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

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

Distinct binding properties of eaeA-negative verocytotoxin-producing Escherichia coli of serotype O113:H21.

Infection of humans with verotoxin-producing Escherichia coli (VTEC) O113:H21 is associated with clinical features comparable to those associated with infection with attaching and effacing VTEC strains including those of serotype O157:H7. We have shown previously that the adhesion phenotype of VTEC O157:H7 is influenced by the presence of a homolog of the chromosomal eaeA (for E. coli attaching and effacing) gene. In contrast, by colony blot hybridization, VTEC O113:H21 is negative for the eaeA gene. Therefore, the aim of this study was to define the adhesion phenotype of VTEC O113:H21 strain CL-15 to both cultured epithelial cells (HEp-2) and rabbit intestine in vivo. Under transmission electron microscopy, areas of microvillus effacement were observed in regions directly beneath the organism in CL-15-infected cells both in vitro and in vivo. However, F-actin adhesion pedestals on the host plasma membrane were absent. Failure of CL-15 to induce polymerization of actin was confirmed by using staining of F-actin with fluorescein-labeled phalloidin. Under indirect immunofluorescence microscopy, CL-15-infected HEp-2 cells also failed to demonstrate the recruitment of another cytoskeletal element, alpha-actinin, below foci of bacterial adhesion. In contrast, VTEC O157:H7 infection of HEp-2 cells was associated with increased alpha-actinin immunofluorescence. These findings suggest that bacterial factors distinct from those of EaeA are necessary for the adhesion phenotype of VTEC O113:H21.     

Signal transduction responses following adhesion of verocytotoxin-producing Escherichia coli.

Attaching and effacing adhesion to epithelial cells is a pathognomonic feature of infection by both enteropathogenic Escherichia coli (EPEC) and certain strains of verocytotoxin-producing E. coli (VTEC). EPEC adhesion to tissue culture epithelial cells results in activation of the phosphatidylinositol pathway, with elevated levels of inositol 1,4,5-triphosphate and cytosolic free calcium. In this report, we show that VTEC also activate this signal transduction pathway in infected epithelial cells. Specifically, increased levels of inositol 1,4,5-triphosphate and intracellular free calcium were observed in HEp-2 cells infected with VTEC of serotype O157:H7. VTEC of serotypes O157:H7 and O113:H21 also induced increases in intracellular calcium levels in the human intestinal crypt-like cell line T84, even with minimal or no attaching and effacing activity as monitored by transmission electron microscopy. In contrast to EPEC, VTEC failed to induce tyrosine phosphorylation of epithelial cell proteins in HEp-2 and T84 cells, as determined by indirect immunofluorescence microscopy. These findings suggest that signal transduction responses to VTEC, including elevated levels of inositol triphosphates and intracellular free calcium, are independent of formation of the attaching and effacing lesion. Our findings also show that VTEC pathogenesis may involve signal transduction pathways that are distinct from those induced by EPEC infection.     

Adherence of Vero cytotoxin-producing Escherichia coli of serotype O157:H7 to human epithelial cells in tissue culture: role of outer membranes as bacterial adhesins

Escherichia coli of serotype O157:H7 are Vero cytotoxin-producing enteric pathogens that have recently been associated with outbreaks of haemorrhagic colitis, sporadic cases of haemorrhagic colitis and with the haemolytic uraemic syndrome. The organisms demonstrate attaching and effacing binding to the caecum and colon of orally infected gnotobiotic piglets, chickens and infant rabbits. E. coli O157:H7 cells adhere to the surface but do not invade the cytoplasm of human epithelial cell lines in tissue culture. Since outer membranes, lipopolysaccharides and flagella have been identified as bacterial adhesins on other enteric pathogens, we evaluated their roles in the binding of non-fimbriated E. coli O157:H7 to HEp-2 cells. Hyperimmune rabbit antisera were prepared to whole cells, outer membranes and flagella of E. coli O157:H7. The presence of antibody to homologous antigen was confirmed by dot blot immunoassays. Both antisera and purified outer membrane and flagellar antigens were co-incubated with bacteria and HEp-2 cells to quantitate inhibition of bacterial attachment. Adherence of E. coli O157:H7 to tissue culture cells was inhibited by rabbit antisera raised to whole cells (76.0 +/- 5.6% inhibition compared with bacterial adherence in the presence of pre-immune rabbit serum) and outer membranes (69.2 +/- 3.4% inhibition). In contrast, inhibition of bacterial attachment to tissue-culture cells was significantly less when two antisera to H7 flagella were co-incubated with E. coli O157:H7 and HEp-2 cells (12.4 +/- 7.6%; 6.0 +/- 3.5% inhibition). Outer-membrane extracts inhibited adherence to E. coli O157:H7 to HEp-2 cells in a concentration dependent manner whereas isolated flagella and lipopolysaccharide antigens did not inhibit bacterial attachment.(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved selective medium for the isolation of Escherichia coli O157

