Serodiagnosis Using Microagglutination Assay during the Food-Poisoning Outbreak in Japan Caused by Consumption of Raw Beef Contaminated with Enterohemorrhagic Escherichia coli O111 and O157

A microagglutination (MA) assay to identify antibodies to Escherichia coli O111 and O157 was conducted in sera collected from 60 patients during a food-poisoning outbreak affecting 181 patients in Japan which was caused by the consumption of contaminated raw beef. Enterohemorrhagic E. coli (EHEC) O111:H8 and/or O157:H7 was isolated from the stools of some of the patients, but the total rate of positivity for antibodies to O111 (45/60, 75.0%) was significantly higher than that for antibodies to O157 (10/60, 16.7%). The MA titers of antibodies to O111 measured in patients with hemolytic-uremic syndrome and bloody diarrhea were higher than those measured in patients with only diarrhea. In patients from whose stool no isolates of E. coli O111 and O157 were obtained, the positive antibody detection rates were 12/19 (63.2%) for O111 and 2/19 (10.5%) for O157, and the MA titers of antibodies to O111 measured were higher than those to O157. Similarly, the MA titers of antibodies to O111 were significantly higher than those to O157, regardless of the other groups, including groups O111, O111 and O157, and O157. These serodiagnosis results suggest that EHEC O111:H8 stx₂ played a primary role in the pathogenesis of this outbreak. Furthermore, our findings suggest that the isolates from the patients'' stool specimens were not always the major causative pathogen in patients with multiple EHEC infections, because the sera from patients from whose stools only O157 was isolated were positive for antibodies to O111. Measuring antibodies to E. coli O antigen is helpful especially in cases with multiple EHEC infections, even with a non-O157 serotype.     

Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection

Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine.     

Cytolethal distending toxin gene cluster in enterohemorrhagic Escherichia coli O157:H- and O157:H7: characterization and evolutionary considerations

We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-), EHEC O157:H7, and E. coli O55:H7/H(-) strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H(-) strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.     

Contribution of the twin arginine translocation system to the virulence of enterohemorrhagic Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli O157:H7 is a major food-borne infectious pathogen. In order to analyze the contribution of the twin arginine translocation (TAT) system to the virulence of E. coli O157:H7, we deleted the tatABC genes of the O157:H7 EDL933 reference strain. The mutant displayed attenuated toxicity on Vero cells and completely lost motility on soft agar plates. Further analyses revealed that the ΔtatABC mutation impaired the secretion of the Shiga toxin 1 (Stx1) and abolished the synthesis of H7 flagellin, which are two major known virulence factors of enterohemorrhagic E. coli O157:H7. Expression of the EDL933 stxAB₁ genes in E. coli K-12 conferred verotoxicity on this nonpathogenic strain. Remarkably, cytotoxicity assay and immunoblot analysis showed, for the first time, an accumulation of the holotoxin complex in the periplasm of the wild-type strain and that a much smaller amount of StxA₁ and reduced verotoxicity were detected in the ΔtatC mutant cells. Together, these results establish that the TAT system of E. coli O157:H7 is an important virulence determinant of this enterohemorrhagic pathogen.     

Immunological Characterization of Escherichia coli O157:H7 Intimin γ1

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin γ1-producing E. coli strains such as E. coli O157:H7.     

Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus.

Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.     

Responses of cattle to gastrointestinal colonization by Escherichia coli O157:H7

Recent research has established that the terminal rectum is the predominant colonization site of enterohemorrhagic Escherichia coli O157:H7 in cattle. The main aim of the present work was to investigate pathological changes and associated immune responses at this site in animals colonized with E. coli O157:H7. Tissue and gastrointestinal samples from a total of 22 weaned Holstein-cross calves challenged with E. coli O157:H7 were analyzed for bacterial colonization and pathology. Five unexposed age-matched calves were used as comparative negative controls. E. coli O157:H7 bacteria induced histopathological alterations of the rectal mucosa with enterocyte remodeling. This was often associated with removal of the colonized epithelial layer. Immunogold labeling and transmission electron microscopy (TEM) showed E. coli O157 bacteria on pedestals, as part of attaching and effacing lesions. These pathological changes induced a local infiltration of neutrophils that was quantified as larger in infected animals. Rectal mucosal immunoglobulin A responses were detected against the E. coli O157:H7 antigen. This work presents evidence that E. coli O157:H7 is not a commensal bacteria in the bovine host and that the mucosal damage produced by E. coli O157:H7 colonization of the terminal rectum induces a quantifiable innate immune response and production of specific mucosal antibodies.     

