Real-time PCR assays to detect 10 Shiga toxin subtype (Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) genes.

To develop subtyping methods for Shiga toxin (Stx)1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g genes for epidemiological analyses of Shiga toxin-producing Escherichia coli (STEC), we developed 10 simplex real-time polymerase chain reaction (PCR) assays with reference to 284 valid stx sequences and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates and recombinant plasmids, respectively. Three stx1 and 5 stx2 subtype genes, except for stx2c and stx2d, were detected with high specificity using STEC isolates. However, some stx2a sequences potentially being close to both Stx2a and Stx2d cluster in neighbor-joining cluster analysis were positive for stx2a and stx2d by real-time PCR. For the stx2c assay, the number of real-time PCR cycles was reduced to avoid unnecessary false-positive results. Based on these considerations, the real-time PCR assays developed here might aid epidemiological investigations of infections or outbreaks caused by STEC harboring any of the stx subtype genes.

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

AB5 protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB5 toxin family were described in Shiga toxin-producing Escherichia coli (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.

Immunological detection assays for recombinant Shiga toxin & Shigella dysenteriae.

Background & objectives: : Shiga toxin (Stx) is produced by Shigella dysenteriae, a Gram-negative, facultative anaerobic bacillus that causes shigellosis, haemolytic uraemic syndrome (HUS) and Reiter's syndrome. The detection methods for shiga toxin needs to be rapid, accurate, reliable and must be extensively evaluated under field conditions. The aim of this study was to develop rapid, sensitive and specific detection method for Stx. Methods: : Mice and rabbits were immunized with purified recombinant Shiga toxin B (rStxB). Using these antibodies dot ELISA, sandwich ELISA and flow through assay were developed. Results: : The high-titre antibodies specifically reacted with purified rStxB. Dot-ELISA, sandwich ELISA and flow-through assay were developed and standardized that could detect StxB with limit of detection (LOD) of 9.75, 9.7 ng/ml and 0.46 µg/cassette, respectively. Interpretation & conclusions: : The rStxB was used to produce antibodies to avoid handling of pathogen. The Flow through assay 'developed was specific, rapid and field amenable.

[Case report of acute encephalopathy caused by enterohemorrhagic Escherichia coli infection in a 24-year-old woman].

Hemolytic uremic syndrome (HUS) and acute encephalopathy caused by enterohemorrhagic Escherichia coli infection occur commonly in children, whereas adult-onset disease is rare. Here we report the case of a 24-year-old woman who developed acute encephalopathy and recovered without sequelae. She initially developed abdominal pain and diarrhea. On day 6, O-157 Shiga toxin was detected in her stool and she developed HUS. On day 11, acute encephalopathy developed and she required artificial ventilation. She was treated with steroid pulse therapy and plasma exchange (PE) and then discharged on day 53 without any sequelae. Globotriaosylceramide, a Shiga toxin receptor, is more frequently present on the cellular membranes of women than on those of men. Therefore, it is conceivable that adult women are at a higher risk of developing acute encephalopathy than men. Steroid pulse therapy and PE may effectively treat acute encephalopathy by reducing inflammatory cytokine levels in the blood; therefore, these treatments should be proactively considered.

Use of an ingredient-based analysis to investigate a national outbreak of Escherichia coli O157, United Kingdom, July 2016.

Public Health England was alerted to a national outbreak of Shiga toxin-producing Escherichia coli O157 PT34 in July 2016. Early investigations suggested that the likely source was a salad item consumed outside of the home. A number of cases reported consuming meals at a staff canteen (Venue A) and a garden café (Venue B). Both venues shared a common salad supplier. An investigation was undertaken to measure associations between salad items and illness using an 'ingredient-based analysis'. A retrospective case-control study was conducted using an online questionnaire to collect information on menu items consumed at each venue. Chefs at both venues were interviewed to identify ingredients contained within each menu item. Both venues were pooled together for multivariable analysis measuring associations at the ingredient level. Among 203 responses, 24 cases were identified (13 confirmed, two probable and nine possible). Case onsets ranged between 7 and 25 June 2016. Multivariable analysis identified strong evidence that only baby mixed-leaf salad from the common supplier was a vehicle of infection (adjusted odds ratio = 13.1; 95% confidence interval: 1.6-106.5). Identifying the specific salad ingredient associated with illness was made possible by using an ingredient-based analysis. We recommend the increased use of ingredient-based analyses.