Sorbitol-MacConkey medium has become widely used for the isolation of verotoxigenic (VT+) Escherichia coli O157. However, many organisms other than VT+ E. coli O157, especially other serogroups of E. coli and Proteus spp., may not ferment sorbitol, and thus may be confused initially with VT+ E. coli O157. Rhamnose is not fermented by VT+ E. coli O157, but is by most sorbitol non-fermenting E. coli of other serogroups. Cefixime is a cephalosporin antibiotic that is more active against Proteus spp. than against E. coli. Inclusion of rhamnose and cefixime in sorbitol-MacConkey agar improves its selectivity for the isolation of VT+ E. coli O157.     

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.     

The StcE protease contributes to intimate adherence of enterohemorrhagic Escherichia coli O157:H7 to host cells

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.     

H7 antiserum-sorbitol fermentation medium: a single tube screening medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis.

Escherichia coli serotype O157:H7 has been isolated from outbreaks and sporadic cases of hemorrhagic colitis. There is convincing evidence that it can cause this diarrheal disease. Because of the interest in hemorrhagic colitis, it has become desirable to detect this particular strain in human feces, which usually contains many other strains of E. coli. Two characteristics of the incriminated E. coli O157:H7 strain have made its isolation and identification easier. It does not ferment D-sorbitol rapidly, in contrast to about 95% of other E. coli strains. In addition, the strain has H antigen 7, but only about 10% of other E. coli strains have this particular antigen. To screen for E. coli O157:H7 we devised H7 antiserum-sorbitol fermentation medium (18 g of enteric fermentation base, 10 g of D-sorbitol, 4 g of agar, 10 ml of Andrade indicator, 989 ml of water; all ingredients were mixed, autoclaved, and cooled; 1 ml of E. coli H7 antiserum was then added). Colonies to be screened were inoculated into this medium. Strains of E. coli O157:H7 gave a characteristic pattern; they did not ferment sorbitol and were immobilized in the semisolid medium because of the reaction of their flagella with the flagella antiserum. Almost all other strains of E. coli gave a different pattern; they fermented sorbitol or were not immobilized by the H7 serum or both. Strains which were presumptive positives (sorbitol negative, H7 positive) were then tested in E. coli O157 serum by slide or tube agglutination. The number of strains which were presumptive positive by H7-sorbitol medium but then were not found to be O157 was less than 1%. A second approach has been helpful in deciding which colonies to screen in H7-sorbitol medium. MacConkey-sorbitol agar (22.2 g MacConkey agar base [which contains no sugar], 10 g of D-sorbitol, 1,000 ml of water) was designed as a plating medium. Stools were plated on MacConkey agar to estimate the number of E. coli colonies and also plated on MacConkey-sorbitol agar to estimate the number of sorbitol-negative colonies of E. coli. These two approaches have proved useful for isolating and identifying E. coli O157:H7 form human feces and from feces of animals infected in the laboratory with this strain. The results suggest that media may be formulated in a similar fashion for detecting other specific strains of E. coli.     

Serotype O157:H7 Escherichia coli from bovine and meat sources.

Serotype O157:H7 Escherichia coli strains from several different bovine and meat (beef) sources were studied to determine the diversity of their virulence properties and to compare their plasmid characteristics. Eighteen strains from cattle feces, 2 from water buffalo feces, 3 from beef samples, and 2 from feces of human hemolytic uremic syndrome cases were examined. All of these strains hybridized with the CVD419 DNA probe which identifies serotype O157:H7 and many other serotypes of verocytotoxin-producing E. coli. Of 15 bovine strains that hybridized with two verocytotoxin DNA probes, 8 hybridized with both verocytotoxin 1 (VT1) and VT2 probes, 5 hybridized with only the VT2 probe, and 2 hybridized with only the VT1 probe. This distribution was similar to that reported for O157:H7 E. coli isolated from humans. All three beef isolates hybridized with both VT1 and VT2 probes. All strains that hybridized with the VT probes were positive in the verocytotoxin assay, and all probe-negative strains were negative in the assay. All the strains possessed large plasmids with molecular sizes ranging from 53 to 64 MDa. Fifteen of the 20 cattle and water buffalo strains had one or more additional small plasmids. Restriction patterns resulting from HindIII, SmaI, and BamHI digestions of the large plasmids were used to compare all possible pairs of five different single plasmid-bearing strains from different countries (Egypt, England, and the United States). The restriction patterns of these strains were distinct, and the mean coefficients of similarity for these comparisons ranged from 71 to 91%, indicating a moderate degree of genetic diversity. This diversity and the presence of multiple plasmids in many bovine and human O157:H7 strains reinforce the usefulness of plasmid analysis in future studies. Only four of the 20 bovine strains and 1 of the 3 beef strains possessed the capability for adherence to HEp-2 and Intestine 407 cells in the presence of mannose, indicating that in vitro expression of localized adherence is not a universal property of O157:H7 strains of bovine origin.     

