Indirect hemagglutination assay for diagnosis of Escherichia coli O157 infection in patients with hemolytic-uremic syndrome.

An indirect hemagglutination assay consisting of sheep erythrocytes coated with lipopolysaccharide (LPS) from Shiga-like toxin-producing Escherichia coli O157 was used for the serological diagnosis of E. coli O157 infections in children with classical (enteropathic) hemolytic-uremic syndrome (HUS). One week after the onset of diarrhea (acute phase of the disease), the E. coli O157 antibody titer was greater than or equal to 1:4,096 in 22 of 27 patients with HUS, compared with 4 of 249 controls, the majority of whom had O157 antibody titers of between 1:4 and 1:256. This antibody response was observed in HUS patients with stool cultures positive and negative for E. coli O157. Selective absorption with homologous LPS and heterologous LPS showed that the antibody response was specific for E. coli O157. Because of its simplicity and ease of interpretation, the indirect hemagglutination assay described in this paper is recommended for the serological diagnosis of E. coli O157 infections in patients with HUS.     

Vero cytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes

Faecal specimens from 66 children with haemolytic uraemic syndrome in the United Kingdom were examined for strains of Escherichia coli producing Vero cytotoxin (VT). Initially, conventional bacteriological methods were used to identify colonies of E. coli which were then tested for VT production. Subsequently, specific DNA probes for VT1 and VT2 were used in hybridisation tests to detect VT-producing E. coli (VTEC). VTEC strains were isolated from 19 cases and in 15 they belonged to serogroup O157. Fourteen of these O157 strains possessed the flagellar antigen H7 and one was non-motile. The VTEC strains from the remaining four cases belonged to serotypes O26:H11, O104:H2, O153:H25, and O163:H19 together with a rough VT+ strain with flagellar antigen H51. The O157 strains hybridised with either the VT2 probe or both VT1 and VT2 probes. The other VTEC strains hybridised with either the VT1 or VT2 probe. Confirmation of the production of VT1 and VT2 in vivo was obtained by the neutralisation of faecal VT with specific antisera raised against these two cytotoxins.     

Neutralizing antibodies to Escherichia coli Vero cytotoxin 1 and antibodies to O157 lipopolysaccharide in healthy farm family members and urban residents.

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Escherichia coli O157 lipopolysaccharide (LPS) was developed with sera from 63 children with confirmed recent E. coli O157 infection and from 256 age-stratified urban controls. The median ELISA values for control and case sera were 0.05 (interquartile range, 0 to 0.20; mean +/- standard deviation [SD], 0.15 +/- 0.22) and 1.41 (interquartile range, 1.11 to 1.59; mean +/- SD, 1.41 +/- 0.53), respectively (P < 0.001). With a breakpoint of 0.59 (mean ELISA value of the control sera + 2 SDs), the assay had a sensitivity, specificity, and positive and negative predictive values of 95, 94, 80, and 98%, respectively, for recent E. coli O157 infection. The O157 LPS assay and Vero cytotoxin (VT) 1-neutralizing-antibody (NAb) assay were used to compare the relative frequencies of O157 LPS antibodies and VT1-NAbs in an age-stratified urban population from Toronto, Ontario, Canada, and in 216 healthy family members from dairy farm in southern Ontario. The frequency of O157 LPS antibodies was about threefold higher in dairy farm residents (12.5%) than in urban residents (4.7%) (P < 0.01). Similarly, the frequency of VT1-NAbs was about sixfold higher in dairy farm residents (42.0%) than in urban residents (7.7%) (P < 0.001). These findings are consistent with a greater level of exposure of dairy farm residents to VT-producing E. coli (VTEC) strains. The high rate of seropositivity to VT1 in farm residents probably reflects the booster effect of repeated VTEC exposures and argues against a sustained generalized immunosuppressive effect of VT1. Seroepidemiological studies may help in assessing the level of exposure of different populations to VTEC strains.     

Antibodies to Escherichia coli O157 in patients with haemorrhagic colitis and haemolytic uraemic syndrome.