Protein Kinase C Mediates Enterohemorrhagic Escherichia coli O157:H7-Induced Attaching and Effacing Lesions

Enterohemorrhagic Escherichia coli serotype O157:H7 causes outbreaks of diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. E. coli O157:H7 intimately attaches to epithelial cells, effaces microvilli, and recruits F-actin into pedestals to form attaching and effacing lesions. Lipid rafts serve as signal transduction platforms that mediate microbe-host interactions. The aims of this study were to determine if protein kinase C (PKC) is recruited to lipid rafts in response to E. coli O157:H7 infection and what role it plays in attaching and effacing lesion formation. HEp-2 and intestine 407 tissue culture epithelial cells were challenged with E. coli O157:H7, and cell protein extracts were then separated by buoyant density ultracentrifugation to isolate lipid rafts. Immunoblotting for PKC was performed, and localization in lipid rafts was confirmed with an anti-caveolin-1 antibody. Isoform-specific PKC small interfering RNA (siRNA) was used to determine the role of PKC in E. coli O157:H7-induced attaching and effacing lesions. In contrast to uninfected cells, PKC was recruited to lipid rafts in response to E. coli O157:H7. Metabolically active bacteria and cells with intact lipid rafts were necessary for the recruitment of PKC. PKC recruitment was independent of the intimin gene, type III secretion system, and the production of Shiga toxins. Inhibition studies, using myristoylated PKCζ pseudosubstrate, revealed that atypical PKC isoforms were activated in response to the pathogen. Pretreating cells with isoform-specific PKC siRNA showed that PKCζ plays a role in E. coli O157:H7-induced attaching and effacing lesions. We concluded that lipid rafts mediate atypical PKC signal transduction responses to E. coli O157:H7. These findings contribute further to the understanding of the complex array of microbe-eukaryotic cell interactions that occur in response to infection.     

Identification and distribution of the enterohemorrhagic Escherichia coli factor for adherence (efa1) gene in sorbitol-fermenting Escherichia coli O157: H-

Sorbitol-fermenting (SF) Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H- strains are emerging as causes of hemorrhagic colitis and the hemolytic-uremic syndrome in Europe. Using subtractive hybridization between SF STEC O157:H- strain 493/89 and STEC O157:H7 strain EDL933, three different fragments, of approximately 700 bp in length, were identified. Each demonstrated > 99% homology to genes encoding the enterohemorrhagic E. coli factor for adherence (efa1) and lymphostatin (lifA). Therefore, a cosmid library was constructed from SF STEC O157:H- strain 493/89, and one clone containing these fragments was sequenced. This sequencing demonstrated a 9669-bp open reading frame (ORF) that had 99.9% sequence homology to efa1 of STEC O111:H- strain E45035 and to lifA of an enteropathogenic E. coli O127:H6 strain E2348/69. In STEC O157:H7 strain EDL933, only small (ca. 3 kb) initial and terminal fragments of this ORF are present. PCR analysis with primers complementary to the efa1/lifA sequence of strain 493/89 indicated that the complete sequence is present in each of 10 SF STEC O157:H- isolates but in none of 10 STEC O157:H7 strains investigated. The presence of the complete efa1/lifA also in both tested E. coli O55:H7 strains supports the hypothesis that SF STEC O157:H- are phylogenetically closer to the proposed E. coli O55:H7 ancestor than STEC O157:H7. Our data demonstrate the presence of a potential virulence gene in SF STEC O157:H- that is only rudimentarily present in STEC O157:H7.     

Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

Isogenic Strain of Escherichia coli O157:H7 That Has Lost both Shiga Toxin 1 and 2 Genes