Isolation and characterization of a beta-D-glucuronidase-producing strain of Escherichia coli serotype O157:H7 in the United States.

A phenotypic variant of Escherichia coli serotype O157:H7 (G5101) was isolated from a patient with bloody diarrhea. Strain G5101 does not ferment sorbitol but is beta-D-glucuronidase and urease positive. Serotyping and colony hybridization using a serotype-specific DNA probe confirmed that the isolate was O157:H7. G5101 produces Shiga-like toxins I and II and contains an eae gene that is highly conserved in the O157:H7 serotype. This strain would have been missed by laboratories that screen for the sorbitol-negative, beta-D-glucuronidase-negative phenotype in isolating E. coli O157:H7 from clinical and food specimens.     

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.     

Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157.

Fluorogenic procedures were used with the substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) to identify Escherichia coli. Most strains produced beta-glucuronidase and, thus, were MUG positive. A 20-min procedure was developed to detect glucuronidase activity in 1,295 bacterial cultures, representing 23 genera, of strains that were isolated from clinical specimens. Very few organisms other than E. coli were MUG positive. Of 682 E. coli strains that were isolated, 630 (92.4%) were MUG positive. When an additional 188 E. coli serotype O157 isolates were examined, 155 E. coli O157:H7, 10 E. coli O157:H-, and 1 E. coli O157:H (rough) isolate were MUG negative. All 166 cultures were verocytotoxin positive. Of the remaining 22 E. coli O157 isolates, 2 isolates were O157:H-, 1 isolate was O157:H (rough), and 19 isolates were other H types (H6, H16, H19, H25, H42, and H45); these 22 isolates were MUG positive. All 22 cultures were verocytotoxin negative. The rapid MUG procedure can be used to predict verocytotoxin-positive isolates of E. coli O157; that is, there is a very good likelihood that MUG-negative E. coli O157 isolates are verocytotoxin positive.     

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.     

Escherichia coli O128 strains from infants with diarrhea commonly show localized adhesion and positivity in the fluorescent-actin staining test but do not hybridize with an enteropathogenic E. coli adherence factor probe.

Twenty-nine strains of Escherichia coli O128 isolated from infants with diarrhea that did not produce heat-stable enterotoxin, heat-labile enterotoxins, or Vero cytotoxin showed localized attachment to HEp-2 cells (LA). Only four strains hybridized with the enteropathogenic E. coli adherence factor (EAF) probe. One of the 25 LA+ EAF- strains attached to 72% of cells, while a plasmid-negative variant attached to 0.5% of cells. LA+ EAF- and LA+ EAF+ strains gave a positive fluorescent-actin staining test that correlates with the ability to cause attaching and effacing lesions in the intestine. The use of the EAF probe alone to detect LA+ strains is inadequate for epidemiological studies.     

Surface properties of the Vero cytotoxin-producing Escherichia coli O157:H7.

Strains of Escherichia coli serotype O157:H7 are Vero cytotoxin-producing enteric pathogens which have been associated with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome in humans. In addition to toxin production, adherence of many pathogenic bacteria to intestinal mucosal surfaces is a critical primary step in the pathogenesis of diarrheal diseases. Although E. coli serotype O157:H7 organisms adhere to intestinal epithelia of orally infected animals in a pattern morphologically identical to that previously described in adherent, effacing E. coli infections, the mechanisms of bacterial adherence are not known. To determine the cell surface adhesins which mediate attachment of E. coli O157:H7 to epithelial surfaces, we evaluated the surface properties of these organisms. Five strains isolated from children with the hemolytic uremic syndrome were grown both in broth cultures and on agar media. Adherence and invasion of E. coli O157:H7 in Intestine 407 and HEp-2 epithelial cell lines was quantitated using an enteroinvasive E. coli strain (serotype O164:NM) as a control. Cell surface properties of E. coli O157:H7 were evaluated by agglutination of a series of erythrocytes, transmission electron microscopy, DEAE-ion-exchange chromatography, and hydrophobic interaction chromatography. E. coli O157:H7 strains adhered to but did not invade either Intestine 407 or HEp-2 cells. Homologous O157:H7 rabbit antiserum blocked attachment of bacteria to tissue culture cells, in contrast to heterologous antiserum and preimmune rabbit serum, which did not inhibit attachment of E. coli O157:H7. None of the five O15:H7 isolates mediated mannose-resistant hemagglutination under any of the in vitro culture conditions. One isolate mediated mannose-sensitive hemagglutination after serial passage in broth cultures. Pili and fibrillae were not visualized by electron microscopy on nonhemagglutinating organisms, but pili were demonstrated on the one isolate which mediated mannose-sensitive hemagglutination. All O157:H7 strains demonstrated high anionic surface charge (DEAE) but low surface hydrophobicity properties (hydrophobic interaction chromatography). The findings suggest that surface structures other than pili can mediate attachment of serotype O157:H7 bacteria to epithelial cells in vitro.     

Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes.

Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains) [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic. E. cole. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:H-, O103:H2, O111:H-, O111:H2, O119:H25, O128:H-, O145:H28, and O157:H-). Only 15 STEC strains, belonging to serotypes O111H:-, O111H:2, O145:H28, and O157:H-, gave amplicons in all three eae-specific PCRs. These data demonstrate that bovine STEC strains are a heterogeneous group of pathogenic bacteria, a lot of which share virulence markers with STEC strains causing infections in humans. However, in contrast to human STEC strains, bovine eae-positive STEC strains are mainly restricted to the stx1 genotype. The observation that espB sequences are not highly conserved might have consequences for the serological recognition of the ESPB protein in patients. Like in human STEC strains, eae-related sequences are closely associated with certain E. coli O groups; however, they are not serotype specific.     

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

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

Isolation from a sheep of an attaching and effacing Escherichia coli O115:H- with a novel combination of virulence factors

Attaching and effacing (AE) lesions were observed in the caecum, proximal colon and rectum of one of four lambs experimentally inoculated at 6 weeks of age with Escherichia coli O157:H7. However, the attached bacteria did not immunostain with O157-specific antiserum. Subsequent bacteriological analysis of samples from this animal yielded two E. coli O115:H(-) strains, one from the colon (CO) and one from the rectum (RC), and those bacteria forming the AE lesions were shown to be of the O115 serogroup by immunostaining. The O115:H(-)isolates formed microcolonies and attaching and effacing lesions, as demonstrated by the fluorescence actin staining test, on HEp-2 tissue culture cells. Both isolates were confirmed by PCR to encode the epsilon (epsilon) subtype of intimin. Supernates of both O115:H(-) isolates induced cytopathic effects on Vero cell monolayers, and PCR analysis verified that both isolates encoded EAST1, CNF1 and CNF2 toxins but not Shiga-like toxins. Both isolates harboured similar sized plasmids but PCR analysis indicated that only one of the O115:H(-) isolates (CO) possessed the plasmid-associated virulence determinants ehxA and etpD. Neither strain possessed the espP, katP or bfpA plasmid-associated virulence determinants. These E. coli O115:H(-) strains exhibited a novel combination of virulence determinants and are the first isolates found to possess both CNF1 and CNF2.     

Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.     

Laboratory investigation of outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7.

A severe outbreak of hemorrhagic colitis occurred in London, Ontario, during the month of September 1985. A total of 55 residents and 18 employees of a nursing home developed diarrhea, and 17 residents (age range, 78 to 99 years) died. Specimens from 38 patients, 37 employees and contacts, and 10 autopsies were investigated for all enteric pathogens. Specimens were also planted on MacConkey-sorbitol agar. Fecal extracts were tested on Vero cells for cytotoxin (FVT). Escherichia coli isolates were serotyped and tested for verotoxin and beta-glucuronidase production. Of the 38 symptomatic patients, 26 were positive for FVT, verotoxin-producing E. coli (VTEC), or both. Of the 105 specimens that were examined from these 38 patients, FVT and VTEC were both positive in 30 specimens, FVT only was positive in 13 specimens, and VTEC only was positive in 4 specimens. None of the 27 specimens from 10 autopsies was positive for FVT or VTEC. No other enteric pathogen was found in any of the cases. All asymptomatic individuals were negative for both FVT and VTEC. Of 19 VTEC strains that were isolated, 18 belonged to serotype O157:H7. These 18 strains and 2 more strains that were obtained from sporadic cases that had occurred within the 2 previous months were found to give similar biochemical reactions in a 36-test identification system. All isolates of serotype O157:H7 were beta-glucuronidase negative and susceptible to the antimicrobial agents that are used to treat E. coli infections. Testing for FVT and VTEC was found to be the most sensitive and specific technique for the laboratory diagnosis of this disease. Negative sorbitol, positive raffinose, and negative beta-glucuronidase tests appeared to be consistent markers for aiding in the detection of E. coli O157:H7.