Twenty four sera from patients with haemolytic uraemic syndrome or haemorrhagic colitis and healthy controls were examined for antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157. Faecal specimens from these patients were also examined for Vero cytotoxin producing E coli (VTEC) by DNA probes, and for faecal Vero cytotoxin. Eight patients with faecal E coli O157:H7 gave a strong antibody response to O157 LPS, shown by immunoblotting and an enzyme linked immunosorbent assay. Six symptomatic patients without evidence of faecal VTEC also gave a strong antibody response to O157 LPS. Sera from the remaining five patients and five healthy controls did not contain antibodies to E coli O157. The results suggest that the testing of sera from patients with haemorrhagic colitis or haemolytic uraemic syndrome by ELISA or immunoblot would prove valuable in addition to the established procedures for detecting VTEC, using DNA probes and testing for faecal Vero cytotoxin.     

Serum antibodies to Escherichia coli serotype O157:H7 in patients with hemolytic uremic syndrome

Sera from 13 patients with hemolytic uremic syndrome (HUS) and 8 healthy control subjects were examined for antibodies specific for bacterial antigens of Eschericia coli serotype O157:H7. Bacterial components, including outer membrane proteins (OMPs), lipopolysaccharide (LPS), and flagella, were reacted with sera by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting and by enzyme-linked immunosorbent assay. All 13 serum samples from HUS patients contained high-titered antibodies of the immunoglobulin M class against O157 LPS and some OMPs. These same sera reacted weakly with some of the major OMPs, but not the LPS, of non-O157 strains of E. coli. Sera from patients did not contain antibodies to non-O157 LPS or H7 flagella. The possibility of using E. coli serotype O157 LPS in an enzyme-linked immunosorbent assay for the routine diagnostic testing of sera from HUS patients for evidence of O157:H7 infection is discussed.     

Experimental infection of infant rabbits with verotoxin-producing Escherichia coli.

To study the pathogenesis of diarrheal disease due to verotoxin (VT)-producing Escherichia coli, 3-day-old rabbits were inoculated intragastrically with live E. coli O157:H7 (high VT producer), E. coli O113:K75:H21 (low VT producer), or O157:H45 (VT negative) and were examined for clinical symptoms, bacterial colonization, presence of detectable free VT in the intestines, and histological changes. Diarrhea developed consistently with 10(8) bacteria of E. coli O157:H7 but was observed only infrequently with even a higher dose of E. coli O113:K75:H21. VT-negative strains failed to cause diarrhea under the same experimental conditions. E. coli O157:H7 was recovered from the colon of infected animals in a significantly higher concentration than from the small intestine, and the clinical symptoms correlated with the presence of detectable free VT in the colon. Histological changes were seen mainly in the mid- and distal colon; these changes were characterized by a vast increase in apoptosis in the surface epithelium, increased mitotic activity in the crypts, mucin depletion, and a mild to moderate infiltration of neutrophils in the lamina propria and epithelium. Multiple foci of attached bacteria were seen on the surface epithelium of the gut-associated lymphoid tissue, cecum, and colon. Bacteria were never seen in epithelial cells or the lamina propria. These mucosal abnormalities as well as clinical symptoms were reproduced in infant rabbits by the intragastric administration of VT alone. These results are consistent with the hypothesis that VT plays a major role in the pathogenesis of diarrhea caused by E. coli O157:H7 and other VT-producing E. coli.     

Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome

Ninety-five household contacts (aged 2 months to 73 years) of patients with enteropathic hemolytic-uremic syndrome (HUS) were investigated for the presence of immunoglobulin (Ig) G antibodies to Shiga toxins Stx2 and Stx1 by Western blot assay. Thirty-one percent of the household contacts and 19% of 327 controls had anti-Stx2 IgG (heavy and light chain [H + L]), 5 and 8%, respectively, had anti-Stx1 IgG (H + L), and 3 and 2%, respectively, had both anti-Stx2 and anti-Stx1 IgG (H + L). The incidence of infections with Stx-producing Escherichia coli (STEC) was determined based on the following diagnostic criteria: STEC isolation, detection of stx gene sequences, free fecal Stx in stool filtrates, and serum IgM antibodies against E. coli O157 lipopolysaccharide. Evidence of STEC infection was observed in 25 household contacts, of whom 18 (72%) were asymptomatic and represented a potential source of infection. Six of 13 (46%) household contacts with Stx2-producing E. coli O157:H7 in stool culture developed anti-Stx2 IgG (H + L), compared to 71% of Stx2-associated HUS cases. In individuals showing anti-Stx2 IgG (H + L), the antibody response was directed against the B subunit in 69% of household contacts and 71% of controls, in contrast to 28% of HUS patients. In this investigation controls had a significant increase of the median of IgM antibodies to O157 lipopolysaccharide (LPS) with age, up to the fifth decade. The lack of disease in household contacts with B subunit-specific antibodies, as well as the significantly higher median of anti-O157 LPS IgM antibodies in controls beyond 4.9 years of age, suggests a protective role for anti-Stx and anti-O157 LPS antibodies.     

