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The interest in the development of nanoscale plasmonic technologies has dramatically increased in recent years. The photonic properties of plasmonic nanopatterns can be controlled and tuned via their size, shape, or the arrangement of their constituents. In this work, we propose a 2D hybrid metallic polymeric nanostructure based on the octupolar framework with enhanced sensing property. We analyze its plasmonic features both numerically and experimentally, demonstrating the higher values of their relevant figures of merit: we estimated a surface-enhanced Raman spectroscopy (SERS) enhancement factor of 9 × 10 7 and a SPR bulk sensitivity of 430 nm/RIU. In addition, our nanostructure exhibits a dual resonance in the visible and near-infrared region, enabling our system toward multispectral plasmonic analysis. Finally, we illustrate our design engineering strategy as enabled by electron beam lithography by the outstanding performance of a SERS-based biosensor that targets the Shiga toxin 2a, a clinically relevant bacterial toxin. To the best of our knowledge, this is the first time that a SERS fingerprint of this toxin has been evidenced.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Almost 5% of STEC infections result from waterborne exposures, yet there is no test listed in the EPA's current Selected Analytical Methods for the detection of active Shiga toxins (Stxs) in water. In this study, a HeLa cell-based assay is validated for the detection of metabolically active Stxs produced by STEC in water, including tap, bottled, and pond water. Active Stxs are detected even when the number of Stx-producing bacteria is less than 0.4 CFU/mL and the assay performance is not affected by background flora or chlorine in the water. This assay is not only as simple and affordable as cell-free assays but also detects active holotoxins without the use of live animals. In addition, the assay is designed for use in multi-well formats, making it ideal for high-throughput screening of water samples and therefore useful for environmental public health surveillance programs to reduce human risk of infection with STEC.

Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study, we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the non-DRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the non-DRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins toward DRMs and failure in interaction with non-DRMs. These results support the hypothesis of preferential binding of Stxs toward microdomains harboring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unraveling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lactosides having either an amino-triethylene glycol or an azido-triethylene glycol were designed and synthesized, and the two derivatives were immobilized onto silicon nitride (SiN) surfaces. When a click reaction was applied for the immobilization of the azido-sugar, a Ricinus communis lectin (RCA 120 ) was detected with a higher response by reflectometric interference spectroscopy (RIfS). When an N-hydroxysuccinimide (NHS) method was applied for the sugar immobilization, the response was less than that of the click one. The response of bovine serum albumin (BSA) as the negative control was negligible, but the lactose-SiN chip prepared by the click method suppressed nonspecific binding more effectively than did the chip from the NHS method. Next, we examined an antibody-immobilized SiN chip prepared by the click reaction. The detection response was, however, lower than that of the lactose-SiN chip, meaning that the sugar-chip by the click reaction was superior to the antibody-chip. Finally, to detect Shiga toxins from Escherichia coli O157:H7, globotrisaccharide (Gb 3 ) with an azido-triethylene glycol was synthesized and immobilized onto the SiN chip by the click reaction. The Gb 3 -SiN chips enabled us to detect the toxins at concentrations less than 100 ng/mL. RCA 120 , horse gram, gorse lectins and BSA showed no response to the Gb 3 -SiN chip, showing a high specificity for the toxin., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aims: To evaluate the performance of the IQ-Check kits and the USDA Microbiology Laboratory Guidebook (MLG) methods for detection of the top seven Shiga toxin-producing Escherichia coli (STEC) (O157:H7, O26, O45, O103, O111, O121 and O145) in ground beef and both STEC and Salmonella in co-inoculated samples., Methods and Results: Ground beef samples inoculated with ~10 CFU of STEC or both STEC and Salmonella Typhimurium were stored at 4°C for 72 h, followed by screening with the IQ-Check and BAX System kit (MLG) methods that employ different enrichment media. STEC and S. Typhimurium were detected after 12 and 18 h and their presence was confirmed by colony isolation., Conclusions: Both methods were able to detect STEC in ground beef after 12 h of enrichment in samples inoculated with low levels of the pathogen. STEC and S. Typhimurium can be detected and isolated in co-inoculated ground beef samples., Significance and Impact of the Study: The IQ-Check methods are comparable to the MLG methods for detection of STEC and simultaneous detection of STEC and S. Typhimurium in seeded ground beef after a short enrichment time, thus the IQ-Check method can be useful for the food industry for rapid detection of these pathogens., (Published 2016. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Functionalization of quantum dots (QDs) with a single biomolecular tag using traditional approaches in bulk solution has met with limited success. DNA polyhedra consist of an internal void bounded by a well-defined three-dimensional structured surface. The void can house cargo and the surface can be functionalized with stoichiometric and spatial precision. Here, we show that monofunctionalized QDs can be realized by encapsulating QDs inside DNA icosahedra and functionalizing the DNA shell with an endocytic ligand. We deployed the DNA-encapsulated QDs for real-time imaging of three different endocytic ligands-folic acid, galectin-3 (Gal3) and the Shiga toxin B-subunit (STxB). Single-particle tracking of Gal3- or STxB-functionalized QD-loaded DNA icosahedra allows us to monitor compartmental dynamics along endocytic pathways. These DNA-encapsulated QDs, which bear a unique stoichiometry of endocytic ligands, represent a new class of molecular probes for quantitative imaging of endocytic receptor dynamics., Competing Interests: Competing financial interests: BD owns NexDot, a for-profit company that provided some of the quantum dots used in this study.

