Enterohemorrhagic Escherichia Coli O157 Strains Research Articles

Comparison of Enterohemorrhagic Escherichia coli (EHEC) O157 and EHEC Non-O157 Isolates from Patients with Diarrhea in Korea.

We compared 47 enterohemorrhagic Escherichia coli (EHEC) O157 isolates with 184 EHEC non-O157 isolates from Korean patients with diarrhea. In the O157 group, the strains harboring both Shiga toxin genes (stx1 and stx2) were detected with highest frequency, whereas the strains harboring only stx1 gene were most frequently detected in the non-O157 group. Eight virulence genes (eaeA, hlyA, ehx, iha, efa1, tir, toxB, and espA) were found to show a higher frequency of occurrence in the O157 group than in the non-O157 group. In addition, the symptom of bloody diarrhea was exhibited at a higher rate in the O157 group (51.1%) than in the non-O157 group (16.8%). Our findings demonstrate that EHEC O157 strains are more frequently implicated in cases of bloody diarrhea in the Korean population than EHEC non-O157 strains.

Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR=16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR=0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR=0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.

Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection.

Quantifying Colonization Potential of EnterohemorrhagicEscherichia coliO157 Using BovineIn VitroOrgan Culture and Immunofluorescent Staining

A robust semiquantitative method for measuring the colonization potential of O157 enterohemorrhagic Escherichia coli (EHEC) strains was developed combining an established ex vivo model infection system, bovine in vitro organ culture, with detection of bacteria attached to tissue sections by immunofluorescent assay (bIVOC-IFA) using Quantum dot(®) nanocrystal technology. The method was tested on ten O157 strains chosen to reflect a diversity of genotypes found in New Zealand based on the novel polymerase chain reaction-binary typing (P-BIT) system. High- and low-colonizing EHEC O157 strains were identified using bIVOC-IFA, with the highest colonizing strain belonging to the pulsed-field gel electrophoresis type most commonly identified from New Zealand beef meat. Furthermore, all of the toxigenic O157 strains exhibiting a low-colonizing phenotype were closely related, belonging to the same P-BIT genotype cluster. Future use of this method to characterize EHEC strains could provide valuable information for risk assessment and risk management interventions aimed at improving food safety along the beef farm to fork continuum.

Antimicrobial Activity of a Combination of Mume Fructus, Schizandrae Fructus, and Coptidis Rhizoma on Enterohemorrhagic Escherichia coli O26, O111, and O157 and Its Effect on Shiga Toxin Releases

The antibacterial activity of an herbal combination composed of Mume Fructus, Coptidis Rhizoma, and Schizandrae Fructus extracts on enterohemorrhagic Escherichia coli (EHEC) was evaluated in the present study. The combination demonstrated antibacterial activity against all EHEC strains tested in this study, including those resistant to multiple antibiotics; minimum inhibitory concentration values ranged from 0.49 to 31.25 mg/mL. In in vivo antibacterial activity assay, the herbal combination was administered to mice after initial E. coli O157 infection and had significant effects on mouse mortality. The effects of the herbal combination on Shiga toxin release from EHEC O26, EHEC O111, and EHEC O157 strains containing the stx1 and stx2 genes were assessed by the reversed passive latex agglutination method, and there was no increased Shiga toxin release in the strain cultures containing the herbal combination. These results suggested that the herbal combination may be a safe and effective remedy for EHEC inhibition.

Differential expression of the outer membrane protein W (OmpW) stress response in enterohemorrhagic Escherichia coli O157:H7 corresponds to the viable but non-culturable state

During an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157, we showed previously that food isolates were resistant to oxidative stress, while patient isolates were sensitive to it. Because food isolates increased stress-sensitivity after mouse passage, this change most likely occurred during passage through patients. Here we demonstrate that the phenotypic change occurring during mouse passage correlates with the stress response of outer membrane protein W (OmpW) in EHEC O157 strains. Upon induction of oxidative stress, OmpW was highly expressed only in the stress-sensitive MP37 strain, obtained by mouse passage of food strain F2, but not in the F2 strain. Western blotting confirmed that expression of OmpW was induced in the viable but non-culturable (VBNC) state. Deletion of ompW in the MP37 strain increased recovery from dormancy, while overexpression of OmpW in the F2 strain decreased recovery when exposed to oxidative stress, suggesting that high levels of OmpW sensitize the bacteria to stress. DNA alignment revealed that the class I integron (int1I) fragments flanking the ompW gene are oriented in opposite directions between stress-resistant and -sensitive strains. All stress-sensitive strains induced ompW under stress. We propose that the different stress response of OmpW was introduced by genetic alteration during in vivo passage.

