Shiga toxin-producing Escherichia coli in children: diagnosis and clinical manifestations of O157:H7 and non-O157:H7 infection

Shiga toxin-producing Escherichia coli (STEC), a cause of food-borne colitis and hemolytic-uremic syndrome in children, can be serotype O157:H7 (O157) or other serotypes (non-O157). E. coli O157 can be detected by culture with sorbitol-MacConkey agar (SMAC), but non-O157 STEC cannot be detected with this medium. Both O157 and non-O157 STEC can be detected by immunoassay for Shiga toxins 1 and 2. The objectives of this study were first to compare the diagnostic utility of SMAC to that of the Premier EHEC enzyme immunoassay (Meridian Diagnostics) for detection of STEC in children and second to compare the clinical and laboratory characteristics of children with serotype O157:H7 STEC and non-O157:H7 STEC infections. Stool samples submitted for testing for STEC between April 2004 and September 2009 were tested by both SMAC culture and the Premier EHEC assay at Children's Hospital Boston. Samples positive by either test were sent for confirmatory testing and serotyping at the Hinton State Laboratory Institute (HSLI). Chart review was performed on children with confirmed STEC infection. Of 5,110 children tested for STEC, 50 (0.9%) had STEC infection confirmed by culture; 33 were O157:H7 and 17 were non-O157:H7. The Premier EHEC assay and SMAC culture detected 96.0% and 58.0% of culture-confirmed STEC isolates (any serotype), respectively, and 93.9% and 87.9% of STEC O157:H7 isolates, respectively. There were no significant differences in disease severity or laboratory manifestations of STEC infection between children with O157:H7 and those with non-O157 STEC. The Premier EHEC assay was significantly more sensitive than SMAC culture for diagnosis of STEC, and O157:H7 and non-O157:H7 STEC caused infections of similar severity in children.     

Detection of Escherichia coli O157:H7 from Musca domestica (Diptera: Muscidae) at a cattle farm in Japan

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 was isolated for the first time from Musca domestica L. A total of 310 fly samples was collected from 4 different farms in Obihiro-City, Hokkaido, in the summer and autumn of 1997;5 samples carried E. coli serotype O157:H7. Using ELISA and Vero cell cytotoxicity assay, 3 isolates from 1 cattle farm produced both active Shiga-toxin type 1 (Stx1) and 2 (Stx2). These isolates also carried hemolysin and eaeA genes and harbored the 90-kb virulence plasmid of EHEC O157:H7. Based on plasmid profiles, antibiotic patterns, polymerase chain reaction (PCR)-based DNA finger printing analysis using random amplified polymorphic DNA, pulsed field gel electrophoresis analysis, and DNA sequences of stx1 and stx2, all 3 isolates from fly samples were identical. These results indicate that the house fly is capable of carrying the toxigenic EHEC O157:H7 involved in human disease.     

Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli

The Shiga toxins (Stx) are critical virulence factors for Escherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.     

Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli of serotypes O157:H7 and O26:H11

A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli O157:H7 and O26:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli O157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli O157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shigella dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype O157:H7 (including strains of serotype O157 but not H7). The E. coli strains (all of serotype O26:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli O157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes O157:H7 and O26:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes O157:H7 and O26:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.     

Polymorphisms within EspA filaments of enteropathogenic and enterohemorrhagic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) possess a filamentous type III secretion system (TTSS) employed to deliver effector proteins into host cells. EspA is a type III secreted protein which forms the filamentous extension to the TTSS and which interacts with host cells during early stages of attaching and effacing (A/E) lesion formation. By immunofluorescence, a polyclonal antibody previously raised to EspA from EPEC strain E2348/69 (O127:H6) stained approximately 12-nm-diameter EspA filaments produced by this strain but did not stain similar filaments produced by EHEC serotype O157:H7. Similarly, an antibody that we subsequently raised to EHEC strain 85-170 (O157:H7) EspA stained approximately 12-nm-diameter EspA filaments produced by strain 85-170 but did not stain E2348/69 EspA filaments. Given such heterogeneity between EPEC and EHEC EspA filaments, we examined polymorphisms of functional EspA filaments among different EPEC and EHEC serotypes. With use of the EPEC EspA antiserum, EspA filaments were observed only with EPEC serotypes O127:H6 and O55:H6, serotypes which encode an identical EspA protein. When stained with the EHEC EspA antiserum, EspA filaments were detected only on EHEC strains belonging to serotype O157:H7; the EHEC antiserum did, however, stain EspA filaments produced by the closely related EPEC serotype O55:H7 but not filaments of any other EPEC serotype tested. Such polymorphisms among functional EspA filaments of EPEC and EHEC would be expected to have important implications for the development of broad-range EspA-based vaccines.     