Rational: Analysis of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) often relies upon sample preparation methods that result in cell lysis, e.g. bead-beating. However, Shiga toxin-producing Escherichia coli (STEC) can undergo bacteriophage-induced cell lysis triggered by antibiotic exposure that may allow greater selectivity of the proteins extracted., Methods: We have developed a sample preparation method for selective extraction of bacteriophage-encoded proteins and specifically Shiga toxins 1 and 2 (Stx1 & 2) expressed from STEC strains induced by DNA-damaging antibiotics. STEC strains were cultured overnight on agar supplemented with ciprofloxacin, mitomycin-C or an iron chelator to induce the bacteriophage lytic cycle with concomitant expression and release of Stx1 and/or Stx2. Sample preparation relied exclusively on bacteriophage lysis for release Stx into the extraction solution., Results: Three clinical STEC strains were analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and top-down proteomics analysis: E. coli O157:H7 strain EDL933, E. coli O91:H21 strain B2F1 and E. coli O26:H11 strain ECRC #05.2217. The B-subunit of Stx1a of EDL933 was detected and identified even though it was ~100-fold less abundant than the B-subunit of Stx2a that had been identified previously for this strain. Two bacteriophage-encoded proteins were also identified: L0117 and L0136. The B-subunits of Stx2d of strain B2F1 and Stx1a of strain ECRC #05.2217 were also detected and identified., Conclusions: Bacteriophage lysis appeared to enhance the detection sensitivity of Stx for these STEC strains compared to previous work using mechanical lysis. Detection/identification of other bacteriophage-encoded proteins (beyond Stx) tends to support the hypothesis of Stx release by bacteriophage cell lysis., (Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.)

A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5' end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3' end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients' stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

Shiga toxin-producing Escherichia coli (STEC) produce shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA) for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD) for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

A peptide aptamer that changes fluorescence upon binding to verotoxin was selected in vitro using ribosome display with a tRNA carrying an environment-sensitive fluorescent probe. The aptamer specifically bound to verotoxin with a dissociation constant (K d) of 3.94 ± 1.6 µM, and the fluorescence decreased by 78% as the verotoxin concentration was increased. The selected peptide can be used for detection of verotoxin.

Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg mL(-1) (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg mL(-1) (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg mL(-1) (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation <20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little cross-reactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

Objective: To evaluate the suitability of stx-PCR, Vero cell assay and commercial enzyme immunoassay for detection of Shiga toxin Escherichia coli and to compare sensitivity and specificity of three different methods for detection of Shiga toxin-producing Escherichia coli., Methods: Using stx-PCR, Vero cell assay and commercial enzyme immunoassay to detect 35 Escherichia coli reference strains and 45 strains isolated from food., Results: The three methods all had good specificity. 31 strains gave positive reaction in the Vero cell assay and in the stx-PCR. The consistency between the Vero cell assay and stx-PCR was 100%. Only 38 strains can be detected by commercial enzyme immunoassay., Conclusion: stx-PCR method can serve as a routine rapid detection method in the laboratory. Vero cell assay is recommended to be the gold standard to determine whether the bacteria had the functionally active toxin. Commercial kit was suitable for preliminary rapid detection during clinical testing and outbreaks of food-borne disease.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of worldwide importance, but a shortage of data exists for STEC isolation from India. Therefore, an epidemiological and environmental study that covers a large geographic area in north India was conducted. Ruminant stool samples (n=650) were collected from 59 dairies. Meat samples (n=450) were collected from local abattoirs and the main slaughterhouse of the region. Additionally, 600 human cases of diarrhea and hemolytic uremic syndrome were screened for STEC. Isolates were characterized for the virulence gene profiles and for the serogroups and were submitted to molecular typing by the multilocus variable-number tandem-repeat analysis (MLVA). Overall, 12.3% of animal stool samples and 6.3% of mutton samples (n=160) were positive for STEC. Additionally, STEC were isolated from 1.7% and 1.6% of watery (n=290) and bloody (n=310) stool specimens, respectively. Animal stool isolates were significantly more prevalent in hilly areas (p<0.05) than in plain areas. Polymerase chain reaction demonstrated the presence of stx1, stx2, hly, espP, saa, toxB, and iha genes in 117 (83.5%), 94 (67.1%), 77 (55%), 33 (23%), 62 (44.2%), 29 (20.7%), and 51 (36%) of the isolates, respectively. Five new serogroups (O55, O33, O173, O165, and O136) are being reported for the first time from India. Four isolates from serogroup O103 were found in mutton and stool specimens of cattle and humans (n=160). One isolate from serogroup O104 was isolated from a mutton sample. MLVA suggested the potential transmission of STEC from contaminated meat and bovine sources. This study confirms the frequent contamination of mutton samples (24%), whereas chicken and pork samples were negative for STEC. This study demonstrates the presence of STEC that carry a large repertoire of virulence genes and the potential transmission of STEC from contaminated mutton and animal stools in north India.