Síndrome urémico hemolítico asociado a diarrea sin trombocitopenia / Postdiarrhoeal haemolytic uraemic syndrome without thrombocytopenia

Antecedentes: La presencia de trombocitopenia es una marca distintiva del síndrome urémico hemolítico asociado a diarrea (SUH D+); sin embargo, puede ser transitoria y, por lo tanto, no ser detectada. Existe limitada información sobre la prevalencia y el curso de la enfermedad en niños con SUH D+ sin trombocitopenia. Objetivo: Determinar la prevalencia de SUH D+ sin trombocitopenia y describir las características clínicas de una serie de niños con esta particularidad. Pacientes y métodos: Fueron revisadas las historias clínicas de los pacientes con SUH D+ internados entre 2000 y 2016 para identificar a aquellos sin trombocitopenia (>150.000mm3). De los casos seleccionados se recolectaron las variables demográficas, clínicas y de laboratorio, las cuales fueron analizadas descriptivamente. Resultados: De 161 pacientes internados durante el periodo de estudio se identificaron 9 sin trombocitopenia (5,6%). La mediana de la edad al diagnóstico fue de 17 meses (7-32) y la de la duración del periodo prodrómico, de 15 días (7-21). Ocho pacientes mantuvieron diuresis normal y uno requirió diálisis. Ningún paciente presentó compromiso extrarrenal severo y/o hipertensión arterial. Conclusiones: La prevalencia de SUH D+ sin trombocitopenia fue del 5,6% y la mayoría de los casos fueron leves; sin embargo, el requerimiento de diálisis en uno de ellos señala que la normalización del recuento de plaquetas no siempre es un marcador preciso de resolución de la enfermedad. Nuestros resultados también confirman que el momento de presentación de los pacientes con SUH D+ sin trombocitopenia está usualmente alejado de los primeros síntomas intestinales, por lo que es necesario un alto índice de sospecha diagnóstica (AU)

Background: Thrombocytopenia is a hallmark of postdiarrhoeal haemolytic uraemic syndrome (D+ HUS), although it can be transient and therefore undetected. There is scarce information regarding the prevalence and the course of the disease in children with D+ HUS without thrombocytopenia. Objective: To determine the prevalence of D+ HUS without thrombocytopenia and to describe the clinical characteristics of a series of children with this condition. Patients and methods: The medical records of patients with D+ HUS hospitalised between 2000 and 2016 were reviewed to identify those without thrombocytopenia (>150,000mm3). Demographic, clinical and laboratory parameters of the selected cases were collected and descriptively analysed. Results: Nine cases (5.6%) without thrombocytopenia were identified among 161 patients hospitalised during the study period. Median age at diagnosis was 17 months (7-32) and median prodromal symptom duration was 15 days (7-21). Eight patients maintained normal urine output while the remaining one required dialysis. No patient presented with severe extrarenal compromise and/or hypertension. Conclusions: The prevalence of non-thrombocytopenic D+ HUS was 5.6% and most cases occurred with mild forms of the disease; however, the need for dialysis in one of them indicated that normalisation of platelet count is not always an accurate marker for disease remittance. Our results also confirm that the time of onset of D+ HUS in patients without thrombocytopenia is usually delayed with respect to the initial intestinal symptoms; thus, heightened diagnostic suspicion is necessary (AU)

Steak and Stupor: seizures and E. coli O157 infection.