Comparison of a direct fecal Shiga-like toxin assay and sorbitol-MacConkey agar culture for laboratory diagnosis of enterohemorrhagic Escherichia coli infection.

A direct fecal Shiga-like toxin assay (DSLTA) was used to prospectively screen 9,449 unselected stool samples, received at the British Columbia Provincial Health Laboratories and the Metropolitan Laboratories of Vancouver, for Shiga-like toxin I and Shiga-like toxin II. The results were compared with results of routine stool culture on sorbitol-MacConkey agar (SMAC) for Escherichia coli O157:H7. Of 80 specimens positive by either method, 59 (74%) and 74 (93%) were positive by SMAC and DSLTA, respectively; 53 (66%) were positive by both methods, 21 (26%) were positive by DSLTA only, and 6 (7%) were positive by SMAC only. On further screening, Shiga-like toxin-producing E. coli were detected in 8 (38%) of the 21 stools positive by DSLTA only, including serotypes O157:H7 (1 stool), O26:K60 (5 stools), O128:K67 (1 stool), and O103:H2 (1 stool). For the remaining 13 stools in which no SLTEC was found but DSLTA was positive, clinical information revealed that 11 of 12 patients had diarrheal illnesses, and 4 of these 11 had bloody diarrhea or hemolytic-uremic syndrome. Stools positive only by SMAC were collected earlier in the illness than stools positive by DSLTA, suggesting that free fecal toxin levels may be too low to detect at this time. Overall we found that DSLTA detected 19% more positive specimens than SMAC and that Shiga-like toxin-producing E. coli serotypes other than E. coli O157:H7 are causing disease in the province of British Columbia, Canada.     

Detection in Escherichia coli of the genes encoding the major virulence factors,the genes defining the O157:H7 serotype,and components of the type 2 Shiga toxin family by multiplex PCR

Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx(1), stx(2), stx(2c), stx(2d), stx(2e), stx(2f), EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.     

Genetic analysis for virulence factors in Escherichia coli O104:H21 that was implicated in an outbreak of hemorrhagic colitis

Isolates of enterohemorrhagic Escherichia coli (EHEC) of serotype O104:H21 implicated in a 1994 outbreak of hemorrhagic colitis in Montana were analyzed for the presence of trait EHEC virulence markers. By using a multiplex PCR that specifically amplifies several genes, the O104:H21 strains were found to carry only the Shiga toxin 2 gene (stx2) and to express Stx2. They did not have the eaeA gene for gamma-intimin, which is typically found in O157:H7, or the alpha- or beta-intimin derivatives, which are common in other EHEC and enteropathogenic E. coli serotypes. Results of the multiplex PCR also indicated that the ehxA gene for enterohemolysin was absent from O104:H21. This, however, was not consistent with the results of a phenotypic assay that showed them to be hemolytic or a PCR analysis with another set of ehxA-specific primers, which indicated the presence of ehxA. To resolve this discrepancy, the ehxA region in O104:H21 and O157:H7 strains, to which the multiplex PCR primers anneal, was cloned and sequenced. Comparison of the sequences showed that the upstream primer binding site in the ehxA gene of O104:H21 was not identical to that of O157:H7. Specifically, there were several base mutations, including an A-to-G substitution at the 3' end of the primer binding site. These base mutations are presumably not unique to O104:H21, since other enterohemolytic serotypes were also not detected with the ehxA primers used in the multiplex PCR. Comparison of the ehxA sequences of O104:H21 strains with those of other Stx-producing E. coli strains showed that they more closely resembled those of O8:H19 strains, which have cluster II ehxA genes, than those of O157:H7 strains, which have cluster I ehxA sequences. By modifying the upstream ehxA primer, the multiplex PCR was able to detect ehxA genes in both O157:H7 and O104:H21 strains.     