Shiga-like toxins (verotoxins) are a class of AB5 holotoxins that are primarily responsible for the virulence associated with Shiga-like toxin producing Escherichia coli (STEC) infections. The holotoxins are composed of a pentamer of identical subunits (B subunit) responsible for delivering the catalytic subunit (A subunit) to a host cell and facilitating endocytosis of the toxin into the cell. The B subunits are not associated with toxicity. We developed a multiple reaction monitoring method based on analyzing conserved peptides, derived from the tryptic digestion of the B subunits. Stable-isotope-labeled analogues were prepared and used as internal standards to identify and quantify these characteristic peptides. We were able to detect and quantify Shiga toxins (Stx), Shiga-like toxin type 1 (Stx1) and type 2 (Stx2) subtypes, and to distinguish among most of the known subtypes. The limit of detection for digested pure standards was in the low attomole range/injection (~10 attomoles), which corresponded to a concentration of 1.7 femtomol/mL. A matrix effect was observed when dilute samples were digested in the buffer, Luria broth, or mouse plasma (LOD ~ 30 attomol/injection = 5 femtomol/mL). In addition, we determined that the procedures necessary to perform our mass spectrometry-based analysis completely inactivate the toxins present in the sample. This is a safe and effective method of detecting and quantitating Stx, Stx1, and Stx2, since it does not require the use of intact toxins.

The immunochromatographic assay, which targets Shiga toxin 1/verotoxin 1 (VT1) and/or Shiga toxin 2/verotoxin 2 (VT2) independently with same test device, was used for easily, rapidly and specifically detecting verotoxin-producing Escherichia coli among E. coli strains from food and fecal materials. All 10 strains of VT 1 and/or VT 2- producing E. coli among E. coli isolates from various sources showed a positive reaction to VT1- or VT2- antibodies, but other gram-negative and positive bacterial species had a negative reaction. Bacterial counts of 10(8) cfu/ml in enrichment broth and food suspension were required for the detection of VT-producing E. coli. The IC assay described here could detect easily and specifically the verotoxin-producing E. coli within 20 min by pure culture.

In Greece, Shiga toxin-producing Escherichia coli (STEC) have only been sporadically reported. The objective of this study was to estimate the prevalence of STEC and Escherichia coli O157:H7 in farm animals, vegetables, and humans in Greece. A total number of 1,010 fecal samples were collected from farm animals (sheep, goats, cattle, chickens, pigs), 667 diarrheal samples from humans, and 60 from vegetables, which were cultured in specific media for STEC isolates. Enzyme-linked immunosorbent assay (ELISA) was used to detect toxin-producing colonies, which, subsequently, were subjected to a multiplex polymerase chain reaction (PCR) for stx1, stx2, eae, rfbE O157, and fliC h7 genes. Eighty isolates (7.9 %) from animal samples were found to produce Shiga toxin by ELISA, while by PCR, O157 STEC isolates were detected from 8 (0.8 %) samples and non-O157 STEC isolates from 43 (4.2 %) samples. STEC isolates were recovered mainly from sheep and goats, rarely from cattle, and not from pigs and chickens, suggesting that small ruminants constitute a potential risk for human infections. However, only three human specimens (0.4 %) were positive for the detection of Shiga toxins and all were PCR-negative. Similarly, all 60 vegetable samples were negative for toxin production and for toxin genes, but three samples (two roman rockets and one spinach) were positive by PCR for rfbE O157 and fliC h7 genes. These findings indicate that sheep, goats, cattle, and leafy vegetables can be a reservoir of STEC and Escherichia coli O157:H7 isolates in Greece, which are still rarely detected among humans.

A functional detection assay was developed for Escherichia coli secreted shiga-like toxin based on antibody capture and visualization with a multivalent galabiose ligand. It was possible to detect verotoxin in medically relevant E. coli samples in a dose dependent fashion. This method is a new step towards measuring functional protein levels in complex mixtures, which can be used for diagnostic purposes in a clinical setting., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

When 3,972 ground beef enrichments with 6 confirmed to contain a non-O157 Shiga toxin-producing intimin-positive Escherichia coli isolate were tested for Shiga toxin, intimin, and O group (O26, O45, O103, O111, O121, and O145) genes, 183 potential positives and only 2 of the 6 confirmed positives were identified.

Conventional enzyme-linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy, and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., Escherichia coli O157) of E. coli isolates.

