistant strain was selected with nalidixic acid in order to facilitate the recovery of this strain from fecal samples. All calves were inoculated using a stomach tube with an exponential-phase culture (10⁹ CFU) of the nalidixic acid-resistant Sakai-215 strain. Fecal samples were collected from the four calves daily for 45 days. Fecal culturing was performed as described previously (2). Eight non-sorbitol-fermenting colonies were selected daily from each animal and identified as EHEC O157:H7 colonies by routine diagnostic methods (25).All animals were clinically normal throughout the experimental period. The EHEC O157:H7-inoculated calves (calves 1 to 4) were culture positive for the organism 24 h after inoculation. Intermittent fecal shedding by the calves was observed until 27, 32, 26, and 39 days postinoculation for calves 1, 2, 3, and 4, respectively (Fig. ​(Fig.1).1). The numbers of EHEC O157:H7 isolates recovered from calves 1, 2, 3, and 4 were 200, 224, 200, and 281, respectively.Open in a separate windowFIG. 1.Changes in PFGE profiles of EHEC O157:H7 isolates recovered from calves 1 (A), 2 (B), 3 (C), and 4 (D). The absence of a bar indicates that no EHEC O157:H7 was detected. The open horizontal bars under the vertical bars indicate that the eight isolates obtained on a day were obtained from the enrichment culture.A total of 905 recovered isolates in addition to the inoculated strain were used for PFGE analysis. Genomic DNA from each EHEC O157:H7 isolate was prepared using the method of Persing (Mayo Clinic, Rochester, MN) described by Rice et al. (20). Agarose-embedded chromosomal DNA was cleaved with XbaI by following the manufacturer''s instructions. PFGE was performed in a 0.85% megabase agarose gel, using a CHEF DR III apparatus (Bio-Rad Laboratories). The pulse time was increased from 12 to 35 s for 18 h. The PFGE profiles of all of the EHEC O157:H7 isolates recovered from the four calves were compared with that of the inoculated strain. The number of band differences was determined by enumerating the loss and addition of fragments (22).Two hundred eighty-nine isolates had PFGE profiles different from that of the inoculated strain, and 12 distinct PFGE profiles were identified for these isolates (Table ​(Table1).1). The fact that only one to three band differences were observed for the 12 profiles suggested that these isolates were closely related (22) and were variants of the inoculated strain. In addition, the pens had individual floor drains and were cleaned twice daily with water and disinfectant, which reduced the likelihood of introduction of novel EHEC O157:H7 strains. We designated the PFGE profiles A to L. PFGE profiles A, C, and H were obtained for all four calves and accounted for 30.4% of the 905 isolates recovered. Different PFGE profiles were obtained for all animals at least 2 days postinoculation (Fig. ​(Fig.1).1). All eight isolates from calf 2 collected on day 15 postinoculation and from calf 3 collected on days 22 and 23 postinoculation had PFGE profiles different from that of the original isolate (Fig. ​(Fig.1).1). The isolates that had the same PFGE profile as the inoculated strain were detected again later.

TABLE 1.

Temporal distribution of PFGE profiles of EHEC O157:H7 isolates recovered from experimentally infected cattle

Concentration and Prevalence of Escherichia coli O157 in Cattle Feces at Slaughter

The concentration and prevalence of Escherichia coli O157 in cattle feces at the time of slaughter was studied over a 9-week period from May to July 2002. Fecal samples (n = 589) were collected from the rectums of slaughtered cattle, and the animal-level prevalence rate was estimated to be 7.5% (95% confidence interval [CI], 5.4 to 9.6%) while the group prevalence was 40.4% (95% CI, 27.7 to 53.2%). Of the 44 infected animals detected, 9% were high shedders that contained E. coli O157 at concentrations of >10⁴ CFU g⁻¹. These 9% represented >96% of the total E. coli O157 produced by all animals tested. All isolates possessed the vt₂ gene, 39 had the eaeA gene, and a further five had the vt₁ gene also. The presence of high-shedding animals at the abattoir increases the potential risk of meat contamination during the slaughtering process and stresses the need for correctly implemented hazard analysis and critical control point procedures.     