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O118 display three distinctive clonal groups of EHEC pathogens

A recent case report of a child infected with enterohemorrhagic Escherichia coli (EHEC) of serotype O118:H16 in Bavaria, in association with the isolation of a bovine O118 strain on the same farm (A. Weber, H. Klie, H. Richter, P. Gallien, M. Timm, and K. W. Perlberg, Berl. Muench. Tieraerztl. Wochenschr. 110:211-213, 1997), prompted us to investigate the relationship between bovine and human strains of serogroup O118. A total of 29 human O118 E. coli strains from Europe (21), Canada (4), and Peru (4) were compared by virulence typing and macrorestriction analysis with 7 bovine O118 EHEC strains isolated in Bavaria. Twenty-five of the human strains were characterized as EHEC. By serotyping and determination of the virulence-associated factors Shiga toxin (stx1 stx2 stx2 variants), intimin (eae), and EHEC hemolysin (Hly(EHEC)), three distinctive groups of O118 human pathogens were identified. Most of the strains belonged to serotype O118:H16, displaying the virulence traits Stx1, intimin, Hly(EHEC), and EspP/PssA (group 1). In addition, we identified strains of serotype O118:H12 (Stx2d only; group 2) and of serotype O118:H30 (Stx2 and intimin; group 3). Macrorestriction analysis with BlnI and XbaI revealed that all strains with a single O118 serotype profile (O118:H12, O118:H16, and O118:H30) belonged to one clonal cluster, irrespective of their origin. Group 1 strains clustered in the same clonal group as the bovine O118:H16 strains. Moreover, four pairs of strains of different origins and indistinguishable by all other methods applied were identified as group 1 strains. Our data support the direct transmission of an EHEC O118:H16 strain from a calf to a 2-year-old boy in the above-mentioned case report. Since bovine and human O118:H16 strains represent the same clones, they must be considered zoonotic EHEC pathogens. In contrast, EHEC strains of serotypes O118:H12 and O118:H30 have been isolated only from humans, indicating a reservoir for certain human O118 EHEC strains other than bovines.     

Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an approximately 90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of lpfA(O113) were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf(O113) may function as an adhesin in LEE-negative isolates of EHEC.     

Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7

The largest EHEC outbreak up to now in Germany occurred in 2011. It was caused by the non-O157:H7 Shiga-toxinogenic enterohemorrhagic E. coli strain O104:H4. This strain encodes in addition to the Shiga toxin 2 (Stx2), responsible for the hemolytic uremic syndrome (HUS), several adhesins such as aggregative adherence fimbriae. Currently, there is no effective prophylaxis and treatment available for EHEC infections in humans. Especially antibiotics are not indicated for treatment as they may induce Stx production, thus worsening the symptoms. Alternative therapeutics are therefore desperately needed. We tested the probiotic Escherichia coli strain Nissle 1917 (EcN) for antagonistic effects on two O104:H4 EHEC isolates from the 2011 outbreak and on the classical O157:H7 EHEC strain EDL933. These tests included effects on adherence, growth, and Stx production in monoculture and co-culture together with EcN. The inoculum of each co-culture contained EcN and the respective EHEC strain either at a ratio of 1:1 or 10:1 (EcN:EHEC). Adhesion of EHEC strains to Caco-2 cells and to the mucin-producing LS-174T cells was reduced significantly in co-culture with EcN at the 1:1 ratio and very dramatically at the 10:1 ratio. This inhibitory effect of EcN on EHEC adherence was most likely not due to occupation of adhesion sites on the epithelial cells, because in monocultures EcN adhered with much lower bacterial numbers than the EHEC strains. Both EHEC strains of serotype O104:H4 showed reduced growth in the presence of EcN (10:1 ratio). EHEC strain EDL933 grew in co-culture with EcN only during the first 2 h of incubation. Thereafter, EHEC counts declined. At 24 h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24 h co-incubation with EcN (EcN:EHEC ratio 10:1) was for all 3 EHEC strains tested significantly reduced in comparison to EHEC monocultures.     