Beef organ meat, such as liver, and beef are major food sources contaminated with Escherichia coli O157. This study investigated the detection method of E. coli O157 in beef liver and carcass. In an experiment with beef liver inoculated with E. coli O157, the direct plating method, plating after the immunomagnetic separation (IMS) method, and Shiga toxin (Stx)-producing E. coli detection and E. coli O157 detection loop-mediated isothermal amplification (LAMP) assays were compared for the detection of Stx-producing E. coli O157. Fifty percent and 45% of samples were positive by Stx-producing E. coli detection LAMP assay and E. coli O157 detection LAMP assay, respectively. Thirty-five percent and 10% of samples were positive by the IMS method and direct plating method, respectively. In an examination of beef swab samples, contamination frequencies with E. coli O157 were analyzed by LAMP assays and the IMS method. E. coli O157 was detected in 12 of 230 samples (5.2%). There was no sample positive for E. coli O157 isolation but negative for LAMP assays for Stx gene and O157 antigen gene. Four samples (1.7%) were positive by both LAMP assays but negative by the IMS method. The result that there was no sample positive for the O157 antigen gene, but not the Stx gene, indicated that the IMS method failed to detect E. coli O157. Twenty-nine samples (12.6%) were positive for the Stx gene but not the O157 antigen gene. The results indicated that screening of Stx gene and O157 antigen gene by LAMP assays is effective in saving time and effort to isolate E. coli O157 by the IMS method because the LAMP assay is more sensitive. This suggested that samples positive for Stx gene and O157 antigen gene should be examined by the IMS method to isolate E. coli O157.

Aims: The Shiga-like toxins (Stx) are critical virulence factors of enterohaemorrhagic Escherichia coli (EHEC). Stx1B subunit plays important roles in EHEC infection. This work aims to generate and characterize monoclonal antibodies (mAbs) against the Stx1B and to investigate their utility in discrimination ELISA., Methods and Results: Two newly identified mAbs (designated 2H8 and 1B10, respectively) against the Stx1B protein were prepared via hybridoma techniques. The immunoreactivity of both mAbs to the Stx1B protein was confirmed in ELISA and Western blot. Moreover, they differentiate EHEC from Salmonella enteritis, non-Stx1-producing E. coli, Mycobacterium tuberculosis, Listeria monocytogenes, Streptococcus agalactiae and Staphylococcus aureus., Conclusions: The anti-STx1B mAbs are valuable diagnostic reagents for distinguishing EHEC from other bacteria., Significance and Impact of the Study: This is the first report regarding the usage of anti-STx1B mAbs in discrimination ELISA. The established ELISA may have potential in clinical surveillance of EHEC infection., (© 2010 The Authors. © 2010 The Society for Applied Microbiology.)

The paper presents the results of etiological interpretation of the outbreak of Escherichia coli O157-induced enteric infection complicated by hemolytic uremic syndrome in Omsk in spring 2009. Seventeen infants aged 5 months to 2.5 years were examined; of them 9 patients were on hemodialysis. The diagnosis was verified in 7 children, by isolating verotoxin after fecal enrichment on the concentrating nutrient RIDA Enrichment Bouillon medium containing bile salts and mitomycin C, E. coli O157:H7 cultures on the sorbitol medium and/or by identifying E. coli O157 DNA. Six (4 having verotoxin) infants were found to have rotavirus and noro-virus antigens or DNA/RNA, which may make a contribution into the development of mixed infection and increase the risk of complications.

Enterohaemorrhagic Escherichia coli O-157 : H7 produces verotoxins called Shiga toxin or Shiga-like toxin. The type 1 and type 2 toxins present different immunological and physicochemical characteristics, and cause symptoms such as watery diarrhea, bloody diarrhea, severe stomach ache, fever and vomiting. The toxins can be assayed by several methods, including ELISA and PCR, with some limitations as to sensitivity. In the present study, we used a ProteoChip, which requires smaller volumes of reagents and allows detection of lower concentrations of the toxins, compared with the conventional assay.

Shiga toxin--producing Escherichia coli (STEC) are a leading cause of bacterial enteric infections in the United States. Prompt, accurate diagnosis of STEC infection is important because appropriate treatment early in the course of infection might decrease the risk for serious complications such as renal damage and improve overall patient outcome. In addition, prompt laboratory identification of STEC strains is essential for detecting new and emerging serotypes, for effective and timely outbreak responses and control measures, and for monitoring trends in disease epidemiology. Guidelines for laboratory identification of STEC infections by clinical laboratories were published in 2006. This report provides comprehensive and detailed recommendations for STEC testing by clinical laboratories, including the recommendation that all stools submitted for routine testing from patients with acute community-acquired diarrhea (regardless of patient age, season of the year, or presence or absence of blood in the stool) be simultaneously cultured for E. coli O157:H7 (O157 STEC) and tested with an assay that detects Shiga toxins to detect non-O157 STEC. The report also includes detailed procedures for specimen selection, handling, and transport; a review of culture and nonculture tests for STEC detection; and clinical considerations and recommendations for management of patients with STEC infection. Improving the diagnostic accuracy of STEC infection by clinical laboratories should ensure prompt diagnosis and treatment of these infections in patients and increase detection of STEC outbreaks in the community.