Waterborne Escherichia coli O157

The waterborne route of Vero cytotoxin-producing E. coli (VTEC) O157 infection was first suggested in two unconnected human cases in 1985. Since then, waterborne VTEC O157 has been identified in sporadic cases and in outbreaks of illness. Recreational waters, private and municipal supplies have been implicated from microbiological, environmental and epidemiological studies of cases. In addition, a research cohort study of farm workers identified exposure to private water supplies as a risk factor for having antibodies to E. coli O157. Sources of contamination are thought to be animal and human faeces or sewage. The presence of low numbers of target organisms in water makes microbiological confirmation difficult, therefore epidemiological evidence has been essential in outbreak investigations. Despite the potential for contamination of water with VTEC O157, waterborne infection is relatively rare largely due to the susceptibility of the organism to water treatment processes. This paper presents the evidence for waterborne VTEC O157 infection, considering current microbiological, environmental and particularly epidemiological information.     

Carriage of stx2a Differentiates Clinical and Bovine-Biased Strains of Escherichia coli O157

Background

Shiga toxin (Stx) are cardinal virulence factors of enterohemorrhagic E. coli O157:H7 (EHEC O157). The gene content and genomic insertion sites of Stx-associated bacteriophages differentiate clinical genotypes of EHEC O157 (CG, typical of clinical isolates) from bovine-biased genotypes (BBG, rarely identified among clinical isolates). This project was designed to identify bacteriophage-mediated differences that may affect the virulence of CG and BBG.

Methods

Stx-associated bacteriophage differences were identified by whole genome optical scans and characterized among >400 EHEC O157 clinical and cattle isolates by PCR.

Results

Optical restriction maps of BBG strains consistently differed from those of CG strains only in the chromosomal insertion sites of Stx2-associated bacteriophages. Multiplex PCRs (stx1, stx2a, and stx2c as well as Stx-associated bacteriophage - chromosomal insertion site junctions) revealed four CG and three BBG that accounted for >90% of isolates. All BBG contained stx2c and Stx2c-associated bacteriophage – sbcB junctions. All CG contained stx2a and Stx2a-associated bacteriophage junctions in wrbA or argW.

Conclusions

Presence or absence of stx2a (or another product encoded by the Stx2a-associated bacteriophage) is a parsimonious explanation for differential virulence of BBG and CG, as reflected in the distributions of these genotypes in humans and in the cattle reservoir.     

Prevalence of Escherichia coli O157:H7 in Gallbladders of Beef Cattle

Gallbladders and rectal contents were collected from cattle (n = 933) at slaughter to determine whether the gallbladder harbors Escherichia coli O157:H7. Both gallbladder mucosal swabs and homogenized mucosal tissues were used for isolation. Only five gallbladders (0.54%) were positive for E. coli O157:H7. Fecal prevalence averaged 7.1%; however, none of the cattle that had E. coli O157:H7 in the gallbladder was positive for E. coli O157:H7 in feces. Therefore, the gallbladder does not appear to be a common site of colonization for E. coli O157:H7 in beef cattle.     

In Vivo Synergy Between Green Tea Extract and Levofloxacin Against Enterohemorrhagic Escherichia coli O157 Infection

We studied the synergistic effects of Japanese green tea extract (JGTE) and levofloxacin (LVFX) against enterohemorrhagic Escherichia coli (EHEC) infection in a gnotobiotic mouse model. Mice fed on JGTE conferred a significant degree of protection against an oral challenge with EHEC. Complete elimination of the bacteria from the mice, was however, difficult. The combination of JGTE and LVFX increased the survival rate and reduced damage to target organs. Thus, dietary supplementation with JGTE improved the therapeutic effects of antibiotic treatment. Received: 28 July 2000 / Accepted: 19 September 2000     

Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.