Enterohemorrhagic Escherichia coli exploits EspA filaments for attachment to salad leaves.

Enterohemorrhagic Escherichia coli (EHEC) strains are important food-borne pathogens that use a filamentous type III secretion system (fT3SS) for colonization of the gut epithelium. In this study we have shown that EHEC O157 and O26 strains use the fT3SS apparatus for attachment to leaves. Leaf attachment was independent of effector protein translocation.

Detection and Quantitation of Enterohemorrhagic Escherichia coli O157, O111, and O26 in Beef and Bovine Feces by Real-Time Polymerase Chain Reaction

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and certain non-O157 EHEC serotypes (such as O26:H11, O26: NM, O111:H8, and O111:NM) have emerged as significant causes of human disease throughout the world. Important virulence attributes of EHEC are the intimin protein (encoded by the eae gene) and Shiga toxins 1 and 2 (encoded by the stx1 and stx2 genes, respectively). Two sets of real-time polymerase chain reaction (R-PCR) assays were developed for the simultaneous detection and quantitation of EHEC through the monitoring of the presence of the eae and stx genes, and these assays were evaluated. In the eaeR-PCR assay, three sets of primers and TaqMan probes were designed for the amplification and real-time detection of a portion of the eae gene specific to the EHEC O26, O111, and O157 serotypes. In the stxR-PCR assay, two sets of primers and TaqMan probes were used to amplify and detect the stx1 and stx2 genes. DNA prepared from 67 bacterial strains carrying known virulence markers was tested to determine the specificities of the two assays. In the eaeR-PCR assay, eaeO157- and eaeO111-specific primer-probe sets identified only EHEC O157 and O111 strains, respectively. The eaeO26-specific primer-probe set identified all EHEC O26 isolates and some Shiga toxin–negative serotypes of enteropathogenic E. coli and rabbit diarrheagenic E. coli. The stxR-PCR assay was able to identify only those strains carrying either or both of the Shiga toxin–encoding genes. The detection range of both R-PCR assays was linear over DNA concentrations corresponding to 103 to 108 CFU/ml of an EHEC strain. Both assays were able to detect and quantify very low levels (1 to 10 CFU/g of food or feces) of EHEC in feces and ground beef enriched for 16 h in a modified Trypticase soy broth. In conclusion, eae- and stx-based R-PCR assays are reliable and sensitive methods for the rapid screening and specific and quantitative detection of important serotypes of EHEC in cattle and in foods of bovine origin.

Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture.

We examined 30 children with classical hemolytic-uremic syndrome (HUS) for the presence of enterohemorrhagic Escherichia coli (EHEC) strains in stool samples and determined the specific immune response to O157 lipopolysaccharide in acute-phase serum samples from these patients. EHEC O157 strains were isolated from stool samples of 18 (60%) of the patients, and non-O157 EHEC strains were isolated from 5 (17%) of the patients. For O157 strain isolation from stools, we introduced a selective enrichment step using O157-specific antibodies attached to paramagnetic particles (immunomagnetic separation [IMS] method). This procedure allowed the detection of O157 strains at 10(2) CFU/g of stool in the presence of 10(7) coliform background flora organisms. By using IMS followed by plating on sorbitol MacConkey (SMAC) agar and cefixime-tellurite SMAC (CT-SMAC) agar, O157 strains were detected in 18 samples, whereas colony hybridization detected a subset of 12 positive samples and direct culture on CT-SMAC or SMAC agar detected only 7. Three of the 18 O157-positive stools were negative by cytotoxicity assay performed with stool filtrates and by direct PCR with DNA extracted from stools. The IMS technique allowed the isolation of O157 strains from 18 of 20 patients with serological evidence for O157 infection. Apart from the increase in sensitivity in O157 detection compared with that of direct culture, the IMS technique also has the advantage of being less labor-intensive and less time-consuming than the molecular methods. IMS can therefore be considered an efficient method for wide-spread use in the detection of O157 strains in clinical microbiology laboratories. However, because a significant number of HUS cases were attributable to non-O157 EHEC serogroups, the use of additional methods besides IMS in the bacteriological diagnosis of HUS is necessary.