An Escherichia coli O157:H7 strain isolated from a patient with hemorrhagic colitis was found to exhibit two slightly different colony morphology types on differential medium. Each morphological type, designated TT12A and TT12B, was isolated, and serological testing using various assays confirmed that both strains carried the O157 and the H7 antigens. Biochemical testing showed that the strains had identical profiles on AP120E analysis and, like typical O157:H7 strains, did not ferment sorbitol or exhibit β-glucuronidase activity. Analysis with a multiplex PCR assay showed that TT12B did not carry the gene for either Shiga toxin 1 (Stx1) or Stx2, whereas these genes were present in TT12A and the toxins were produced. Apart from that, both strains carried the +93 gusA mutation, the cluster I ehxA gene for enterohemolysin, and the eae gene for γ-intimin, which are all characteristics of the O157:H7 serotype. Phenotypic assays confirmed that both strains exhibited enterohemolysin activity and the attachment and effacing lesion on HeLa cells. Multilocus enzyme electrophoresis analysis showed that the strains are closely related genetically and belong in the same clonal group. Pulsed-field gel electrophoresis (PFGE) typing of XbaI-digested genomic DNA revealed that the two strains differed by two bands but shared 90% similarity and clustered in the same clade. All other non-Stx-producing O157:H7 strains examined clustered in a major clade that was distinct from that of Stx-producing O157:H7 strains. The findings that TT12B was identical to TT12A, except for Stx production, and its PFGE profile is also more closely related to that of Stx-producing O157:H7 strains suggest that TT12B was derived from TT12A by the loss of both stx genes.     

Foodborne transmission of sorbitol-fermenting Escherichia coli O157:[H7] via ground beef: an outbreak in northern France, 2011

Sorbitol-fermenting Escherichia coli O157:[H7] is a particularly virulent clone of E. coli O157:H7 associated with a higher incidence of haemolytic uraemic syndrome and a higher case fatality rate. Many fundamental aspects of its epidemiology remain to be elucidated, including its reservoir and transmission routes and vehicles. We describe an outbreak of sorbitol-fermenting E. coli O157:[H7] that occurred in France in 2011. Eighteen cases of paediatric haemolytic uraemic syndrome with symptom onset between 6 June and 15 July 2011 were identified among children aged 6 months to 10 years residing in northern France. A strain of sorbitol-fermenting E. coli O157:[H7] stx2a eae was isolated from ten cases. Epidemiological, microbiological and trace-back investigations identified multiply-contaminated frozen ground beef products bought in a supermarket chain as the outbreak vehicle. Strains with three distinct pulsotypes that were isolated from patients, ground beef preparations recovered from patients' freezers and from stored production samples taken at the production plant were indistinguishable upon molecular comparison. This investigation documents microbiologically confirmed foodborne transmission of sorbitol-fermenting of E. coli O157 via beef and could additionally provide evidence of a reservoir in cattle for this pathogen.     

Host Inflammatory Response Inhibits Escherichia coli O157:H7 Adhesion to Gut Epithelium through Augmentation of Mucin Expression

Escherichia coli O157:H7, a major Shiga toxin-producing pathogen, has a low infectious dose and causes serious illness in humans. The gastrointestinal tract of cattle is the primary reservoir of E. coli O157:H7, and thus, it is critical to eliminate or reduce E. coli O157:H7 gut colonization. Given that E. coli O157:H7 produces effectors that attenuate inflammatory signaling, we hypothesized that the host inflammatory response acts to perturb E. coli O157:H7 intestinal colonization. Tumor necrosis factor alpha (TNF-α) treatment of HT-29 cells resulted in increased expression of inflammatory cytokine interleukin 1β (IL-1β), IL-8, and TNF-α genes and increased IL-8 protein and resulted in decreased adhesion of E. coli O157:H7. Similarly, E. coli O157:H7 adhesion to cattle colonic explants was reduced by TNF-α treatment. Irrespective of the presence of E. coli O157:H7, TNF-α enhanced activation of p65, the key mediator of NF-κB inflammatory signaling, whereas E. coli O157:H7 infection suppressed this pathway by inhibiting p65 activation in HT-29 cells. To further explore the mechanisms linking the inflammatory response to attenuated E. coli O157:H7 adhesion, mucin 2 (MUC2) expression was analyzed, considering that the intestinal mucus layer is the first defense against enteric pathogens and MUC2 is the major secretory mucin in the intestine. MUC2 expression in HT-29 cells was increased by TNF-α treatment and by E. coli O157:H7 infection. However, reducing mucin expression by blocking mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinases 1/2 (ERK1/2) and/or phosphatidylinositol 3-kinase (PI3K)/Akt signaling increased E. coli O157:H7 adherence to HT-29 cells. These data suggest that the inflammatory cytokine response acts to protect host epithelial cells against E. coli O157:H7 colonization, at least in part, by promoting mucin production.     

