Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves.

Cattle are an important reservoir of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains, foodborne pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome in humans. EHEC O157:H7 strains are not pathogenic in calves >3 weeks old. Our objective was to determine if EHEC O157:H7 strains are pathogenic in neonatal calves. Calves <36 h old inoculated with EHEC O157:H7 developed diarrhea and enterocolitis with attaching and effacing (A/E) lesions in both the large and small intestines by 18 h postinoculation. The severity of diarrhea and inflammation, and also the frequency and extent of A/E lesions, increased by 3 days postinoculation. We conclude that EHEC O157:H7 strains are pathogenic in neonatal calves. The neonatal calf model is relevant for studying the pathogenesis of EHEC O157:H7 infections in cattle. It should also be useful for identifying ways to reduce EHEC O157:H7 infections in cattle and thus reduce the risk of EHEC O157:H7 disease in humans.     

Detection of toxB, a plasmid virulence gene of Escherichia coli O157, in enterohemorrhagic and enteropathogenic E. coli

The virulence plasmid of Escherichia coli O157 strain EDL933 carries a 10-kb putative virulence gene designated toxB. Little is known about the distribution of this gene among E. coli O157 strains or its presence in other enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) strains. We developed PCR and hybridization tools for the detection of the entire toxB sequence and investigated its presence in a collection of EHEC O157 strains and other EHEC and EPEC strains belonging to different serogroups and isolated from different sources. The EHEC O157 strains reacted with all of the PCR primers and probes used, thus indicating the presence of a complete toxB gene regardless of the human or bovine origin of the isolates. Similar positive reactions were observed for about 50% of the EHEC O26 strains tested and a few other EHEC and EPEC strains. However, the size of the DNA fragments hybridizing with the toxB probes differed from that of the positive fragments from EHEC O157, suggesting a polymorphism in the toxB genes present in the different E. coli serogroups. Moreover, several EHEC and EPEC strains belonging to different serogroups reacted with only some of the genetic tools used, suggesting either the existence of major variants of toxB or the presence of fragments of the gene. Southern blotting analysis showed that toxB sequences were located on large plasmids in EHEC and EPEC O26 as well.     

The established intimin receptor Tir and the putative eucaryotic intimin receptors nucleolin and beta1 integrin localize at or near the site of enterohemorrhagic Escherichia coli O157:H7 adherence to enterocytes in vivo

For enterohemorrhagic Escherichia coli (EHEC) O157:H7 to adhere tightly to the intestinal epithelium and produce attach and efface (A/E) lesions, the organism must express the adhesin intimin and insert the bacterially encoded translocated intimin receptor Tir into the plasma membrane of the host enterocyte. Additionally, some reports based on tissue culture experiments indicate that intimin has affinity for the eucaryotic proteins nucleolin and beta1 integrin. To address the potential biological relevance of these eucaryotic proteins in the infection process in vivo, we sought to compare the proximity of Tir, nucleolin, and beta1 integrin to regions of EHEC O157:H7 attachment in intestinal sections from three different inoculated animals: piglets, neonatal calves, and mice. Piglets and neonatal calves were chosen because intimin-mediated adherence of EHEC O157:H7 and subsequent A/E lesion formation occur at high levels in these animals. Mice were selected because of their ease of manipulation but only after we first demonstrated that in competition with the normal mouse gut flora, an EHEC O157:H7 strain with a nonpolar deletion in the intimin gene was cleared faster than strains that produced wild-type or hybrid intimin. In all three animal species, we noted immunostained Tir beneath and stained nucleolin closely associated with adherent bacteria in intestinal sections. We also observed immunostained beta1 integrin clustered at locations of bacterial adherence in porcine and bovine tissue. These findings indicate that nucleolin and beta1 integrin are present on the luminal surface of intestinal epithelia and are potentially accessible as receptors for intimin during EHEC O157:H7 infection.     

The Cloned Locus of Enterocyte Effacement from Enterohemorrhagic Escherichia coli O157:H7 Is Unable To Confer the Attaching and Effacing Phenotype upon E. coli K-12

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic Escherichia coli (EHEC) O157:H7 possesses the same genes in identical order and orientation as the LEE of enteropathogenic E. coli (EPEC) O127:H6 but is unable to form attaching and effacing (A/E) lesions or to secrete Esp proteins when it is cloned in an E. coli K-12 background. The A/E phenotype could not be restored by trans complementation with a variety of cloned EPEC LEE fragments, suggesting functional and/or regulatory differences between the LEE pathogenicity islands of EPEC O127:H6 and EHEC O157:H7.     

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.     

Cloning, expression, and characterization of fimbrial operon F9 from enterohemorrhagic Escherichia coli O157:H7

Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.     