Neurological complications of haemolytic uraemic syndrome (HUS) include altered states of consciousness, seizures, ischaemic stroke and encephalopathy. Adult-onset HUS is uncommon, and there is only a limited literature reporting neurological complications in this population. We report an adult with Shiga toxin-associated HUS complicated by focal-onset non-convulsive status epilepticus, who made a full neurological recovery.

Shiga Toxin-Producing Escherichia coli O157, England and Wales, 1983-2012.

We evaluated clinical Shiga toxin-producing Escherichia coli O157 infections in England and Wales during 1983-2012 to describe changes in microbiological and surveillance methods. A strain replacement event was captured; phage type (PT) 2 decreased to account for just 3% of cases by 2012, whereas PT8 and PT21/28 strains concurrently emerged, constituting almost two thirds of cases by 2012. Despite interventions to control and reduce transmission, incidence remained constant. However, sources of infection changed over time; outbreaks caused by contaminated meat and milk declined, suggesting that interventions aimed at reducing meat cross-contamination were effective. Petting farm and school and nursery outbreaks increased, suggesting the emergence of other modes of transmission and potentially contributing to the sustained incidence over time. Studies assessing interventions and consideration of policies and guidance should be undertaken to reduce Shiga toxin-producing E. coli O157 infections in England and Wales in line with the latest epidemiologic findings.

Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit.

Shiga toxin family comprises toxins belonging to two major groups, Stx1 and Stx2, produced by the bacteria Shigella dysenteriae and some strains of Escherichia coli. Shiga toxins are the leading cause of diarrhea associated with life threatening hemolytic uremic syndrome (HUS). StxA is a ribosome inactivating protein (RIP) which inhibits the protein synthesis in most species of prokaryotes and eukaryotes. An in vitro expression system has not been reported to produce full-length biological active StxA subunit; hence substantial progress has been hampered. In the present study, a DNA fragment (955 bp Gene Bank Accn No HM017965) encoding for subunit A of Stx was amplified from Shigella dysenteriae type 1 and subsequently cloned in pGEX-5X-2 vector. We successfully produced recombinant StxA as GST fusion protein in Escherichia coli using pGEX-5X-2-STXA construct under IPTG induction. For the purpose of immunization the GST-tag was removed by factor Xa mediated endoproteolytic cleavage from GST-StxA. Antisera raised against rStxA in mice reacted with recombinant purified protein of rStxA and lysate of Shiga toxin. It was shown that antisera produced against rStxA significantly recognized Stx producing strains of S. dysenteriae and E. coli. The antiserum produced effectively neutralized the Shiga toxin's cytotoxicity towards Vero cells.

Glycolipid binding preferences of Shiga toxin variants.

The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx), an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h) significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA) to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) in the presence of cell membrane components such as phosphatidylcholine (PC), cholesterol (Ch) and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

Multiple-aetiology enteric infections involving non-O157 Shiga toxin-producing Escherichia coli--FoodNet, 2001-2010.

We describe multiple-aetiology infections involving non-O157 Shiga toxin-producing Escherichia coli (STEC) identified through laboratory-based surveillance in nine FoodNet sites from 2001 to 2010. A multiple-aetiology infection (MEI) was defined as isolation of non-O157 STEC and laboratory evidence of any of the other nine pathogens under surveillance or isolation of >1 non-O157 STEC serogroup from the same person within a 7-day period. We compared exposures of patients with MEI during 2001-2010 with those of patients with single-aetiology non-O157 STEC infections (SEI) during 2008-2009 and with those of the FoodNet population from a survey conducted during 2006-2007. In total, 1870 non-O157 STEC infections were reported; 68 (3.6%) were MEI; 60 included pathogens other than non-O157 STEC; and eight involved >1 serogroup of non-O157 STEC. Of the 68 MEI, 21 (31%) were part of six outbreaks. STEC O111 was isolated in 44% of all MEI. Of patients with MEI, 50% had contact with farm animals compared with 29% (P < 0.01) of persons with SEI; this difference was driven by infections involving STEC O111. More patients with non-outbreak-associated MEI reported drinking well water (62%) than respondents in a population survey (19%) (P < 0.01). Drinking well water and having contact with animals may be important exposures for MEI, especially those involving STEC O111.