A total of 1602 stool samples from healthy employees in a slaughter company were screened by PCR for Shiga toxin (Stx)-producing Escherichia coli (STEC). The PCR product of Stx-encoding genes was detected in 90 (5.6%) of 1602 stool samples. Among the 90 stx-positive workers, the Residual Products Handlers and Slaughterers had rates of 8.0% and 6.0%--higher than Inspectors, Grading Testers and Livestock Hygiene Controllers at 3.3%, 2.0% and 3.5%, respectively. Forty-nine (54.4%) were shown to have stx2; 25 (27.7%) carried stx1 and 16 (17.7%) had both stx1 and 2. Distribution of the stx PCR-positive workers by age revealed an increase in STEC infection with age (P<0.05). Phenotypic and genotypic traits of nine STEC strains isolated from eight slaughter plant workers were characterized. A variety of serotypes, five O serogroups (O8, O54, O59, O103 and O153) and two H serogroups (H7 and H32) were found, but none of the strains belonged to the serogroup O157. Eight Vero cell cytotoxicity assay-positive strains were isolated from the workers and these workers were asymptomatic and healthy. The results of the study show that slaughter plant workers are at high risk of STEC infection.

In this study, 100 raw meat samples were collected from 15 local Moroccan butcheries in five different areas of the city of Rabat during a period of 4 months. Overall, 7 of 15 butcheries from three areas of the city yielded strains of Escherichia coli O157. Single isolates from 9 (9%) of 100 raw meat samples were biochemically and serologically confirmed as E. coli O157. Using molecular techniques, two strains were positive for the Shiga toxin, with two additional strains containing an attaching-effacing gene. All potentially virulent serotypes isolated from these meat samples showed distinct pulsed-field gel electrophoresis profiles. Based on antibiotic susceptibility testing, more than 70% of the isolates were resistant to ampicillin and clavulanic acid-amoxicillin. Moreover, one strain was resistant to more than three antibiotics. Our study represents the first survey of E. coli O157 and related serotypes in raw meat products in Morocco.

Enterohemorrhagic Escherichia coli serotype O157:H7 is a pathotype of diarrheagenic E. coli that produces one or more Shiga toxins, forms a characteristic histopathology described as attaching and effacing lesions, and possesses the large virulence plasmid pO157. The bacterium is recognized worldwide, especially in developed countries, as an emerging food-borne bacterial pathogen, which causes disease in humans and in some animals. Healthy cattle are the principal and natural reservoir of E. coli O157:H7, and most disease outbreaks are, therefore, due to consumption of fecally contaminated bovine foods or dairy products. In this review, we provide a general overview of E. coli O157:H7 infection, especially focusing on the bacterial characteristics rather than on the host responses during infection.

An alternating layer-by-layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb(2)), beta-lactoside (beta-Lac), or alpha-D-mannoside (alpha-Man) residues were prepared, and used for the detection of Shiga toxins, Stx-1 and Stx-2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx-1 and Stx-2 show binding specificity for the Gb(2) glycochip as well as a weak affinity for the beta-Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb(2) polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43 %) in the Gb(2) glycopolymer. The maximal affinity constants of Stx-1 and Stx-2 (K(a)=10(8)-10(9) M(-1)) enabled highly sensitive and facile analysis (10 ng mL(-1), 30 min). When Gb(2) glycopolymers were used as competitors, Stx-1 and Stx-2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins.

We present an outbreak of E. coli O157:H7 diarrhea in an urban child care center. Eleven of 45 attendees with diarrhea had positive tests (stool culture or shiga-like toxin assay) for E. coli O157:H7. Two of these 11 (18%) progressed to hemolytic uremic syndrome. Diarrheal illness in child care centers should be considered a public health risk. Staff education, hand washing, and cohorting or exclusion of attendees with diarrhea must be performed to help control infectious outbreaks.

Rapid detection of enterohemorrhagic Escherichia coli is important for its successful treatment. We have evaluated the immunochromatographic Duopath Verotoxin-test for detection of Shiga toxins, in comparison with enzyme-linked immunosorbent assay and polymerase chain reaction, on 240 clinical human stool samples. The Duopath-test showed a lower sensitivity and specificity.

In this report we describe the detection and duration of fecal shedding of Shiga toxin-producing Escherichia coil (STEC) O157 and non-O157 in symptomatic and asymptomatic cases during four events occurred among children in day-care centers in Argentina. In each event, the cases were identified among children, family contacts and staff members of the Institution. The isolates were characterized by pheno-genotyping and subtyping methods. The STEC fecal shedding was prolonged and intermittent. Strains O157:H7 (1st event); O26:H11 (2nd event); O26:H11 (3rd event) and O145:NM (4th event) were shed during 23-30, 37, 31 and 19 days, respectively. Considering the possibility of STEC intermittent long-term shedding, symptomatic and asymptomatic individuals should be excluded from the Institution until two consecutive stool cultures obtained at least 48 h apart, test negative.

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-gamma1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx(1), stx(2), and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity >85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.

