Interlaboratory Profiency Testing trial on the Detection of Staphylococcal Enterotoxins types SEA to SEE in food in Germany 2013.

In the autumn 2013, the National Reference Laboratory for coagulase positive staphylococci (CPS) including Staphylococcus (S.) aureus (NRL-Staph) at the Federal Institute for Risk Assessment has organized its first interlaboratory profiency testing (ILPT) trial for the detection of staphylococcal enterotoxins (SE) types SEA to SEE in food. The purpose of the ILPT was to assess the analytical competence of the official laboratories of the Federal German "Länder"authorities. Moreover, it was the intention to gain an overview of the standard methods implemented in the participating laboratories for the purpose of SE detection in food. Five cream cheese samples at three different contamination levels (blank, low, and high) were sent to each participant. In total, 15 laboratories participated to the ILPT: 14 laboratories from 11 Federal German "Länder", and the European Reference Laboratory for CPS including S. aureus (EU-RL for CPS). Data sets from 14 participating laboratories were included in the analysis. Overall, a specificity of 100% (14/14 true negative results), a sensitivity of 55% (31/56 true positive results), and an accuracy of 64% (45/60 true results) was achieved. The majority of participants (9/15) used other analytical methods for the detection of SE in food than the suggested European Screening Method (ESM) v5. To conclude on the ILPT in general it is to state that the majority of participating laboratories failed to correctly identify SE-low-contaminated samples. Further efforts are necessary to improve the analytical capacity and sensitivity as regards the detection of SE in food in Germany.

[The importance of wildlife as reservoir of antibiotic-resistant bacteria in Bavaria--first results]. / Die Bedeutung von Wildtieren als Reservoir für antibiotikaresistente Erreger in Bayern--erste Ergebnisse.

The use of antimicrobial agents is responsible for the emergence and spread of antibiotic resistant bacteria. Nevertheless, multiresistant bacteria have been found in animals that have never been exposed to antimicrobial agents. Wild animals that are carriers of methicillin-resistant organisms represent a hazard since they can transmit their bacteria to other animals and to humans. In the hunting season 2009/2010 nasal swabs of 98 red deer and 109 wild boars were examined for the presence of methicillin-sensitive and methicillin-resistant staphylococci. From each wild boar methicillin-susceptible staphylococci (Staphylococcus aureus in 28% and Staphylococcus spp. in 72% of the animals) were isolated. In red deer the detection rate of Staphylococcus (S.) aureus and methicillin-susceptible staphylococci was 49% and 17%, respectively. The occurrence of S. aureus was significantly higher (p < 0.05) in red deer than in wild boars. Methicillin-resistant staphylococci were not found. However, in one third of the red deer, methicillin-resistant bacteria of the genus Enterococcus spp. and Bacillus spp. were isolated. The results of the present study indicate that wildlife, especially red deer are an important reservoir for S. aureus and that the upper respiratory tract of red deer is regularly colonised with methicillin-resistant bacteria such as Bacillus spp. and Enterococcus spp. Primarily, commensal bacteria are harmless to human health, however, red deer may be a reservoir for antibiotic-resistant bacteria.

Assessment of Shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans.

A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx(2), stx(2d), and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.

Characterization of attaching and effacing Escherichia coli (AEEC) isolated from pigs and sheep.

BACKGROUND: Attaching and effacing Escherichia coli (AEEC) are characterized by their ability to cause attaching-and-effacing (A/E) lesions in the gut mucosa of human and animal hosts leading to diarrhoea. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports data on the occurrence of eae positive E. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated AEEC strains. RESULTS: Faecal samples from 198 finished pigs and 279 sheep were examined at slaughter. The proportion of eae positive samples was 89% for pigs and 55% for sheep. By colony dot-blot hybridization, AEEC were isolated from 50 and 53 randomly selected porcine and ovine samples and further characterized. Strains of the serotypes O2:H40, O3:H8 and O26:H11 were found in both pigs and sheep. In pigs O2:H40, O2:H49, O108:H9, O145:H28 and in sheep O2:H40, O26:H11, O70:H40, O146:H21 were the most prevalent serotypes among typable strains. Eleven different intimin types were detected, whereas gamma2/theta was the most frequent, followed by beta1, epsilon and gamma1. All but two ovine strains tested negative for the genes encoding Shiga toxins. All strains tested negative for the bfpA gene and the EAF plasmid. EAST1 (astA) was present in 18 of the isolated strains. CONCLUSION: Our data show that pigs and sheep are a source of serologically and genetically diverse intimin-harbouring E. coli strains. Most of the strains show characteristics of atypical enteropathogenic E. coli. Nevertheless, there are stx-negative AEEC strains belonging to serotypes and intimin types that are associated with classical enterohaemorrhagic E. coli strains (O26:H11, beta1; O145:H28, gamma1).

Production and characterization of rabbit polyclonal sera against Shiga toxins Stx1 and Stx2 for detection of Shiga toxin-producing Escherichia coli.