Attaching and effacing lesions caused by Escherichia coli O157:H7 in experimentally inoculated neonatal lambs

Four 6-day-old conventionally reared lambs were inoculated orally with a total of 10(9) cfu comprising equal numbers of four enterohaemorrhagic Escherichia coli (EHEC) O157:H7 strains. All animals remained clinically normal. Tissues were sampled under terminal anaesthesia at 12, 36, 60 and 84 h post inoculation (hpi). EHEC O157:H7 was cultured from most gastrointestinal tract sites. Small, sparse attaching and effacing (AE) lesions were found in the caecum at 12 and 36 hpi and in the terminal colon and rectum at 84 hpi. Organisms in the lesions were labelled specifically by an O157 antiserum. The results indicate that the well-characterised mechanisms for intimate attachment encoded by the locus for enterocyte effacement (LEE) of EHEC O157:H7 may contribute to the initial events, at least, of colonisation of sheep.     

Rectoanal mucosal swab culture is more sensitive than fecal culture and distinguishes Escherichia coli O157:H7-colonized cattle and those transiently shedding the same organism

Enrichment and direct (nonenrichment) rectoanal mucosal swab (RAMS) culture techniques were developed and compared to traditional fecal culture for the detection of Escherichia coli O157:H7 in experimentally infected and naturally infected cattle. Holstein steers (n = 16) orally dosed with E. coli O157:H7 were sampled after bacterial colonization starting 15 days postinoculation. Enrichment RAMS cultures (70.31% positive) were more sensitive than enrichment fecal cultures with 10 g of feces (46.88% positive) at detecting E. coli O157:H7 (P < 0.01). Holstein bull calves (n = 15) were experimentally exposed to E. coli O157:H7 by penning them with E. coli O157:H7-positive calves. Prior to bacterial colonization (1 to 14 days postexposure), enriched fecal cultures were more sensitive at detecting E. coli O157:H7 than enriched RAMS cultures (P < 0.01). However, after colonization (40 or more days postexposure), the opposite was true and RAMS culture was more sensitive than fecal culture (P < 0.05). Among naturally infected heifers, enriched RAMS or fecal cultures were equally sensitive (P = 0.5), but direct RAMS cultures were more sensitive than either direct or enriched fecal cultures at detecting E. coli O157:H7 (P < 0.01), with 25 of 144, 4 of 144, and 10 of 108 samples, respectively, being culture positive. For both experimentally and naturally infected cattle, RAMS culture predicted the duration of infection. Cattle transiently shedding E. coli O157:H7 for <1 week were positive by fecal culture only and not by RAMS culture, whereas colonized animals (which were culture positive for an average of 26 days) were positive early on by RAMS culture. RAMS culture more directly measured the relationship between cattle and E. coli O157:H7 infection than fecal culture.     

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.     

Invasion of epithelial cells by locus of enterocyte effacement-negative enterohemorrhagic Escherichia coli

The majority of enterohemorrhagic Escherichia coli (EHEC) strains associated with severe disease carry the locus of enterocyte effacement (LEE) pathogenicity island, which encodes the ability to induce attaching and effacing lesions on the host intestinal mucosa. While LEE is essential for colonization of the host in these pathogens, strains of EHEC that do not carry LEE are regularly isolated from patients with severe disease, although little is known about the way these organisms interact with the host epithelium. In this study, we compared the adherence properties of clinical isolates of LEE-negative EHEC with those of LEE-positive EHEC O157:H7. Transmission electron microscopy revealed that LEE-negative EHEC O113:H21 was internalized by Chinese hamster ovary (CHO-K1) epithelial cells and that intracellular bacteria were located within a membrane-bound vacuole. In contrast, EHEC O157:H7 remained extracellular and intimately attached to the epithelial cell surface. Quantitative gentamicin protection assays confirmed that EHEC O113:H21 was invasive and also showed that several other serogroups of LEE-negative EHEC were internalized by CHO-K1 cells. Invasion by EHEC O113:H21 was significantly reduced in the presence of the cytoskeletal inhibitors cytochalasin D and colchicine and the pan-Rho GTPase inhibitor compactin, whereas the tyrosine kinase inhibitor genistein had no significant impact on bacterial invasion. In addition, we found that EHEC O113:H21 was invasive for the human colonic cell lines HCT-8 and Caco-2. Overall these studies suggest that isolates of LEE-negative EHEC may employ a mechanism of host cell invasion to colonize the intestinal mucosa.     

toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell adherence phenotype

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is critical for initiation of a bacterial infection. An in vitro infection study previously indicated that EHEC bacteria initially adhere diffusely and then proliferate to develop MC, a process that is mediated by various secreted proteins, such as EspA, EspB, EspD, Tir, and intimin, as well as other putative adherence factors. In the present study, we investigated the role of a large 93-kb plasmid (pO157) in the adherence of O157:H7 (O157Sakai) and found the toxB gene to be involved in the full adherence phenotype. A pO157-cured strain of O157Sakai (O157Cu) developed microcolonies on Caco-2 cells; however, the number of microcolonies was lower than that of O157Sakai, as were the production and secretion levels of EspA, EspB, and Tir. Introduction of a mini-pO157 plasmid (pIC37) composed of the toxB and ori regions restored full adherence capacity to O157Cu, including production and secretion of the proteins. In contrast, introduction of a pO157 mutant possessing toxB::Km into O157Cu could not restore the full adherence phenotype. Expression of truncated versions of His-tagged ToxB also promoted EspB production and/or secretion by O157Cu. These results suggest that ToxB contributes to the adherence of EHEC to epithelial cells through promotion of the production and/or secretion of type III secreted proteins.     