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., Methods and Results: The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains., Conclusions: The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants., Significance and Impact of the Study: This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

This work aims to provide a strategy for rapidly screening food raw materials of bovine origin for the presence of the most frequent O-serogroups of Shiga toxin-encoding Escherichia coli (STEC) involved in food poisoning outbreaks. The prevalence of highly pathogenic serogroups of STEC was surveyed in 25 g portions of minced meat and raw milk using PCR-ELISA and multiplex real-time PCR assays. The prevalence of STEC in raw milk (n=205) and meat samples (n=300) was 21% and 15%, respectively. Contamination by the main pathogenic E. coli O-serogroups representing a major public health concern, including O26, O103, O111, O145, and O157, was potentially around 2.6% in minced meat and 4.8% in raw milk. The MPN values showed an overall contamination ranging from 1 to 2 MPN cells from highly pathogenic serogroups/kg. This survey would indicate that the human pathogenic potential of STEC present in these samples probably remains limited. No conclusion can be drawn at the moment concerning a potential risk for consumers. This rapid screening approach for evaluating the potential presence of highly pathogenic serogroups of STEC in food raw materials should help to improve risk assessment of food poisoning outbreaks.

The contamination of beef carcasses with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC) obtained from a slaughter plant in Guadalajara, Mexico was investigated. A total of 258 beef carcasses were sampled during a 12-month period. All samples were assayed for STEC by selective enrichment in modified tryptone soy broth supplemented with cefixime, cefsulodin and vancomycin, followed by plating on Sorbitol MacConkey Agar supplemented with cefixime and tellurite (CT-SMAC). Simultaneously, all samples were assayed by immunomagnetic separation (IMS) and plated on CT-SMAC and CHROMagar. The presence of the stx1, stx2, eaeA and hly933 genes, recognized as major virulence factors of STEC, was tested for O157:H7 and non-O157 E. coli isolates by multiplex polymerase chain reaction (PCR). STEC was detected in two (0.8%) samples. One of these STEC isolates corresponded to the serotype O157:H7 showing stx2, eaeA and hyl933 genes. The other isolate corresponded to non-O157 STEC and only had the stx1 gene. Thirteen carcasses (5%) were positive for nonmotile E. coli O157 and 7 (2.7%) were positive for E. coli O157:H7. The presence of O157:H7 and non-O157 STEC on beef carcasses in this slaughter plant in Guadalajara, Mexico, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.

Escherichia coli O157:H7 and other strains of E. coli that produce Shiga toxin are collectively known as Shiga toxin-producing E. coli (STEC). The current outbreak of STEC O157 infections associated with eating fresh spinach illustrates the importance of obtaining isolates to identify the source of the infections. Laboratory methods that do not require bacterial culture of stool specimens to identify STEC are being used increasingly by clinical diagnostic laboratories, sometimes without subsequent confirmation of a strain by isolating it in culture. This report describes findings from outbreaks of gastroenteritis in 2005 in New York and North Carolina in which clinical diagnostic laboratories initially used only non-culture methods to detect Shiga toxin (Stx). The findings highlight the importance of confirmation of Stx-positive stool specimens by bacterial culture for timely and reliable identification of STEC infections, including E. coli O157 and non-O157 STEC, to enable implementation of appropriate public health actions. An important part of that identification is determining the serotype of all STEC isolates and the subtype of STEC O157 strains so that outbreaks can be detected and traced back to sources.

In order to study the effects of the fermentation-drying procedure and subsequent in vitro digestion on Shiga toxins (Stx) production by Escherichia coli O157:H7, dry sausages were inoculated during the formulation step with pure cultures of strains 5-1 and ATCC 43895. The inoculated sausages were submitted to a minimum (30 min, pH between 3.1 and 3.5) or a maximum (120 min at stepwise adjusting the pH downward) gastric challenge followed by a 240-min pancreatic challenge at pH 8.0 and 37 degrees C. Production of toxins by the overnight culture controls, assessed using the Vero cell assay, was dependent on the pathogen cell concentration. The effect of cell concentration was not relevant in sausage samples and data showed: (a) higher Stx production in contaminated sausage samples than in overnight cultures; (b) the lowest Stx levels were detected with undigested sausage samples; (c) the maximum gastric challenge enhanced Stx production, compared to minimally digested and undigested samples. Reverse transcriptase polymerase chain reaction (RT-PCR) performed on extracts from inoculated, digested (4.5-6 h process) and undigested sausages produced amplicons for both stx1 and stx2 mRNA, suggesting that post-stress expression of stx genes had occurred. Our data suggest that sub-lethal stresses imposed by the fermentation-drying procedure and subsequent digestion of ingested food (i.e. contaminated sausages) may affect the degree to which the surviving E. coli O157:H7 cells express their virulence in vivo.

Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) infections are a threat to public health. VTEC O157 has been isolated from gulls but evidence of transmission to humans from birds has not been reported. We recount an incident of VTEC O157 infection affecting two sibling children who had no direct contact with farm animals. An outbreak control team was convened to investigate the source of infection, its likely mode of transmission, and to advise on control measures. Human and veterinary samples were examined and the human isolates were found to be identical to an isolate from a sample of bird (rook) faeces. Cattle, rabbit and environmental samples were negative. This report provides evidence that birds may act as intermediaries for human infection with VTEC O157.

This study investigated the prevalence and level of Escherichia coli O157 on samples of beef trimmings (n=1351), beef carcasses (n=132) and bovine head meat (n=132) in a beef slaughter plant in Ireland. The survey also included an assessment of the prevalence of virulence genes in the E. coli O157 isolates obtained. Samples were examined for the presence of E. coli O157 by direct plating on SMAC-CT and by enrichment/immunomagnetic separation (IMS) with plating of recovered immunobeads onto SMAC-CT agar. Presumptive E. coli O157 isolates were confirmed by PCR targeting a range of genes i.e. vt1, vt2, eaeA, hlyA, fliC(h7) and portions of the rfb (O-antigen encoding) region of E. coli O157. Enterobacteriaceae on head meat samples were estimated by direct plating onto Violet Red Bile Glucose agar. E. coli O157 was recovered from 2.4% (32/1351) of beef trimmings samples, at concentrations ranging from<0.70-1.61 log10 cfu g(-1). Of the 32 positive isolates, 31 contained the eaeA and hylA genes while 30/32 contained the fliC(h7) gene and 31/32 contained vt1 or vt2, or both vt genes. E. coli O157 was recovered from 3.0% (4/132) of carcass samples, at concentrations ranging from <0.70-1.41 log10 cfu g(-1). All of the carcass isolates contained the eaeA, hylA and fliC(h7) genes. E. coli O157 was recovered from 3.0% (3/100) of head meat samples, at concentrations of 0.7-1.0 log10 cfu g(-1). All of the head meat isolates contained the eaeA, hylA, fliC(h7) and vt2 genes. No head meat isolates contained the vt1 gene. Head meat samples (n=100) contained Enterobacteriaceae, at concentrations ranging from 0.70-3.0 log10 cfu g(-1). Overall, the qualitative and quantitative data obtained for E. coli O157 on beef trimming samples in this study could be employed as part of a quantitative risk assessment model.

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.

Escherichia coli O157:H7 (HEC), E. coli O157:H7 rpoS mutant (HEC-RM), and nonpathogenic E. coli (NPEC) were step-wise adapted to trisodium phosphate (TSP) by incubation in broths of increasing concentration, from 0% to 0.6%, at 37 degrees C for 24 h. After incubation at each concentration, each population was examined for acid resistance (D value) in simulated gastric fluid of pH 1.5, cell envelope membrane lipid composition, and intracellular and extracellular verotoxin concentrations. The ratio of cis-vaccenic acid (18:1omega7c) to palmitic acid (16:0) increased, indicating increased membrane fluidity with increasing TSP concentration up to 0.4%, but decreased at 0.6%. HEC and HEC-RM adapted at 0.4% TSP had the highest verotoxin concentrations of 1805 and 1879 ng/ml, respectively. In addition, with HEC the ratio of extracellular to intracellular verotoxin concentration decreased at higher TSP concentrations. In contrast, the ratio for HEC-RM increased at 0.4% TSP. HEC adapted to 0.4% TSP had the greatest survival in gastric fluid (58 min D value) among all treatments. For HEC, the increase in membrane fluidity was associated with increased acid resistance and extracellular verotoxin concentration for cells adapted to 0.4% TSP. In contrast, the increase in membrane fluidity was associated with decreased acid resistance of TSP adapted HEC-RM although the extracellular verotoxin concentration increased. Therefore, the deletion of the rpoS gene appeared to affect the changes in verotoxin concentration and acid resistance of TSP adapted E. coli O157:H7.

Of 45 Escherichia coli O157 isolates from cattle feces, which were collected between May 2000 and September 2003 in Korea, 32 were resistant to at least 1 antibiotic and 28 were resistant to 4 or more antibiotics, with 32, 30 and 30 of the isolates being resistant to streptomycin, tetracycline and sulfisoxazole, respectively. Two isolates were resistant to fluoroquinolones and to 10 or more of the 22 other antimicrobial agents that were tested. Thirteen antimicrobial resistant patterns were observed. The most frequent resistance type, which was found for 11 isolates, was streptomycin-tetracycline-kanamycin-ampicillin-piperacillin-cephalothin-sulfisoxazole-ticarcillin. Polymerase chain reaction (PCR) analysis of the isolates for E. coli O157 virulence markers revealed that 25 of the resistant E. coli O157 isolates tested positive for stx2 or both stx1 and stx2 genes. These findings suggest that many of the resistant E. coli O157 isolates might cause disease in humans.