Escherichia coli O157:H7 and O157:H− Strains That Do Not Produce Shiga Toxin: Phenotypic and Genetic Characterization of Isolates Associated with Diarrhea and Hemolytic-Uremic Syndrome

We have isolated one sorbitol-nonfermenting (SNF) Escherichia coli O157:H7 isolate and five sorbitol-fermenting (SF) E. coli O157:H(-) isolates that do not contain Shiga toxin (Stx) genes (stx). Isolates originated from patients with diarrhea (n = 4) and hemolytic-uremic syndrome (HUS) (n = 2). All isolates harbored a chromosomal eae gene encoding gamma-intimin as well as the plasmid genes E-hly and etp. The E. coli O157:H7 isolate was katP and espP positive. Respective sera obtained from the patient with HUS contained antibodies to the O157 lipopolysaccharide antigen. The stx-negative E. coli O157:H7 isolate is genetically related to stx-positive SNF E. coli O157:H7. All stx-negative SF E. coli O157:H(-) isolates belong to the same genetic cluster and are closely related to stx-positive SF E. coli O157:H(-) isolates. Our data indicate that stx-negative E. coli O157:H7/H(-) variants may occur at a low frequency and cannot be recognized by diagnostic methods that target Stx.     

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7.

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.     

Production and characterization of monoclonal antibodies specific for the lipopolysaccharide of Escherichia coli O157.

Identification of the O157 antigen is an essential part of the detection of Escherichia coli O157:H7, which is recognized as a major etiologic agent of hemorrhagic colitis. However, polyclonal antibodies produced against E. coli O157:H7 lipopolysaccharide (LPS) may react with several other bacteria including Brucella abortus, Brucella melitensis, Yersinia enterocolitica O9, Escherichia hermannii, and Stenotrophomonas maltophilia. We produced eight monoclonal antibodies (MAbs) specific for the LPS of E. coli O157. Western blots (immunoblots) of both the phenol phase (smooth) and the aqueous phase (rough) of hot phenol-water-purified LPS indicated that three of the MAbs were specific for the O antigen and five were reactive with the LPS core. The eight MAbs could be further differentiated by their reactivities to Salmonella O30 LPS (group N), which is reported to be identical to the E. coli O157 antigen. All eight MAbs reacted strongly to all of the 64 strains of E. coli O157 tested, which included 47 isolates of O157:H7 and 17 other O157 strains. None of the eight MAbs cross-reacted with any of the 38 other E. coli serotypes tested, which consisted of 29 different O-antigen serotypes, or with 38 strains (22 genera) of non-E. coli gram-negative enteric bacteria.     

The kinetics of antibody production to antigens of Escherichia coli O157 in a pregnant woman with haemolytic uraemic syndrome

Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E. coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M). Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens. In the mother, antibodies of the IgM class, specific for E. coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later. High levels of IgG class antibodies, specific for E. coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18. Serum antibodies of the IgA class to E. coli O157 LPS were not detected. Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18. Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood. This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E. coli O157.     

Prothrombotic coagulation abnormalities preceding the hemolytic-uremic syndrome.