Protein expression, crystallization and preliminary X-ray crystallographic analysis of the isolated Shigella flexneri VapC toxin.

Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNA(fMet) in the anticodon region. Recombinant Shigella flexneri VapC(D7A) harbouring an active-site mutation was overexpressed in Escherichia coli, purified to homogeneity and crystallized by the vapour-diffusion technique. A preliminary X-ray crystallographic analysis shows that the crystals diffracted to at least 1.9 Å resolution at a synchrotron X-ray source and belonged to the trigonal space group in the hexagonal setting, H3, with unit-cell parameters a = b = 120.1, c = 52.5 Å, α = ß = 90, γ = 120°. The Matthews coefficient is 2.46 Å(3) Da(-1), suggesting two molecules per asymmetric unit and corresponding to a solvent content of 50.0%.

Outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 associated with romaine lettuce consumption, 2011.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the causal agent for more than 96,000 cases of diarrheal illness and 3,200 infection-attributable hospitalizations annually in the United States. MATERIALS AND METHODS: We defined a confirmed case as a compatible illness in a person with the outbreak strain during 10/07/2011-11/30/2011. Investigation included hypothesis generation, a case-control study utilizing geographically-matched controls, and a case series investigation. Environmental inspections and tracebacks were conducted. RESULTS: We identified 58 cases in 10 states; 67% were hospitalized and 6.4% developed hemolytic uremic syndrome. Any romaine consumption was significantly associated with illness (matched Odds Ratio (mOR) = 10.0, 95% Confidence Interval (CI) = 2.1-97.0). Grocery Store Chain A salad bar was significantly associated with illness (mOR = 18.9, 95% CI = 4.5-176.8). Two separate traceback investigations for romaine lettuce converged on Farm A. Case series results indicate that cases (64.9%) were more likely than the FoodNet population (47%) to eat romaine lettuce (p-value = 0.013); 61.3% of cases reported consuming romaine lettuce from the Grocery Store Chain A salad bar. CONCLUSIONS: This multistate outbreak of STEC O157:H7 infections was associated with consumption of romaine lettuce. Traceback analysis determined that a single common lot of romaine lettuce harvested from Farm A was used to supply Grocery Store Chain A and a university campus linked to a case with the outbreak strain. An investigation at Farm A did not identify the source of contamination. Improved ability to trace produce from the growing fields to the point of consumption will allow more timely prevention and control measures to be implemented.

Pathogenic bacteria in Finnish bulk tank milk.

While the quality of raw cow milk in Finland is known for its high hygienic standard, with the national average total bacterial count being below 10(4) CFU/mL annually, the prevalence of pathogenic bacteria in Finnish raw milk is underreported. The aim of this study was to determine the occurrence of Listeria monocytogenes, thermophilic Campylobacter spp., Salmonella spp., stx-positive Escherichia coli (STEC), coagulase-positive staphylococci, Yersinia spp., and Bacillus cereus group in raw cow milk samples collected from bulk tanks at 183 Finnish farms. Additionally, the hygienic quality of the milk was studied by determining the total bacterial and E. coli counts. L. monocytogenes was detected in 5.5% of the milk samples, with concentrations varying from <1 to 30 CFU/mL. Thermophilic Campylobacter spp. or Salmonella spp. were not detected in any of the samples. STEC with Shiga toxin-encoding stx2 was detected in 2.7% of the samples. Yersinia enterocolitica was detected in 7.7% of the samples; however, all isolates were negative for ail, suggesting that they were non-pathogenic. Coagulase-positive staphylococci were detected in 34.4% of the samples, with an average concentration of 25 CFU/mL in the positive samples. Members of the B. cereus group were detected in 20.8% of the samples, with an average concentration of 1 CFU/mL in the positive samples. No relationship was detected between E. coli or the total bacterial count and the presence of pathogenic bacteria, which suggests that pathogens can be present also in farms with excellent production hygiene. Although the concentration of pathogenic bacteria in fresh raw milk was mainly relatively low, it should be borne in mind that some of the pathogenic bacteria can survive and multiply at refrigeration temperatures and may cause a disease with a very low infectious dose. Thus, consumption of raw milk and related products poses a potential risk for food poisoning.