The role of intimin in the adherence of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 to HEp-2 tissue culture cells and to bovine gut explant tissues

Intimin, an outer membrane protein encoded by eaeA, is a key determinant for the formation of attaching and effacing (AE) lesions by enterohaemorrhagic Escherichia coli (EHEC). To investigate the role of intimin in adherence, the eaeA gene was insertionally inactivated in three EHEC O157:H7 strains of diverse origin. The absence or presence of intimin did not correlate with the extent of adhesion of mutant or wild-type O157:H7 in tissue culture and neonatal calf gut tissue explant adherence assays. Adherence of the eaeA mutants to HEp-2 cells was diffuse with no evidence of intimate attachment whereas wild-type bacteria formed microcolonies and AE lesions. Intimin-independent adherence to neonatal calf gut explants was demonstrated by eaeA mutants and wild-type strains which adhered in the greatest numbers to colon but least well to rumen tissue. These results confirm that intimin is necessary for intimate attachment and that additional adherence factors are involved in intimin-independent adherence.     

Decreased adherence of enterohemorrhagic Escherichia coli to HEp-2 cells in the presence of antibodies that recognize the C-terminal region of intimin

Antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intimin(O157)) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intimin(O157) serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intimin(O157) antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intimin(O157) could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC.     

Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-

The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H- strain 493/89, each of 107 SF EHEC O157:H- clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H- strains which contained eae, which encodes gamma-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H-. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H- strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H-.     

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.     

Identification and characterization of lpfABCC'DE,a fimbrial operon of enterohemorrhagic Escherichia coli O157:H7

The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC'DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC'DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim(-)) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation.     

A mosaic pathogenicity island made up of the locus of enterocyte effacement and a pathogenicity island of Escherichia coli O157:H7 is frequently present in attaching and effacing E. coli

Enteropathogenic Escherichia coli (EPEC) and enterohemorragic E. coli (EHEC) possess a pathogenicity island (PAI), termed the locus of enterocyte effacement (LEE), which confers the capability to cause the characteristic attaching and effacing lesions of the brush border. Due to this common property, these organisms are also termed attaching and effacing E. coli (AEEC). Sequencing of the EHEC O157 genome recently revealed the presence of other putative PAIs in the chromosome of this organism. In this article, we report on the presence of four of those PAIs in a panel of 133 E. coli strains belonging to different pathogroups and serotypes. One of these PAIs, termed O122 in strain EDL 933 and SpLE3 in strain Sakai, was observed in most of the AEEC strains examined but not in the other groups of E. coli. It was also found to contain the virulence-associated gene efa1/lifA. In EHEC O157, PAI O122 is located 0.7 Mb away from the LEE. Conversely, we demonstrated that in many EHEC non-O157 strains and EPEC strains belonging to eight serogroups, PAI O122 and the LEE are physically linked to form a cointegrated structure. This structure can be considered a mosaic PAI that could have been acquired originally by AEEC. In some clones, such as EHEC O157, the LEE-O122 mosaic PAI might have undergone recombinational events, resulting in the insertion of the portion referred to as PAI O122 in a different location.     

Distribution of the urease gene cluster among and urease activities of enterohemorrhagic Escherichia coli O157 isolates from humans

Enterohemorrhagic Escherichia coli (EHEC) O157 strains belong to two closely related major groups, which are differentiated by their sorbitol fermentation phenotypes. Here we studied the conservation of urease genes and their expression in sorbitol-fermenting (SF) and non-SF EHEC O157 isolates. PCR targeting ure genes (ureA, -B, -C, -D, -E, -F, and -G) demonstrated that each of these genes was present in 58 of 59 EHEC O157:H7 isolates. In contrast, none of 82 SF EHEC O157:NM (nonmotile) isolates contained any of the ure genes. Hence, the absence of the urease genes distinguishes SF EHEC O157:NM strains from EHEC O157:H7, but this absence demonstrates that the urease genes are not useful genetic targets for the detection of EHEC strains, because SF EHEC O157:NM strains are missed by such a strategy. When examined for urease activity on Christensen agar and in the API 20E system, only one O157:H7 strain displayed urease activity and produced elevated levels of ammonia, which was subsequently confirmed by ammonia electrode measurement. Because the ure genes were absent from each of nine strains of E. coli O55:H7, the proposed progenitor of EHEC O157, we hypothesize that EHEC O157:H7 diverged from the evolutionary pathway at an early stage and then acquired the O islands carrying the ure gene cluster.