Escherichia coli encoding Shiga toxin 2f as an emerging human pathogen.

Escherichia coli harbouring the stx2f gene have been previously reported in pigeons. Here we demonstrate the presence of this allele in human diarrhoeagenic E. coli strains originally classified as atypical enteropathogenic E. coli (aEPEC). Thirty-two stx2f-positive E. coli serotyped as O63:H6, O128:H2, O132:H34, O145:H34, and O178:H7 were found to belong to a large number of clonal groups due to their different MLST-, PFGE- and virulence patterns. The appearance of various stx2f-positive clonal lineages among E. coli reveals emerging clinical significance. Therefore, it seems to be prudent to include stx2f into the diagnostic scope employed for laboratory investigation of enteric infections.

CsrA and CsrB are required for the post-transcriptional control of the virulence-associated effector protein AvrA of Salmonella enterica.

The virulence-associated effector protein AvrA of Salmonella enterica is an ubiquitin-like acetyltransferase/cysteine protease, which interferes with the first line of immune response of the target organism. In contrast to translation of the AvrA protein in S. enterica strains, which takes place either constitutively (class 1 strains), or after acid induction (class 2 strains), or not at all (class 3 strains); the constitutive transcription of the respective avrA genes occurs regardless of these defined expression classes. When the number of avrA genes and mRNA molecules is raised experimentally using plasmids carrying the respective cloned avrA genes together with their promoter regions, the translation of avrA mRNA takes place very strongly in all respective AvrA expression classes. This kind of copy-dependent, post-transcriptional control of AvrA was shown to be dependent on the regulatory action of the CsrA/CsrB system since the deletion of both genes completely abolished the translation in the tested S. enterica strains, whereas the transcription remained unaffected. Moreover, AvrA production in strains carrying the cloned avrA genes on plasmids remained dependent on the presence of CsrA but unaffected in csrB mutant strains. On the other hand, overproduction of the regulatory molecules CsrA and CsrB in S. enterica strains carrying cloned csrA and csrB genes on plasmids ceased the expression of AvrA again. Therefore, the expression of avrA is suggested to be regulated in a post-transcriptional manner by critical and effective concentrations of CsrA (see-saw regulation), which is achieved through the sequestering activity of CsrB.

Antimicrobial drug resistance in human nontyphoidal Salmonella isolates in Europe 2000-2004: a report from the Enter-net International Surveillance Network.

A 5-year survey, from 2000 to 2004, of results of antimicrobial susceptibility testing for 11 antimicrobials for 134,310 isolates of nontyphoidal salmonellas from cases of human infection in 10 European countries has demonstrated an overall increase in the occurrence of resistance, from 57% to 66% over the period of study. In contrast, multiple resistance (to four or more antimicrobial drugs) has declined from 18% to 15%. The most significant increase in resistance has been to nalidixic acid (14% to 20%), particularly in Salmonella enterica serovar Enteritidis (10% to 26%), the most common serovar. For England and Wales this increase has for the most part been attributed to infections linked to contaminated eggs originating outside the United Kingdom. For Salmonella Typhimurium, the second most prevalent serovar, there has been an overall decline in the occurrence of resistance to ampicillin, chloramphenicol, and tetracyclines, attributed to a decline in the occurrence of multiresistant Salmonella Typhimurium DT 104. For Salmonella Virchow, a serotype with a predilection for invasive disease, there has been a substantive increase in resistance to most antimicrobials, attributed to the spread of drug-resistant strains associated with poultry. Because of the widespread importation of foods, it is important that controls to reduce the emergence and spread of drug-resistant strains of Salmonella are internationally implemented.

Prevalence, virulence profiles, and clinical significance of Shiga toxin-negative variants of enterohemorrhagic Escherichia coli O157 infection in humans.