Molecular Characterization of Human Atypical Sorbitol-Fermenting Enteropathogenic Escherichia coli O157 Reveals High Diversity

Alongside the well-characterized enterohemorrhagic Escherichia coli (EHEC) O157:H7, serogroup O157 comprises sorbitol-fermenting typical and atypical enteropathogenic E. coli (EPEC/aEPEC) strains that carry the intimin-encoding gene eae but not Shiga toxin-encoding genes (stx). Since little is known about these pathogens, we characterized 30 clinical isolates from patients with hemolytic uremic syndrome (HUS) or uncomplicated diarrhea with respect to their flagellin gene (fliC) type and multilocus sequence type (MLST). Moreover, we applied whole-genome sequencing (WGS) to determine the phylogenetic relationship with other eae-positive EHEC serotypes and the composition of the rfbO157 region. fliC typing resulted in five fliC types (H7, H16, H34, H39, and H45). Isolates of each fliC type shared a unique ST. In comparison to the 42 HUS-associated E. coli (HUSEC) strains, only the stx-negative isolates with fliCH7 shared their ST with EHEC O157:H7/H⁻ strains. With the exception of one O157:H⁻_fliCH16 isolate, HUS was exclusively associated with fliCH7. WGS corroborated the separation of the fliCH7 isolates, which were closely related to the EHEC O157:H7/H⁻ isolates, and the diverse group of isolates exhibiting different fliC types, indicating independent evolution of the different serotypes. This was also supported by the heterogeneity within the rfbO157 region that exhibited extensive recombinations. The genotypic subtypes and distribution of clinical symptoms suggested that the stx-negative O157 strains with fliCH7 were originally EHEC strains that lost stx. The remaining isolates form a distinct and diverse group of atypical EPEC isolates that do not possess the full spectrum of virulence genes, underlining the importance of identifying the H antigen for clinical risk assessment.     

Variation in the persistence of Escherichia coli O157:H7 in experimentally inoculated 6-week-old conventional lambs

Six-week-old lambs were inoculated orally with 10(9) cfu of an antibiotic-resistance marked four-strain mixture of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 to investigate faecal excretion and intestinal colonisation. In the first experiment, three E. coli O157:H7 isolates were not detected in the faeces of any lambs beyond day 8 post inoculation (pi), or from any of the tissues derived from inoculated animals. One strain, 140065 Nal(r), was isolated from the caecum and colon of one lamb on day 9 pi, from the rectum of another on day 22 pi and persisted in the faeces for up to 28 days pi. All animals remained clinically normal throughout the study period and histological evidence of adhesion of E. coli O157:H7 to the intestinal mucosa was not found. In a separate experiment, four 6-week-old lambs were inoculated orally with 10(9) cfu of E. coli O157:H7 strain 140065 Nal(r) alone. Faecal samples were positive for this strain until the end of the experiment (day 19 pi). This strain was also recovered from the gastrointestinal tract of lambs on days 6, 18 and 19 pi, but was not isolated at day 17 pi. When sampled separately, rectum and terminal colon contents contained higher numbers of the inoculated strain than the intestinal tissue at these sites. Animals inoculated with O157:H7 strain 140065 Nal(r) alone produced soft faeces from day 5 pi onwards. Although attaching and effacing lesions were observed in the caecum, proximal colon and rectum in one animal on day 18 pi, the adherent bacteria did not stain with antiserum raised against the O157 antigen.     

Reduction of Carriage of Enterohemorrhagic Escherichia coli O157:H7 in Cattle by Inoculation with Probiotic Bacteria