BACKGROUND: The hemolytic-uremic syndrome is a thrombotic complication of Escherichia coli O157:H7 infection. It is not known whether the coagulation abnormalities precede, and potentially cause, this disorder. METHODS: In 53 children infected with E. coli O157:H7, we measured a panel of markers indicating activation of the clotting cascade and renal function within four days after the onset of illness. These markers were measured again in as many as possible of the 16 children in whom the hemolytic-uremic syndrome developed. RESULTS: The children in whom the hemolytic-uremic syndrome subsequently developed had significantly higher median plasma concentrations of prothrombin fragment 1+2, tissue plasminogen activator (t-PA) antigen, t-PA-plasminogen-activator inhibitor type 1 (PAI-1) complex, and D-dimer than children with uncomplicated infection. These abnormalities preceded the development of azotemia and thrombocytopenia. When the hemolytic-uremic syndrome developed, the urinary concentrations of beta2-microglobulin and N-acetyl-beta-glucosaminidase rose significantly (P=0.03 for both increases); the plasma concentrations of t-PA antigen, t-PA-PAI-1 complex, D-dimer, and plasmin-antiplasmin complex also increased significantly. The concentration of t-PA antigen correlated with that of the t-PA-PAI-1 complex in a linear regression model (squared correlation coefficient, 0.80; P<0.001). CONCLUSIONS: In the hemolytic-uremic syndrome, thrombin generation (probably due to accelerated thrombogenesis) and inhibition of fibrinolysis precede renal injury and may be the cause of such injury.     

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-.     

Mucosal and systemic antibody responses to the lipopolysaccharide of Escherichia coli O157 in health and disease

Mucosal immunity in the gastrointestinal (GI) tract is a primary defence against GI pathogens. We hypothesise that a mucosal response to lipopolysaccharide (LPS), especially to the common (core) determinants of GI pathogenic Escherichia coli strains, is protective. The aims of this study were to investigate the specificities, levels and development of humoral responses in health and GI disease to the R3 LPS core and O-polysaccharide of E. coli O157. The purpose was to try to predict whether vaccination or passive immunisation might induce protection. Wherever possible, paired whole gut lavage fluid (WGLF) and serum samples were collected for comparison of the mucosal and systemic responses. Matched saliva samples were also collected from some study groups. The patient groups included those with acute E. coli O157 disease (serum only), patients convalescing after E. coli O157 infections, and patients undergoing routine investigation for GI conditions but subsequently shown to be immunologically normal. Some samples of WGLF from patients with Crohn's disease (CRO) and ulcerative colitis (UC) were included to allow comparisons with patients with inflammatory conditions known to alter antibody secretion in the GI tract. The healthy groups from whom serum and saliva only were taken included blood donors, healthy volunteers and a group of slaughterhouse workers. This latter group was likely to have been exposed regularly to faecal bacteria from animals and antibody specificities might have been expected to be different from other healthy individuals. Levels and classes of antibodies were determined by ELISA with microtitration plates coated with polymyxin complexes of whole LPS extracted from E. coli O157 and LPS from the E. coli R3 rough mutant. Antibodies of IgG and IgM classes were measured in serum and IgA was measured in WGLF and saliva. IgG antibodies to the O157 LPS and the R3 core oligosaccharide were detected in the serum of healthy blood donors. Patients with acute E. coli O157 disease showed elevated levels of serum IgM to O157 LPS and R3, with IgG levels raised only to R3. In serum from convalescent patients, IgG to O157 LPS was significantly above the control groups only in the period 6-16 weeks after infection. Total IgA levels were similar in WGLF specimens from all groups, except the patients with UC, whose levels were much higher. Specific IgA levels were higher in the E. coli O157 convalescent group, but there were no significant correlations overall. UC patients had significantly lower levels of IgA to O157 and CRO patients had higher O157 IgA levels than UC patients and healthy volunteers. In serum, inhibition of ELISA showed that the response to the O157 LPS was due in part to a response to the R3 oligosaccharide component. This response was much more pronounced in the healthy and non-O157 groups than in convalescent patients. There was no correlation between specific IgA antibody levels in saliva and matched specimens of WGLF, and levels in sequential saliva specimens fluctuated widely. The significant IgG and IgA responses to the R3 core suggest that there is immunological memory to this oligosaccharide LPS component which may have a role in protection against E. coli LPS both systemically and locally in the GI tract. Boosting of this mucosal response to the LPS core, either naturally through exposure or by active or passive immunisation, may confer protection. Finally, antibody responses to E. coli O157 must be interpreted with caution, as the response detected is a sum of responses to the O-specific polysaccharide and the R3 core.     

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.     

Serum antibodies to verotoxin-producing Escherichia coli (VTEC) strains in patients with haemolytic uraemic syndrome.