BACKGROUND: Escherichia coli O157, of the H7 clone, exists in humans and in the environment as Shiga toxin (Stx)-positive and Stx-negative variants. Stx production by infecting organisms is considered to be a critical requirement for the development of hemolytic uremic syndrome (HUS), which occurs in approximately 15% of E. coli O157-infected patients. It is unknown if loss of the stx gene during the early stage of an enterohemorrhagic E. coli infection prevents HUS, or if absence of the stx gene from E. coli O157 reduces or ablates virulence. METHODS: We determined the frequency of stx-positive and stx-negative E. coli O157 isolates in stool samples obtained from patients who experienced sporadic cases of diarrhea or HUS, as well as the frequency in samples obtained during outbreaks, and investigated the clinical course of the disease. RESULTS: Among E. coli O157 isolates obtained from samples related to sporadic cases of diarrhea, stx-negative strains accounted for 4%. The proportion of stx-negative strains was significantly higher among sorbitol-fermenting, nonmotile E. coli O157 isolates (12.7%) than among non-sorbitol-fermenting E. coli O157:H7 or nonmotile isolates (0.8%; P<.001). stx-Negative sorbitol-fermenting E. coli O157 isolates were also observed in samples related to 3 HUS outbreaks and 1 outbreak of diarrhea caused by sorbitol-fermenting, nonmotile enterohemorrhagic E. coli O157; additionally, they were the only pathogens that were isolated in 2 other outbreaks of diarrhea without HUS. CONCLUSIONS: Strains of stx-negative E. coli O157 isolated from stool samples of patients are either inherently stx-negative strains that cause mostly uncomplicated diarrhea, or strains that descended from enterohemorrhagic E. coli O157 by the loss of the stx gene during infection; the latter strains may still cause severe disease.

Enterohemorrhagic Escherichia coli in human infection: in vivo evolution of a bacterial pathogen.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) cause most cases of the hemolytic uremic syndrome (HUS) worldwide. To investigate genetic changes in EHEC during the course of human infection, we analyzed consecutive stool samples and shed isolates from patients with HUS, focusing on the genes encoding Shiga toxin (stx) and intimin (eae). METHODS: Sequential stool samples from 210 patients with HUS were investigated for the persistence of E. coli strains harboring stx and/or eae. Initial stool samples were collected during the acute phase of HUS, and subsequent stool samples were collected 3-16 days later (median interval, 8 days). RESULTS: Organisms that were stx and eae positive (stx+/eae+ strains; n=137) or stx negative and eae positive (stx-/eae+ strains; n=5) were detected in the initial stool samples from 142 patients. Subsequently, the proportion of those who shed stx+/eae+ strains decreased to 13 of 210 patients, whereas the proportion of those who shed strains that were stx-/eae+ increased to 12 of 210 patients. Seven patients who initially excreted strains that were stx+/eae+ shed, at second analysis, stx-/eae+ strains of the same serotypes; they had no free fecal Shiga toxin at follow-up. Comparison of the initial and follow-up isolates from these patients with use of molecular-epidemiological methods revealed loss of stx genes and genomic rearrangement. CONCLUSIONS: We demonstrate the loss of a critical bacterial virulence factor from pathogens during very brief intervals in the human host. These genetic changes have evolutionary, diagnostic, and clinical implications. Generation of stx- mutants might contribute to subclonal evolution and evolutionary success.

Expression of cellulose and curli fimbriae by Escherichia coli isolated from the gastrointestinal tract.

Escherichia coli colonizes the gastrointestinal tract of humans; however, little is known about the features of commensal strains. This study investigated whether expression of the biofilm extracellular matrix components cellulose and curli fimbriae is found among commensal isolates. Fifty-two E. coli strains were isolated from faecal samples and, as a control, 24 strains from urinary tract infections were also used. Faecal isolates were characterized by serotyping and phylogenetically grouped by PCR. The genotype was determined by PFGE and the presence of virulence factors was assessed. Co-expression of cellulose and curli fimbriae at 28 degrees C and 37 degrees C was typical for faecal isolates, while urinary tract infection strains typically expressed the extracellular matrix components at 28 degrees C only. Knockout studies in a representative faecal isolate revealed that the response regulator CsgD regulated cellulose and curli fimbriae, as found previously in Salmonella enterica. In contrast to S. enterica, at 37 degrees C pellicle formation occurred in the absence of cellulose and curli fimbriae. The gastrointestinal tract represents a source of biofilm-forming bacteria, which can spread to susceptible sites.