Bacteria inhibitory to Escherichia coli O157:H7 were isolated from cattle and evaluated for their potential for reducing carriage of E. coli O157:H7 in calves. Eighteen of 1,200 bacterial isolates from cattle feces and intestinal tissue samples were screened and determined to inhibit the growth of E. coli O157:H7 in vitro. Seventeen of the isolates were E. coli and one was Proteus mirabilis. None produced Shiga toxin. Genomic DNA fingerprinting by pulsed-field gel electrophoresis revealed 13 distinguishable profiles among the 18 isolates. Two calves inoculated perorally with a mixture of all 18 isolates (10¹⁰ CFU) appeared to be normal and did not develop signs of clinical disease throughout a 25- to 27-day observation period. These bacteria colonized segments of the gastrointestinal tract and were in feces at the termination of the experiment (25 and 27 days postinoculation) at levels of 50 to 200 CFU/g. Fifteen cannulated calves were studied to determine the efficiency of the probiotic bacteria in reducing or eliminating the carriage of E. coli O157:H7. Nine calves served as controls, with each animal receiving perorally 10¹⁰ CFU of E. coli O157:H7. E. coli O157:H7 was detected intermittently in the rumen samples from all control animals throughout 3 weeks postinoculation, whereas E. coli O157:H7 was shed at various levels in feces continuously throughout the experiment (mean, 28 days). E. coli O157:H7 was isolated from the rumens and colons of eight of nine and nine of nine calves, respectively, at the termination of the study. Six calves each received perorally 10¹⁰ CFU of probiotic bacteria and then 2 days later received 10¹⁰ CFU of E. coli O157:H7. E. coli O157:H7 was detected in the rumen for only 9 days postinoculation in two animals, for 16 days in one animal, for 17 days in two animals, and for 29 days in one animal. E. coli O157:H7 was detected in feces for only 11 days postinoculation in one animal, for 15 days in one animal, for 17 days in one animal, for 18 days in one animal, for 19 days in one animal, and for 29 days in one animal. At the end of the experiment (mean, 30 days), E. coli O157:H7 was not recovered from the rumen of any of the six animals treated with probiotic bacteria; however, E. coli O157:H7 was recovered from the feces of one of the animals. This animal was fasted twice postinoculation. These studies indicate that selected probiotic bacteria administered to cattle prior to exposure to E. coli O157:H7 can reduce the level of carriage of E. coli O157:H7 in most animals.     

Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7.

Escherichia coli of the serotype O157:H7 is an enterohemorrhagic human pathogen which demonstrates attaching and effacing adhesion to colonocytes in vivo and to epithelial cells grown in tissue culture. Transposon TnphoA mutants of E. coli O157:H7 strain CL-8 were produced. Two of 300 alkaline phosphatase positive mutants, designated JB6 and JB27, did not express O157 side chains as assessed by agglutination with specific polyclonal O157 antiserum, silver staining of lipopolysaccharide extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblots with polyclonal O157-specific antiserum. Both O157-negative mutants and the parent strain demonstrated localized adherence to HEp-2 cells when examined by Giemsa staining and bright-field microscopy. Furthermore, both O157-negative mutants showed enhanced adherence to HEp-2 cells compared with the parent strain when assessed by quantification of adherent bacterial CFUs. The parent strain, CL-8, and both of the mutants produced fluorescent foci when adherent bacteria and HEp-2 cells were stained with fluorescein isothiocyanate-labelled phalloidin. Transmission electron microscopy confirmed attaching and effacing adherence of strain CL-8 and the OO7-negative mutants to HEp-2 cells. These findings indicate that mutants deficient in O157 polysaccharide repeats exhibit adherence to tissue culture cells in vitro and that O157 polysaccharide repeats are not required to produce the attaching and effacing lesion.     

Immunological characterization of Escherichia coli O157:H7 intimin gamma1.

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a+ expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a+. Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin types alpha (O serogroups 86, 127, and 142), beta1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), gamma 1 (O serogroups 55, 145, and 157), gamma 2 (O serogroups 111 and 103), and epsilon (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intgamma1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin gamma 1-producing E. coli strains such as E. coli O157:H7.     

Comparative pathogenicity of Escherichia coli O157 and intimin-negative non-O157 Shiga toxin-producing E coli strains in neonatal pigs

We compared the pathogenicity of intimin-negative non-O157:H7 Shiga toxin (Stx)-producing Escherichia coli (STEC) O91:H21 and O104:H21 strains with the pathogenicity of intimin-positive O157:H7 and O157:H(-) strains in neonatal pigs. We also examined the role of Stx2d-activatable genes and the large hemolysin-encoding plasmid of O91:H21 strain B2F1 in the pathogenesis of STEC disease in pigs. We found that all E. coli strains that made wild-type levels of Stx caused systemic illness and histological lesions in the brain and intestinal crypts, whereas none of the control Stx-negative E. coli strains evoked comparable central nervous system signs or intestinal lesions. By contrast, the absence of intimin, hemolysin, or motility had little impact on the overall pathogenesis of systemic disease during STEC infection. The most striking differences between pigs inoculated with non-O157 STEC strains and pigs inoculated with O157 STEC strains were the absence of attaching and effacing intestinal lesions in pigs inoculated with non-O157:H7 strains and the apparent association between the level of Stx2d-activatable toxin produced by an STEC strain and the severity of lesions.