Gladys Krause

Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated from Human Patients in Germany over a 3-Year Period

ABSTRACT 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 α1, β1, γ1, ε, θ, and η. 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 α1 (one serotype), β1 (eight serotypes), ε (two serotypes), and η (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.

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.

ABSTRACT 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). stx1 or variant genes were detected in 88 (40.2%) strains and stx2 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 (stx1, stx1c, and stx1d) and the Stx2 (stx2, stx2d, stx2-O118, stx2e, and stx2g) 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 stx2 and/or mucus-activatable stx2d 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. stx2 and stx2d STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx2e), lamb, and wildlife meat (stx1c). 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.

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

ABSTRACT 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 (stx2, stx2d, 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.

Identification of Escherichia coli O114 O-Antigen Gene Cluster and Development of an O114 Serogroup-Specific PCR Assay

ABSTRACT 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.

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources

Aims:  To evaluate the suitability of the commercially distributed Ridascreen® Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity.

Evaluation of Major Types of Shiga Toxin 2e-Producing Escherichia coli Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans

ABSTRACT 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 stx2e genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx2e genes. DNA sequences specific for the int, Q, dam, and S genes of the stx2e 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 stx1 or stx2 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.

TccP2 of O157:H7 and Non-O157 Enterohemorrhagic Escherichia coli (EHEC): Challenging the Dogma of EHEC-Induced Actin Polymerization

ABSTRACT 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, TirEHEC O157 is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspFU, 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, β-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 ΔtccP 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.

Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhoea in Germany.

Aims:  To investigate the prevalence of traditional and emerging types of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) strains in stool samples from children with diarrhoea and to characterize their virulence genes involved in the attaching and effacing (A/E) phenotype.

Characteristics of shiga toxin-producing Escherichia coli isolated from Swiss raw milk cheese within a 3-year monitoring program.

Food is an important vehicle for transmission of Shiga toxin-producing Escherichia coli (STEC). To assess the potential public health impact of STEC in Swiss raw milk cheese produced from cows, goats, and ewes milk, 1,422 samples from semihard or hard cheese and 80 samples from soft cheese were examined for STEC, and isolated strains were further characterized. By PCR, STEC was detected after enrichment in 5.7% of the 1,502 raw milk cheese samples collected at the producer level. STEC-positive samples comprised 76 semihard, 8 soft, and 1 hard cheese. By colony hybridization, 29 STEC strains were isolated from 24 semihard and 5 soft cheeses. Thirteen of the 24 strains typeable with O antisera belonged to the serogroups O2, O22, and O91. More than half (58.6%) of the 29 strains belonged to O:H serotypes previously isolated from humans, and STEC O22:H8, O91:H10, O91:H21, and O174:H21 have also been identified as agents of hemolytic uremic syndrome. Typing of Shiga toxin genes showed that stx(1) was only found in 2 strains, whereas 27 strains carried genes encoding for the Stx(2) group, mainly stx(2) and stx(2vh-a/b). Production of Stx(2) and Stx(2vh-a/b) subtypes might be an indicator for a severe outcome in patients. Nine strains harbored hlyA (enterohemorrhagic E. coli hemolysin), whereas none tested positive for eae (intimin). Consequently, semihard and hard raw milk cheese may be a potential source of STEC, and a notable proportion of the isolated non-O157 STEC strains belonged to serotypes or harbored Shiga toxin gene variants associated with human infections.

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

BackgroundAttaching 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.ResultsFaecal 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 γ2/θ was the most frequent, followed by β1, ε and γ1. All but two ovine strains tested negative for the genes encoding Shiga toxins. All strains tested negative for the bfp A gene and the EAF plasmid. EAST1 (ast A) was present in 18 of the isolated strains.ConclusionOur 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, β1; O145:H28, γ1).