International outbreak of Salmonella Oranienburg due to German chocolate.

BACKGROUND: This report describes a large international chocolate-associated Salmonella outbreak originating from Germany. METHODS: We conducted epidemiologic investigations including a case-control study, and food safety investigations. Salmonella (S.) Oranienburg isolates were subtyped by the use of pulsed-field gel electrophoresis (PFGE). RESULTS: From 1 October 2001 through 24 March 2002, an estimated excess of 439 S. Oranienburg notifications was registered in Germany. Simultaneously, an increase in S. Oranienburg infections was noted in other European countries in the Enter-net surveillance network. In a multistate matched case-control study in Germany, daily consumption of chocolate (matched odds ratio [MOR]: 4.8; 95% confidence interval [CI]: 1.3-26.5), having shopped at a large chain of discount grocery stores (MOR: 4.2; CI: 1.2-23.0), and consumption of chocolate purchased there (MOR: 5.0; CI: 1.1-47.0) were associated with illness. Subsequently, two brands from the same company, one exclusively produced for that chain, tested positive for S. Oranienburg. In two other European countries and in Canada chocolate from company A was ascertained that also contained S. Oranienburg. Isolates from humans and from chocolates had indistinguishable PFGE profiles. No source or point of contamination was identified. Epidemiological identification of chocolate as a vehicle of infections required two months, and was facilitated by proxy measures. CONCLUSIONS: Despite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing Salmonella-containing products. Particularly in diffuse outbreak-settings, clear associations with surrogates of exposure should suffice to trigger public health action. Networks such as Enter-net have become invaluable for facilitating rapid and appropriate management of international outbreaks.

Prevalence and molecular diversity of pHS-2 plasmids, marker for arthritogenicity, among clinical Escherichia coli Shigella isolates.

Reactive arthritis can occur after numerous bacterial infections, including bacillary dysentery caused by Escherichia coli Shigella. A major risk factor for the disease is the HLA B27 phenotype in the human host. By comparison between plasmid profiles of arthritogenic vs. nonarthritogenic Shigella strains, the pHS-2 plasmid has been previously associated with the arthritogenic capacity of Shigella isolates. However, the prevalence of this plasmid in the various Shigella biotypes and serotypes is largely unknown. On this background, 188 clinical isolates from intestinal disease representing all 46 Shigella serogroups were studied for the presence of the pHS-2 plasmid, using PCR, dot blot and Southern blot techniques and by analysis of restriction fragment length polymorphisms. The pHS-2 plasmid was found in nine of 14 E. coli Flexneri serogroups, in E. coli Dysenteriae 1 and in E. coli Boydii 16. In addition, we show marked variability of this plasmid in E. coli Flexneri 3A and 4A strains. Major biological diversity of the pHS-2 plasmid was found to be strictly related to Shigella serogroups. The prevalence pattern of the pHS-2 plasmid matches published data on arthritogenic Shigella isolates, providing additional indirect evidence for the potential validity of this plasmid as a marker for arthritogenicity.

Expression profiles of effector proteins SopB, SopD1, SopE1, and AvrA differ with systemic, enteric, and epidemic strains of Salmonella enterica.

The presence and expression of sopB, sopD1, sopE1, and avrA genes encoding virulence associated effector proteins were studied comparatively in 405 Salmonella enterica strains. They belong to different serovars and clonal types (genotypes, phage types) and originated from different clinical (systemic infection, focal enteritis, enterocolitis) and epidemic sources (epidemics, sporadic cases). The sopB and sopD1 determinants were commonly prevalent, but sopE1 and avrA genes only in 55% and 80%, respectively. A correlation of this pattern of absence and presence of the respective genes to the epidemic and clinical origin could not be detected. In contrast, the expression of the respective genes appeared differently: SopB and SopE1 proteins are well produced, but SopD1 and AvrA proteins only rarely under the applied standard culture conditions. However, using a range of different environmental signals (temperature, pH, cations, etc.) some of the S. enterica nonproducer strains (e. g., S. Agona, S. Bovismorbificans, S. Virchow, etc.) begin to produce AvrA and SopD1. They turned now into an expression profile which was found typically for the epidemic strains of S. Typhimurium and S. Enteritidis. Also S. enterica strains from systemic infections could be characterized by their strong SopB and SopE1 expression while SopD1 and AvrA proteins were missing. Although it is premature to outline generally a correlation of these expression profiles and the clinical and epidemiological potency of Salmonellae, the reported results allow a first understanding how a fine tuning of their virulence will take place.