Shiga toxin-encoding genes (stx genes) in human faecal samples.

The aim of the two studies reported here was to investigate the distribution of stx genes in human faecal samples from volunteers and in faecal samples submitted to a regional microbiology hospital laboratory, and to isolate and characterize STEC from stx-positive samples. In total, faecal samples from 13.9% of 165 volunteers and 36.1% of 416 swabs from the regional microbiology hospital laboratory were positive for stx genes after screening by PCR. Isolation of STEC and of E. coli O157 from stx-positive faecal samples was performed by a filter-hybridization protocol and by automated immunomagnetic separation, respectively, and isolates were further characterized by serotyping, virulence typing by PCR and toxin production by the Vero cell assay. STEC were isolated from two samples only, an O146:H21 isolate from one of the volunteers and an O157:H7 isolate from a human case of diarrhoea. To conclude; the results show that it is not unusual to detect stx genes in faecal samples from humans in Norway, both from asymptomatic people and from people with gastrointestinal illness. This finding emphasizes the importance of correct diagnostic criteria for interpretation of the finding of an occasional stx-positive sample or an STEC isolate when searching for an aetiological agent of human cases of diarrhoea.

[Epidemic of EHEC (Escherichia coli O104:H4) in Europe in 2011--clinical and therapeutic problems]. / Epidemia EHEC (Escherichia coli O104:H4) w Europie w 2011 roku--problemy kliniczne i terapeutyczne.

Large outbreak of bloody diarrhoea complicated by haemolytic uraemic sundrome (HUS) has been observed in north Germany since May 2011. Epidemy spreaded throughout Germany and other countries and ceased at the end of July 2011. The WHO and German authorities confirmed that this epidemy was related to infection by new, unusual enteroaggregative Shiga toxin/verotoxin-producing Escherichia coli 014:H4 strain.

Development of a simple fed-batch process for the high-yield production of recombinant shiga toxin B-chain protein.

Shiga toxins are one of the very potent agents for causing dysentery, diarrhoea and haemolytic uremic syndrome with very low LD50. For better understanding of their biology, detection and neutralization, the components of toxins are needed to be expressed and purified in bulk amounts. However, following traditional expression procedures, this task is very tedious as the yield of the toxin is very low. In this manuscript, we have described the optimization of media for enhanced production of recombinant Shiga toxin B (rStx-B) chain protein in Escherichia coli. This protein is known to have neutralization ability against shiga toxins. Furthermore, fed-batch cultivation process in E. coli was also developed in the optimized medium. Expression was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG). The purification of protein involved Ni-NTA affinity chromatography under native conditions followed by gel filtration chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell weight and purified protein of about 19.41 g/l (dry cell weight, 11.38 g/l) and 30 mg/l of culture, respectively. The purity of the recombinant StxB protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Reactivity of this protein was determined by Western blotting as well as indirect ELISA using specific antibodies. These results establish the application of this protein for diagnosis of shiga toxin infection or for neutralizing the toxicity.

Attaching and effacing Escherichia coli and Shiga toxin-producing E. coli in children with acute diarrhoea and controls in Teresina/PI, Brazil.