Serological evidence of infection with verotoxin-producing Escherichia coli (VTEC) was sought in 28 patients suffering from haemolytic uraemic syndrome (HUS) and 25 age- and sex-matched controls. ELISA was used to detect anti-lipopolysaccharide (LPS) antibodies to E. coli strains O157, O111, O26 and NCTC 10418, a non-VTEC strain, and Shigella dysenteriae O1. Sera from 19 of the HUS patients but from none of the 25 controls had significant antibody levels to the verotoxin-producing bacteria. Sera from 13 patients reacted with only one LPS of the four verotoxin-producing bacteria; sera from six reacted with more than one LPS antigen but not with LPS of E. coli NCTC 10418. Paired sera taken 2-3 weeks apart were obtained from 20 HUS patients; 14 of these had high levels of antibody in the acute phase sample. Analysis of antibody levels in the convalescent sera showed that one patient had an increase, one was unchanged and 12 patients had a decrease in antibody to the verotoxin-producing bacteria.     

Direct detection of verotoxin genes in stool samples by polymerase chain reaction in hemolytic uremic syndrome patients in France

Objective: To reassess the occurrence of verocytotoxin-producing Escherichia coli (VTEC) in French hemolytic uremic syndrome (HUS) patients.
Method: From March 1991 to January 1995, direct detection of verotoxin genes (VT) by the polymerase chain reaction (PCR) was performed on stool samples from 169 patients suffering from HUS.
Results: Fifty-one were PCR positive (30.1%); one was positive for the VT1 gene and the others for the VT2 gene. VTEC was isolated from only 32 of the 51 PCR-positive samples. E. coli O157:H7 was isolated from five patients. E. coli O111 was isolated from seven patients during an outbreak of HUS. Among the other VT2 E. coli strains, only four were serotypable. Of the 51 PCR-positive stools, 19 were culture negative for VTEC.
Conclusions: This study provides evidence that in France E. coli O157 and other VTEC serotypes are involved in HUS.     

Purified verotoxins of Escherichia coli O157:H7 decrease prostacyclin synthesis by endothelial cells

Two immunologically distinct verotoxins purified from Escherichia coli C600, lysogenized with distinct temperate phages from E. coli strain 933 of serotype O157:H7, were compared by SDS-PAGE and different biological assays. The two toxins termed verotoxin 1 (VT1) and verotoxin 2 (VT2) differing in molecular weight exhibited similar biological activities. Both preparations were toxic for HeLa cells and lethal for mice. Epidemiological evidence of verotoxinogenesis in some cases of hemolytic-uremic syndrome (HUS) and the recent observations of inadequate prostacyclin production by endothelial cells associated with HUS prompted us to study the effect of purified verotoxins on prostacyclin synthesis in rat aortic tissue. Our results demonstrate a significant reduction of prostacyclin by both toxins at picomolar levels. The suppression of prostacyclin release by a lower concentration of VT2 as compared with VT1 reflects the relative potencies of these toxins in HeLa cell toxicity and mouse lethality. The results suggest an effect of verotoxins on endothelial cells and support the concept of these toxins as virulence factors in E. coli.     

Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H(-)

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H(-), pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H(-) strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H(-).     

Microbiological aspects of infection with verotoxin-producing E. coli strains in children with the hemolytic-uremic syndrome

The authors examined the faeces of five children with haemolyticuraemic syndrome for the presence of E. coli producing verotoxin (VTEC) and free verotoxin (VT). For detection of strains of serotype O157:H7 they used sorbitol MacConkey agar in combination with biochemical tests and typing of sorbitol negative strains. For detection of strains belonging to the other VTEC serogroups they used serotyping of 24 E. coli colonies from End media. VT was assessed in lysates and supernatants of cultures, on cell cultures of Vero cells, and the antigenic VT type was assessed by neutralization experiments. Strains corresponding as to serotype or O group to VTEC were detected in faeces of all five children. Two strains belonged to the serotype O157:H7 and had a typical biotype; another four strains belonged to serogroups 026 (2 strains), 05 and 01. All strains produced verotoxin, either VT1 or VT2 or both. In the faecal filtrates of all five children free VT was present. In conjunction with the haemolytic-uraemic syndrome in children in the CSSR E. coli producing verotoxin are found, incl. strains of serotype O157:H7, the detection of which was not described hitherto.