Shiga toxin-producing Escherichia coli (STEC) cause sporadic cases and outbreaks of nonbloody and bloody diarrhea, and hemolytic uremic syndrome (HUS). E. coil O157:H7 is the most prevalent STEC serotype. However, other serotypes (O26:H11; O103:H2; O111:NM; O121:H19; O145:NM, among others) can cause a similar disease spectrum. Shiga toxins (Stx1, Stx2, and their variants), intimin, and enterohemolysin are the main virulence factors. Three different diagnostic criteria are used to determine the frequency of STEC infection: 1) isolation and characterization of STEC strains; 2) detection of specifically neutralizable free fecal Stx; and 3) Serological tests to detect Stx-antibodies. The surveillance of the STEC strains is performed using subtyping techniques: a) genotyping of Stx and eae by PCR-RFLP; b) phage typing of E. coil O157 strains; and c) pulsed-field gel electrophoresis. STEC O157 and non-O157 strains are recovered from clinic, animal, food and environmental samples, and E. coli O157:H7, a Stx2 and Stx2c producer, harboring eae and ehxA genes, is the most common serotype. During a prospective case-control study conducted to evaluate risk factors for sporadic STEC infection in Mendoza Province and Buenos Aires City and its surroundings during 2001-2002, exposures associated with risk included eating undercooked beef, contact with a child < 5 years with diarrhea and living in or visiting a place with farm animals. Both washing hands after handling raw beef, and eating fruits and vegetables were frequently protective. Strategies of prevention and control are necessary to decrease the incidence of STEC infections in Argentina.

Faecal samples from 222 healthy dairy goats on 12 farms in Spain, as well as bulk tank milk samples of these farms, were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and enteropathogenic E. coli (EPEC). VTEC and EPEC were isolated in 47.7 and 7.7% of the animals, respectively. VTEC were isolated more frequently from adults and replacement animals than from goat kids. In contrast, EPEC were detected more frequently from goat kids than from replacement animals and adults. VTEC or EPEC strains were not detected in the bulk tank milk samples. Although a selective enrichment protocol was used, the serotype O157:H7 was not detected. The most frequent serotypes among the 106 VTEC strains isolated from goats were O5:H-, O76:H19, O126:H8, O146:H21, ONT:H- and ONT:H21. None VTEC strain was eae-positive. The absence of the eae gene in the VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, 16% of VTEC strains isolated from healthy goats belonged to serotypes associated with haemolytic uraemic syndrome in humans. The ehxA gene was detected in 84.9 and 52.9% of the VTEC and EPEC from goats, respectively. The beta1, theta/gamma2 and zeta were the most frequent intimin types among the 17 EPEC strains studied and the most prevalent serotypes of these strains were O156:H25 and O177:H11. Our data show that in Spain healthy goats are an important reservoir of VTEC and EPEC, and a potential source of infection for humans.

A total of 722 Shiga toxin-producing Escherichia coli (STEC) isolates recovered from humans, cattle, ovines and food during the period from 1992 to 1999 in Spain were examined to determine antimicrobial resistance profiles and their association with serotypes, phage types and virulence genes. Fifty-eight (41%) out of 141 STEC O157:H7 strains and 240 (41%) out of 581 non-O157 STEC strains showed resistance to at least one of the 26 antimicrobial agents tested. STEC O157:H7 showed a higher percentage of resistant strains recovered from bovine (53%) and beef meat (57%) than from human (23%) and ovine (20%) sources, whereas the highest prevalence of antimicrobial resistance in non-O157 STEC was found among isolates recovered from beef meat (55%) and human patients (47%). Sulfisoxazole (36%) had the most common antimicrobial resistance, followed by tetracycline (32%), streptomycin (29%), ampicillin (10%), trimethoprim (8%), cotrimoxazole (8%), chloramphenicol (7%), kanamycin (7%), piperacillin (6%), and neomycin (5%). The multiple resistance pattern most often observed was that of streptomycin, sulfisoxazole, and tetracycline. Ten (7%) STEC O157:H7 and 71 (12%) non-O157 strains were resistant to five or more antimicrobial agents. Most strains showing resistance to five or more antimicrobial agents belonged to serotypes O4:H4 (4 strains), O8:H21 (3 strains), O20:H19 (6 strains), O26:H11 (8 strains eae-beta1), O111:H- (3 strains eae-gamma2), O118:H- (2 strains eae-beta1), O118:H16 (5 strains eae-beta1), O128:H- (2 strains), O145:H8 or O145:H- (2 strains eae-gamma1), O157:H7 (10 strains eae-gamma1), O171:H25 (3 strains), O177:H11 (5 strains eae-beta1), ONT:H- (3 strains/1 eae-beta1) and ONT:H21 (2 strains). Interestingly, most of these serotypes, i.e., those indicated in bold) were found among human STEC strains isolated from patients with hemolytic uremic-syndrome (HUS) reported in previous studies. We also detected, among non-O157 strains, an association between a higher level of multiple resistance to antibiotics and the presence of the virulence genes eae and stx(1). Moreover, STEC O157:H7, showed an association between certain phage types, PT21/28 (90%), PT23 (75%), PT34 (75%), and PT2 (54%), with a higher number of resistant strains. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in animals is highly recommended.