We have investigated 677 Shiga toxin-producing Escherichia coli (STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (eae) genes were present in 62.6% of the strains and subtyped into intimins ␣1, ␤1, ␥1, , , and . Shiga toxin types 1c and 2d were present only in eae-negative STEC strains, and type 2 was significantly (P < 0.001) more frequent in eae-positive STEC strains. Enterohemorrhagic E. coli hemolysin was associated with 96.2% of the eae-positive strains and with 65.2% of the eae-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with eae-positive STEC were significantly (P < 0.001) more frequent in children under 6 years of age than in other age groups, whereas eae-negative STEC infections dominated in adults. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins ␣1 (one serotype), ␤1 (eight serotypes), (two serotypes), and (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.The association of Shiga (Vero) toxin production in Escherichia coli with human pathogenicity was first described in 1979 (82, 85). However, it was the investigation of an outbreak caused by Shiga toxin-producing E. coli (STEC) O157 which provided the major impetus to study these pathogens (65). In the following years, STEC strains were increasingly isolated from humans with diarrhea and hemolytic-uremic syndrome (HUS) and from farm animals, which serve as a natural reservoir for STEC (52,86). Today, more than 200 different E. coli O:H serotypes are known to be associated with the production of Shiga toxins (86; K. A. Bettelheim's VTEC table, May 2003 update, www.sciencenet.com.au/vtectable.htm). Certain STEC strains belonging to serogroups O26, O103, O111, O145, and O157 were more frequently isolated from humans with severe diseases such as hemorrhagic colitis and HUS. Accordingly, these highly virulent STEC strains were also designated as enterohemorrhagic E. coli (EHEC) (42, 52). The search for additional virulence markers in these pathogens revealed that most EHEC strains carry a plasmid which encodes a hemolysin (EHEC hemolysin) and the chromosomally located locus of enterocyte effacement (LEE) pathogenicity island (16,43,70,84). The genes carried by the LEE enable the bacteria to produce attaching and effacing lesions in the host intestinal mucosa cells, which increases the virulence of the bacteria for humans (35,44,60). Intimate attachment of bacteria to the host cell is mediated by the binding of intimin, the product of the eae gene...

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Fecal isolates of Escherichia coli which were collected from human patients in different parts of Germany between 1985 and 1992 were examined for production of verotoxins (VT). Among 2165 isolates 54 (2.5%) verotoxigenic E. coli (VTEC) were found. The 54 VTEC belonged to 13 different serotypes, 46 (85.2%) of these were enterohemorrhagic E. coli (EHEC) types as O157:H7, O157:H-, O145:H-, O111:[H8] and O26:[H11]. Of the 54 VTEC 50 (92.6%) hybridized with one or both of the DNA probes specific for VT1 and VT2. The 4 VTEC strains which were negative for VT1 and VT2 differed from all other VTEC by many phenotypical trains such as serotype, production of alpha-hemolysin and absence of EHEC-plasmid and "attaching and effacing" (eae)-specific DNA sequences. In contrast, VTEC which were positive for VT1, VT2 or both were frequently positive for eae sequences (92.0%), EHEC-plasmids (90.0%) and for production of enterohemolysin (88.0%). With enterohemolysin as an epidemiological marker more VTEC strains (81.5%) could be identified than with others such as the absence of beta-glucuronidase activity (61.1%) or non-fermentation of sorbitol (48.1%). Case reports were available for 42 of the 54 VTEC strains. The clinical presentation of 42 cases with VTEC ranged from uncomplicated diarrhea to severe diseases as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). However, bloody diarrhea, HC and HUS were more associated with the O157 group than with other VTEC groups.

By combining the enterohemolysin test and the VTEC-RPLA test (specific for the detection of Shiga-like toxin I [SLT-I], SLT-II, and SLT-IIc), single colonies of SLT-producing Escherichia coli were found to constitute between 0.03 and 68.1% of the coliform flora in human stool cultures and were isolated and characterized within 72 to 96 h.

Fecal samples from healthy children under 2 years of age living in Berlin, Germany (205 infants), and Melbourne, Australia (184 infants), were investigated for the presence of attaching and effacing (AE) Escherichia coli (AEEC) strains by screening for eae (intimin) genes. Twenty-seven AEEC strains were isolated from 14 children (7.6%) from Melbourne and from 12 children (5.9%) from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (one strain, producing Shiga toxin 1), typical enteropathogenic E. coli (EPEC) (one strain carrying an EPEC adherence factor [EAF] plasmid), and atypical EPEC (25 strains negative for Shiga toxins and EAF plasmids). The AEEC were divided into 18 different serotypes, O-nontypeable and O-rough strains. Typing of their intimin genes revealed the presence of intimin ␣ in 6 strains, intimin ␤ in 11 strains, intimin ␥ in 7 strains, intimin in 2 strains, and intimin in one strain. Analysis of HEp-2 cell adherence showed diffuse adherence or localized adherence-like patterns in 26 AEEC strains; local adherence was found only with the EAF-positive strain. Ten AEEC strains showed an AE property with the fluorescent actin staining (FAS) test. The introduction of an EAF plasmid (pMAR7) converted 11 FAS-negative AEEC strains to FAS positive and increased the FAS reaction in six FAS-positive AEEC strains, indicating that the genes needed for the AE phenotype were functional in these strains. Our finding indicates that atypical EPEC strains could play a double role as strains that naturally immunize against intimin in humans and as reservoirs for new emerging human pathogenic EPEC strains.The ability to cause attaching and effacing (AE) lesions in cells of the intestinal mucosa was identified as an important pathogenicity factor of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) strains (42,64). The AE lesion is generated by intimate attachment of bacteria to the enterocytes, which is followed by aggregation of the cytoskeletal actin and effacement of microvilli (34). Actin aggregation caused by AE E. coli (AEEC) strains results in a typical picture which can be visualized by a fluorescent actin staining (FAS) test which is used for detection of EPEC and EHEC (34,35,58). The genes coding for the AE lesion are located on a pathogenicity island termed "locus of enterocyte effacement" (LEE) in EPEC and EHEC strains. The 34-kb core region of the LEE is highly conserved in human and animal EPEC and EHEC strains and contains polycistronic operons harboring genes for type III secretion and adhesion proteins (18,40,51,68). Intimate attachment of bacteria to the eucaryotic cell is mediated by intimin, the product of the eae gene, which is incorporated in the bacterial membrane. Intimin binds to the translocated intimin receptor (Tir), which is presented on the surface of the eucaryotic target cell (for a review, see reference 64). The detection of the intimin gene was taken as an indicator for the presence of functional LEE genes because it was shown that ...