Identification of unconventional intestinal pathogenic Escherichia coli isolates expressing intermediate virulence factor profiles by using a novel single-step multiplex PCR.

Intestinal pathogenic Escherichia coli represents a global health problem for mammals, including humans. At present, diarrheagenic E. coli bacteria are grouped into seven major pathotypes that differ in their virulence factor profiles, severity of clinical manifestations, and prognosis. In this study, we developed and evaluated a one-step multiplex PCR (MPCR) for the straightforward differential identification of intestinal pathotypes of E. coli. The specificity of this novel MPCR was validated by using a subset of reference strains and further confirmed by PCR-independent pheno- and genotypic characterization. Moreover, we tested 246 clinical E. coli isolates derived from diarrhea patients from several distinct geographic regions. Interestingly, besides strains belonging to the defined and well-described pathotypes, we identified five unconventional strains expressing intermediate virulence factor profiles. These strains have been further characterized and appear to represent intermediate strains carrying genes and expressing factors associated with enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli alike. These strains represent further examples of the extraordinary plasticity of the E. coli genome. Moreover, this implies that the important identification of specific pathotypes has to be based on a broad matrix of indicator genes. In addition, the presence of intermediate strains needs to be accounted for.

Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans.

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

Enterohaemorrhagic Escherichia coli O157 and non-O157 serovars differ in their mechanisms for iron supply.

Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.

Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111.

We analysed 72 clinical isolates of enterohaemorrhagic Escherichia coli (EHEC) O111 from patients with diarrhoea or haemolytic uraemic syndrome (HUS) isolated during the period from 1955 to 2005 and identified six motile strains (flagellar antigens 8, 10 and 11); the remaining 66 (92%) were nonmotile (NM) and could not be typed by conventional H serotyping. To improve subtyping methodologies, we determined genotypes of the flagellin-encoding fliC. Three fliC genotypes were found which were identical to those of motile EHEC O111 with H antigens 8, 10 and 11 and designated fliC(H8), fliC(H10) and fliC(H11). The IS629 insertion element was present, identically located, in six epidemiologically unrelated isolates with fliC(H8). The prevalence of the fliC genotypes in the 72 EHEC O111 strains were fliC(H8) (89%), fliC(H10) (7%) and fliC(H11) (4%). Within these fliC genotypes, a high degree of homogeneity for the presence of disease-associated genes was found. The adhesins-encoding genes eae and efa-1 were present in all strains with fliC(H8) and fliC(H11), but absent from strains with fliC(H10). The latter strains have not been reported previously. Strains with fliC(H10) and fliC(H11), but not those with fliC(H8), retained intact cadA and cadC loci and decarboxylated lysine. Three different stx genotypes including stx(1), stx(2) and stx(1)/stx(2) were determined among the 72 EHEC O111. We observed a significant increase over time in the frequency of strains harbouring both stx(1) and stx(2). The presence of stx(2) both alone and in combination with stx(1) was significantly (chi(2)=23.16, P<0.00001, CI(95) [2.29; 9.76]) associated with HUS. Therefore, the emergence of EHEC O111 should be monitored carefully. We conclude that EHEC O111 strains can be differentiated using specific loci required for motility, adherence, Stx production, and lysine decarboxylation. The divergence within EHEC O111 makes it possible to subtype these emerging pathogens in the laboratory thereby providing a basis for further investigations into their ecological niches and survival capabilities.

Shiga toxin-producing Escherichia coli infection in Germany: different risk factors for different age groups.