An in-vitro model for studying the interaction of Escherichia coli O157:H7 and other enteropathogens with bovine primary cell cultures

Sections of kidney, trachea, ileum, colon, rectum and rumen were removed at post mortem from a neonatal calf and, with the exception of the rumen, primary cell lines were established for each of the cell types. The adherence of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, enteropathogenic E. coli (EPEC) serotype O111, E. coli K12 (a laboratory adapted non-pathogenic strain) and Salmonella enterica serotype Typhimurium was assayed on each cell type. For all adherence assays on all cell lines, EHEC O157:H7 adhered to a significantly greater extent than the other bacteria. S. Typhimurium and EPEC O111 adhered to a similar extent to one another, whereas E. coli K12 was significantly less adherent by 100-fold. In all cell types, >10% of adherent S. Typhimurium bacteria invaded, whereas c. 0.01-0.1% of adherent EHEC O157:H7 and EPEC O111 bacteria invaded, although they are regarded as non-invasive. EHEC O157 generated actin re-arrangements in all cell types as demonstrated by fluorescent actin staining (FAS) under densely packed bacterial micro-colonies. EPEC O111 readily generated the localised adherent phenotype on bovine cells but generated only densely packed micro-colonies on HEp-2 cells. The intensity of actin re-arrangements induced in bovine cells by EPEC O111 was less than that induced by EHEC O157:H7. The intimate attachment on all cell types by both EHEC O157:H7 and EPEC O111 was clearly demonstrated by scanning electron microscopy.     

Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-xi)

A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx(1) genes, 278 (54%) possessed stx(2) genes, and 135 (26%) possessed both stx(1) and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx(2) eae-gamma1 ehxA (46 isolates) was the most common, followed by O157:H7 stx(1) stx(2) eae-gamma1 ehxA (34 isolates), O113:H21 stx(2) (25 isolates), O22:H8 stx(1) stx(2) ehxA (15 isolates), O26:H11 stx(1) eae-beta1 ehxA (14 isolates), and O77:H41 stx(2) ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin beta1, 82 O157:H7 isolates possessed intimin gamma1, three O111:H- isolates had intimin type gamma2, one O49:H- strain showed intimin type delta, 13 (six of serotype O103:H2) isolates had intimin type epsilon and eight (four of serotype O156:H-) isolates had intimin zeta. We have identified a new variant of the eae intimin gene designated xi (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.     

Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.     

Molecular characterization of a serotype O121:H19 clone,a distinct Shiga toxin-producing clone of pathogenic Escherichia coli

Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon ) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.     

Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.     

Enzyme-linked immunosorbent assay for products of the 60-megadalton plasmid of Escherichia coli serotype O157:H7.

Eighty strains of pathogenic Escherichia coli, representing each of the major diarrheal disease-causing groups, were examined by direct enzyme-linked immunosorbent assay (ELISA) for the presence of proteins associated with a 60-MDa plasmid from E. coli serotype O157:H7. Antiserum specific for plasmid-encoded proteins was prepared by immunizing a rabbit with a wild-type E. coli O157:H7 strain (strain 7785) and absorbing the serum with a plasmid-cured derivative (strain 2-45). Use of this antiserum in Western immunoblot analysis detected two proteins of 82 and 92 kDa in strain 7785 but not in strain 2-45. All 16 wild-type E. coli O157:H7 strains and all 10 Shiga-like toxin (SLT)-producing E. coli strains of serotypes other than O157 were ELISA positive. Thirteen of 14 enterotoxigenic and all of 24 enteroinvasive E. coli strains were ELISA negative, as were all of 16 E. coli strains isolated from healthy persons. Of 16 traditional enteropathogenic E. coli (EPEC) serotypes, 10 were ELISA positive, including 10 of 12 strains carrying the EPEC adherence factor gene. Absorption of the serum with an EPEC adherence factor-positive EPEC eliminated EPEC reactivity. This study demonstrates that two plasmid-mediated proteins are common to E. coli O157:H7 strains and to SLT-producing strains of other serotypes. Detection of these proteins by ELISA provides a sensitive and specific screening test for identifying SLT-producing E. coli of both O157 and non-O157 serotypes. Identification of the cross-reactive proteins found in EPEC could provide the basis for a single assay to detect both EPEC and SLT-producing E. coli.     