This 3.5-year prospective study was conducted to ascertain the level of attaching and effacing Escherichia coli (AEEC) associated diarrhoea in children from Teresina, a northeastern state of Brazil. Passed faecal specimens from 400 patients (250 with and 150 without diarrhoea) up to 60 months of age attending from 2004 to 2007 at two public hospitals were investigated. Conventional microbiology methods and PCR were employed. Escherichia coli was isolated from 390 children, 240 of them with diarrhoea. A total of 117 AEEC strains were cultivated from specimens from 63 children, 37 with and 26 without diarrhoea. No association between AEEC and diarrhoea was observed. Atypical enteropathogenic E. coli (a-EPEC) (79.4%) was more commonly found than typical EPEC (t-EPEC). Association between EPEC and EPEC subtypes and diarrhoea was not detected. Mixed infection by t-EPEC and a-EPEC and infection by Shiga toxin-producing E. coli (STEC) were rare. Enteropathogenic E. coli was more common in males and in children aged less than 12 months. Correlation between serotyping and PCR results was 0.19. High resistance rates of AEEC to ampicillin, cephalotin, and trimethoprim-sulfamethoxazole were found. In conclusion, EPEC is very common in children with diarrhoea and controls in the population we studied, with a-EPEC predominating. This diarrhoeagenic E. coli (DEC) pathotype is more common in infant males and is resistant to drugs frequently used in clinical practice.

Detection of stx and stx genes in Pennsylvanian white-tailed deer.

Shiga toxin-producing E. coli carrying the stx(1) and/or stx(2) genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low percentage of samples that have tested positive for Stx-producing microbes, suggesting that while deer may contain these microbes, their overall abundance in deer is low. In this study, quantitative PCR (qPCR) was utilized to test for the presence of stx genes in white-tailed deer fecal matter in western Pennsylvania. In this culture independent screening, nearly half of the samples tested positive for the stx(2) gene, with a bias towards samples that were concentrated with stx(2). This study, while limited in scope, suggests that deer may be a greater reservoir for stx than was previously thought.

Characteristics of the Shiga-toxin-producing enteroaggregative Escherichia coli O104:H4 German outbreak strain and of STEC strains isolated in Spain.

A Shiga-toxin-producing Escherichia coli (STEC) strain belonging to serotype O104:H4, phylogenetic group B1 and sequence type ST678, with virulence features common to the enteroaggregative E. coli (EAEC) pathotype, was reported as the cause of the recent 2011 outbreak in Germany. The outbreak strain was determined to carry several virulence factors of extraintestinal pathogenic E. coli (ExPEC) and to be resistant to a wide range of antibiotics. There are only a few reports of serotype O104:H4, which is very rare in humans and has never been detected in animals or food. Several research groups obtained the complete genome sequence of isolates of the German outbreak strain as well as the genome sequences of EAEC of serotype O104:H4 strains from Africa. Those findings suggested that horizontal genetic transfer allowed the emergence of the highly virulent Shiga-toxin-producing enteroaggregative E. coli (STEAEC) O104:H4 strain responsible for the outbreak in Germany. Epidemiologic investigations supported a linkage between the outbreaks in Germany and France and traced their origin to fenugreek seeds imported from Africa. However, there has been no isolation of the causative strain O104:H4 from any of the samples of fenugreek seeds analyzed. Following the German outbreak, we conducted a large sampling to analyze the presence of STEC, EAEC, and other types of diarrheagenic E. coli strains in Spanish vegetables. During June and July 2011, 200 vegetable samples from different origins were analyzed. All were negative for the virulent serotype O104:H4 and only one lettuce sample (0.6%) was positive for a STEC strain of serotype O146:H21 (stx1, stx2), considered of low virulence. Despite the single positive case, the hygienic and sanitary quality of Spanish vegetables proved to be quite good. In 195 of the 200 samples (98%), <10 colony-forming units (cfu) of E. coli per gram were detected, and the microbiological levels of all samples were satisfactory (<100 cfu/g). The samples were also negative for other pathotypes of diarrheagenic E. coli (EAEC, ETEC, tEPEC, and EIEC). Consistent with data from other countries, STEC belonging to serotype O157:H7 and other serotypes have been isolated from beef, milk, cheese, and domestic (cattle, sheep, goats) and wild (deer, boar, fox) animals in Spain. Nevertheless, STEC outbreaks in Spain are rare.