The authors conducted a matched case-control study in Germany to identify risk factors for sporadic illness associated with Shiga toxin-producing Escherichia coli (STEC) infection, regardless of serogroup. From April 2001 through March 2003, cases were prospectively enrolled through a laboratory-based sentinel surveillance system located in 14 of the 16 German federal states. One control was identified per case, matched by age and region. Conditional logistic regression was used in the analysis, which was conducted separately for three age groups (<3 years, 3-9 years, and > or =10 years). The median age of the 202 enrolled cases was 2.5 years (range, 3 months-89 years). Hemolytic uremic syndrome developed in five patients. Non-O157 strains accounted for 85% of the isolated STEC. In children under 3 years of age, having touched a ruminant had the highest odds of disease, and raw milk was the only food identified as a risk factor. In contrast, in persons aged 10 years or older, only food items (i.e., lamb meat, raw spreadable sausages) were significantly associated with illness. In this study, risk factors were age-specific. Direct transmission through food played a lesser role in children under 3 years of age, the population at greatest risk of both acquiring STEC infection and developing hemolytic uremic syndrome.

Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM.

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.

The expression of the virulence-associated effector protein gene avrA is dependent on a Salmonella enterica-specific regulatory function.

Although the avrA gene is prevalent among 80% of the Salmonella enterica serovars, only a small number of them usually express the respective virulence-associated effector protein AvrA. However, under culture conditions below pH 6.0 many of the AvrA non-producer strains (e.g. S. Agona, S. Bovismorbificans, S. Virchow) begin to produce AvrA, while some others remain silent. Four phenotypical classes of S. enterica were identified under defined standard culture conditions: class 1 comprises strains with a constitutive synthesis of AvrA; class 2 comprises strains with an acid induction of AvrA; class 3 comprises strains with silent avrA genes; and the fourth class comprises strains which do not contain the avrA gene (class 0 strains). The expression of avrA was found to be controlled by a Salmonella-specific mechanism because cloned avrA genes from classes 1, 2, and 3 strains remain silent in Escherichia coli strains, while easily expressed in S. enterica strains. The expression of AvrA in classes 1, 2, and 3 strains does not coincide with the nucleotide sequences of the respective promoter or structural regions of the avrA genes, but depends directly on this Salmonella-specific regulatory system which appears to be differently modulated in the distinct Salmonella serovars.

Cytolethal distending toxin in Escherichia coli O157:H7: spectrum of conservation, structure, and endothelial toxicity.

We identified the cytolethal distending toxin V (CDT-V) gene cluster in 19 (4.9%) of 391 enterohemorrhagic Escherichia coli O157:H7. cdt-V+ strains belonged to five phage types (PTs) and were most frequent within PTs 14 and 34. CDT-V was expressed in all but two cdt-V+ strains and was lethal to cultured endothelial cells. Subtyping schemes should include cdt-V as a marker to differentiate E. coli O157:H7 even within the same phage type.

Emerging Shiga toxin-producing Escherichia coli serotypes in Europe: O100:H- and O127:H40.

Novel and as yet rare non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) serotypes are emerging in Europe. Two different sorbitol-fermenting STECs, O100:H- carrying the virulence gene stx2 and O127:H40 carrying stx1 and eae genes (found in two related subjects), were isolated from patients' stool samples. Non-O157 STEC infections in humans are currently under-diagnosed. This report highlights the need for, and importance of, screening for Shiga toxins or serotypes other than just O157.

Population structure of Francisella tularensis.

We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis.

Diversity of virulence patterns among shiga toxin-producing Escherichia coli from human clinical cases-need for more detailed diagnostics.

Intestinal infections due to shiga toxin-producing Escherichia coli bacteria (STEC) reveal a broad range of clinical symptoms and a large scale of virulence properties of the respective pathogens. The question whether all STEC variants or only a particular group of them need to be considered for clinical and epidemiological purposes was answered throughout this study. Using the PCR technique for the identification of 25 different virulence-associated genes, 266 E. coli strains belonging to 81 different E. coli serotypes from various clinical origins were investigated. A great genetic diversity of the virulence properties and a broad range of virulence marker combinations have been identified. However, distinct virulence marker combinations (e.g. Stx2/LEE/pO157 as well as Stx2dac/pO113) were found to be associated with the same notified clinical symptoms (e.g. HUS). Such an association speaks either for the "shiga toxin-only concept" or for several redundant, but clinically or epidemiologically important virulence properties.