Human Stx2-Specific Monoclonal Antibodies Prevent Systemic Complications of Escherichia coli O157:H7 Infection

Hemolytic-uremic syndrome (HUS) is a serious complication predominantly associated with infection by enterohemorrhagic Escherichia coli (EHEC), such as E. coli O157:H7. EHEC can produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), both of which are exotoxins comprised of active (A) and binding (B) subunits. In piglets and mice, Stx can induce fatal neurological symptoms. Polyclonal Stx2 antiserum can prevent these effects in piglets infected with the Stx2-producing E. coli O157:H7 strain 86-24. Human monoclonal antibodies (HuMAbs) against Stx2 were developed as potential passive immunotherapeutic reagents for the prevention and/or treatment of HUS. Transgenic mice bearing unrearranged human immunoglobulin (Ig) heavy and kappa light chain loci (HuMAb___Mouse) were immunized with formalin-inactivated Stx2. Thirty-seven stable hybridomas secreting Stx2-specific HuMAbs were isolated: 33 IgG1kappa A-subunit-specific and 3 IgG1kappa and 1 IgG3kappa B-subunit-specific antibodies. Six IgG1kappa A-subunit-specific (1G3, 2F10, 3E9, 4H9, 5A4, and 5C12) and two IgG1kappa B-subunit-specific (5H8 and 6G3) HuMAbs demonstrated neutralization of > 95% activity of 1 ng of Stx2 in the presence of 0.04 microg of HuMAb in vitro and significant prolongation of survival of mice given 50 microg of HuMAb intraperitoneally (i.p.) and 25 ng of Stx2 intravenously. When administered i.p. to gnotobiotic piglets 6 or 12 h after infection with E. coli O157:H7 strain 86-24, HuMAbs 2F10, 3E9, 5H8, and 5C12 prolonged survival and prevented development of fatal neurological signs and cerebral lesions. The Stx2-neutralizing ability of these HuMAbs could potentially be used clinically to passively protect against HUS development in individuals infected with Stx-producing bacteria, including E. coli O157:H7.     

Enterohaemorrhagic E. coli in veterinary medicine

The emergence of enterohaemorrhagic Escherichia coli (EHEC) as major human pathogens began with the identification of serotype O157:H7 in the early 1980s as the cause of various food-borne outbreaks of severe intestinal disease. The key virulence factors include verocytotoxins (Vt) and effectors and adhesins associated with type III secretion systems. Tracing the origins of human outbreaks reveals that the primary source of this organism is the ruminant gastro-intestinal tract and a variety of transmission routes to humans have been identified. The epidemiology of E. coli O157:H7 within cattle and other ruminants has been studied extensively and the prevalence of non-O157:H7 serotypes contrasts with the observed dominance of E. coli O157:H7 amongst human EHEC isolates. Although there is some evidence that EHEC cause disease in young animals, the high prevalence of Vt within healthy ruminants suggests that this is not a virulence factor within these species. An understanding of the mechanisms underpinning EHEC persistence within their natural reservoir hosts and the development of a molecular understanding of EHEC biology and evolution could eventually allow a reduction in the incidence of human disease and may reduce future threats. The use of animal models to replicate and study human EHEC pathogenesis is described.     

Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 is a recently recognized human pathogen associated with hemorrhagic colitis. Unlike most E. coli strains, E. coli O157:H7 does not ferment sorbitol. Therefore, the efficacy of MacConkey agar containing sorbitol (SMAC medium) instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined in comparison with MacConkey agar. The relative frequency of non-sorbitol-fermenting (NSF) organisms other than E. coli O157:H7 in feces was low at 10 to 20% (95% confidence limits), and NSF organisms also occurred mostly in small numbers. In a field trial involving over 1,000 diarrheal stools, E. coli O157:H7 was isolated from 18 stools, all of which were from patients with bloody diarrhea. In every instance, the growth of E. coli O157:H7 on SMAC medium was heavy and occurred in almost pure culture as colorless NSF colonies in contrast to fecal flora, which are mostly sorbitol fermenting and hence appear pink on this medium, whereas on MacConkey agar cultures, the growth of E. coli O157:H7 was indistinguishable from fecal flora. SMAC medium permitted ready recognition of E. coli O157:H7 in stool cultures. Detection of E. coli O157:H7 on SMAC medium had a sensitivity of 100%, a specificity of 85%, and an accuracy of 86%. SMAC medium stool culture is a simple, inexpensive, rapid, and reliable means of detecting E. coli O157:H7, and we recommend routine use of SMAC medium especially for culturing bloody stools.