STEC has emerged as an important group of enteric pathogens worldwide. In this study, rabbit polyclonal Stx1 and Stx2 antisera were raised and employed in the standardization of immunoassays for STEC detection. Using their respective antisera, the limit of detection of the toxin was 35.0 pg for Stx1 and 5.4 pg for Stx2. By immunoblotting, these antisera recognized both toxin subunits. Cross-reactivity was observed in the A subunit, but only Stx2 antiserum was able to neutralize the cytotoxicity of both toxins in the Vero cell assay. Six stx-harboring E. coli isolates were analyzed for their virulence traits. They belonged to different serotypes, including the O48:H7, described for the first time in Brazil. Only three strains harbored eae, and the e-hly gene and hemolytic activity was detected in five strains. Three isolates showed new stx2 variants (stx(2v-ha) and stx(2vb-hb)). The ELISA assay detected all six isolates, including one VCA-negative isolate, while the immunodot assay failed to detect one isolate, which was VCA-positive. In contrast, the colony-immunoblot assay detected only one VCA-positive isolate. Our results demonstrate that among the immunoassays developed in this study, the immunodot, and particularly the ELISA, appear as perspective for STEC detection in developing countries.

Evaluation of major types of Shiga toxin 2E-producing Escherichia coli bacteria present in food, pigs, and the environment as potential pathogens for humans.

Shiga toxin 2e (Stx2e)-producing strains from food (n = 36), slaughtered pigs (n = 25), the environment (n = 21), diseased pigs (n = 19), and humans (n = 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx(2e) genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx(2e) genes. DNA sequences specific for the int, Q, dam, and S genes of the stx(2e) bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx(1) or stx(2) variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes.

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.

TccP2-mediated subversion of actin dynamics by EPEC 2 - a distinct evolutionary lineage of enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott-Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157 : H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspF(U) (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H-), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2 %) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck-/- cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin-polymerization signalling cascades.

Characterization of tccP2 carried by atypical enteropathogenic Escherichia coli.

Atypical enteropathogenic Escherichia coli (EPEC) comprise an important group of paediatric pathogens. Atypical EPEC have reservoirs in farm and domestic animals where they can be either commensal or pathogenic; serogroup O26 is dominant in humans and animals. Central to intestinal colonization by EPEC is the translocation of the type III secretion system effector Tir into enterocytes, which following phosphorylation (Tir-Yp) recruits Nck to activate the N-WASP actin signalling cascade. The authors have recently shown that typical EPEC strains, belonging to the EPEC-2 lineage, carry a tir gene encoding Tir-Yp and can also use the alternative TccP2 actin-signalling cascade. The aim of this study was to determine if tccP2 is found in atypical EPEC isolated from human and farm animals. tccP2 was found at a frequency of 41% in non-O26 EPEC isolates and in 82.3% of the O26 strains. TccP2 of human and animal strains show high level of sequence identity. It is shown that most strains carry a tir gene encoding Tir-Yp. In addition the authors identified two new variants of tir genes in EPEC O104:H12 and NT:H19 strains.

Relationship between O-antigen subtypes, bacterial surface structures and O-antigen gene clusters in Escherichia coli O123 strains carrying genes for Shiga toxins and intimin.

Escherichia coli O123 strains express a broad spectrum of phenotypes, H serotypes and virulence markers and are able to colonize and to cause disease in different hosts including humans. In this study, two subtypes of E. coli O123 antigen (group I and group II) have been identified based on their cross-reactions with other E. coli O antigens. Investigation of the relationship between O123 group I and group II strains by O serotyping and Fourier transform infrared spectroscopy of whole bacteria revealed surface structural differences between these two groups of E. coli O123 strains. Nucleotide sequence analysis of the O-antigen gene clusters of two E. coli O123 strains representing O123 group I and group II revealed no change at the amino acid level. These findings indicate that the differences in the surface structures of group I and group II strains are not related to genetic heterogeneity in their O-antigen gene clusters. A PCR assay based on O123 antigen-specific wzx and wzy genes was developed and found to be suitable for reliable detection of all subtypes of E. coli O123 strains, which bears an advantage over traditional serological detection.

TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.

Molecular markers for detection of pathogenic Escherichia coli strains belonging to serogroups O 138 and O 139.

Escherichia coli strains belonging to O-serogroup 138 and 139 are important as disease agents in pigs causing post-weaning diarrhea and edema disease. Several types of shiga toxin-producing O 138 and O 139 strains were isolated from diarrheic humans and from cattle and food of bovine origin. Serotyping is the current method for detection of O 138 and O 139 strains but its applicability can be limited due to the presence of capsules and capsular-like bacterial surface antigens and in the case of rough LPS. To overcome these difficulties for diagnosis, we have developed a specific PCR method suitable for detection of different types of O 138 and O 139 strains. The O-antigen gene clusters of E. coli O 138 and O 139 type strains were sequenced, and the genes were identified on the basis of homology. By screening against 186 E. coli and Shigella type strains, two genes specific to each of E. coli O 138 and O 139 were identified, respectively, and were tested on 15 clinical and environmental isolates of those two serogroups in a double-blind test. The sensitivity of the PCR assays was determined, and the detection limits were 2 pg per mul of chromosomal DNA and 2 CFU per 10 g of water or pork samples. PCR-based detection of O-antigen specific genes of E. coli O 138 and O 139 was shown to be accurate, highly sensitive and rapid, and is suggested as a new diagnostic tool for investigations of infections and outbreaks with these strains in animals and humans and for control of food.

Development of PCR assays targeting the genes involved in synthesis and assembly of the new Escherichia coli O 174 and O 177 O antigens.

Escherichia coli O 174 and O 177 are newly described O serogroups which were reported as human pathogens. Identification of these strains by serotyping has been restricted, as the required sera are not commercially available. In this study, a collection of 13 E. coli O 174 strains and 12 E. coli O 177 strains was studied on the O:H serotypes and virulence markers. The O-antigen gene clusters of E. coli O 174 and O 177 were sequenced, and associated genes were assigned functions on the basis of homology. Two genes, each specific for E. coli O 174 and O 177, were identified. PCR assays based on the O-antigen-specific genes were developed and tested on 25 clinical and environmental isolates of those two serogroups as well as 26 isolates of other O serogroups. As little as 1 pg per mul of chromosomal DNA and as few as 0.1 CFU per g of pork and water samples were detected for either strain. The PCR assays established in this study were shown to be highly sensitive and reliable and could be the method of choice for detection of these two human pathogens from clinical, food, and other environmental samples.

Characterization of two major groups of diarrheagenic Escherichia coli O26 strains which are globally spread in human patients and domestic animals of different species.

Twenty-three Escherichia coli O26 strains from humans, cattle, sheep, pigs and chicken were investigated for virulence markers and for genetic similarity by pulsed field gel electrophoresis and multi locus sequence typing. Two groups of genetically closely related O26 strains were defined. One group is formed by enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli strains, which do not ferment rhamnose and dulcitol and most of these carry a plasmid encoding enterohemolysin. The other group consists of rhamnose and dulcitol fermenting EPEC strains, which carry plasmids encoding alpha-hemolysin. Multiple species of domestic animals were shown to serve as a reservoir for human pathogenic O26 EPEC and EHEC strains.

Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types.

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -(eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin beta being present in 51.5% of the strains, followed by intimins theta (23.7%), epsilon (6.2%), kappa (5.2%), zeta (5.2%), alpha, eta and iota (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.

Sequence analysis of the Escherichia coli O15 antigen gene cluster and development of a PCR assay for rapid detection of intestinal and extraintestinal pathogenic E. coli O15 strains.

A collection of 33 Escherichia coli serogroup O15 strains was studied with regard to O:H serotypes and virulence markers and for detection of the O-antigen-specific genes wzx and wzy. The strains were from nine different countries, originated from healthy or diseased humans and animals and from food, and were isolated between 1941 and 2003. On the basis of virulence markers and clinical data the strains could be split into different pathogroups, such as uropathogenic E. coli, enteropathogenic E. coli, Shiga toxin-producing E. coli, and enteroaggregative E. coli. H serotyping and genotyping of the flagellin (fliC) gene revealed 11 different H types and a close association between certain H types, virulence markers, and pathogroups was found. Nucleotide sequence analysis of the O-antigen gene cluster revealed putative genes for biosynthesis of O15 antigen. PCR assays were developed for sensitive and specific detection of the O15-antigen-specific genes wzx and wzy. The high pathotype diversity found in the collection of 33 O15 strains contrasted with the high level of similarity found in the genes specific to the O15 antigen. This might indicate that the O15 determinant has been spread by horizontal gene transfer to a number of genetically unrelated strains of E. coli.

Identification of Escherichia coli O114 O-antigen gene cluster and development of an O114 serogroup-specific PCR assay.

Screening for the Escherichia coli O serotype is the traditional test for identification of E. coli clones. The O-antigen gene cluster of the E. coli O114 type strain was sequenced, and 12 open reading frames were assigned functions on the basis of homology. By screening against all 186 E. coli and Shigella O serotypes, five genes specific to E. coli O114 were identified. A PCR assay based on the O-antigen-specific genes was developed and tested on 41 clinical isolates of E. coli O114. The PCR assay was shown to be highly specific and sensitive. When tested with pork and water samples, as few as 0.12 CFU of E. coli O114 g(-1) were detected. Thus, the PCR assays established in this study can be used to reliably identify E. coli O114 strains and may also be used to detect E. coli O114 strains in food, water, and other environmental samples.

Characterization of Shiga toxin-producing Escherichia coli strains isolated from human patients in Germany over a 3-year period.

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 alpha1, beta1, gamma1, epsilon, theta, and eta. 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. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (fliC) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. 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 alpha1 (one serotype), beta1 (eight serotypes), epsilon (two serotypes), and eta (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.