Rapid and Label-Free Immunosensing of Shiga Toxin Subtypes with Surface Plasmon Resonance Imaging.

Shiga toxin-producing Escherichia coli (STEC) are responsible for gastrointestinal diseases reported in numerous outbreaks around the world as well as in the United States. Current detection methods have limitation to implement for rapid field-deployable detection with high volume of samples that are needed for regulatory purposes. Surface plasmon resonance imaging (SPRi) has proved to achieve rapid and label-free screening of multiple pathogens simultaneously, so it was evaluated in this work for the detection of Shiga toxins (Stx1a and Stx2a toxoids were used as the less toxic alternatives to Stx1 and Stx2, respectively). Multiple antibodies (Stx1pAb, Stx1-1mAb, Stx1-2mAb, Stx1d-3mAb, Stx1e-4mAb, Stx2pAb, Stx2-1mAb, Stx2-2mAb, and Stx2-10mAb) were spotted one by one by programed microarrayer, on the same high-throughput biochip with 50-nm gold film through multiple crosslinking and blocking steps to improve the orientation of antibodies on the biochip surface. Shiga toxins were detected based on the SPRi signal difference (ΔR) between immobilized testing antibodies and immunoglobulin G (IgG) control. Among the antibodies tested, Stx1pAb showed the highest sensitivity for Stx1 toxoid, with the limit of detection (LOD) of 50 ng/mL and detection time of 20 min. Both Stx2-1mAb and Stx2-2mAb exhibited high sensitivity for Stx2 toxoid. Furthermore, gold nanoparticles (GNPs) were used to amplify the SPRi signals of monoclonal antibodies in a sandwich platform. The LOD reached the level of picogram (pg)/mL with the help of GNP-antibody conjugate. This result proved that SPRi biochip with selected antibodies has the potential for rapid, high-throughput and multiplex detection of Shiga toxins.

Mass spectrometry-based Shiga toxin identification: A clinical validation.

After we published our preliminary study on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and curated E. coli toxin databases on the identification of E. coli Shiga toxins (Stxs) in the Journal of Proteomics in year 2018, we were encouraged to further refine the method and test clinical isolates. In this study, different concentrations of mitomycin C (MMC) and ciprofloxacin (CF), two common antibiotic/chemotherapy agents capable of stimulating Stx production, were first tested and compared on three reference strains and eight clinical isolates to observe the toxin induction and subsequent identification. Notably, no differences were observed between the two agents other than the concentrations applied. Seventeen more clinical isolates were then tested using fixed MMC and CF concentrations and sample amount. This study confirms that the majority of stx2-positive E. coli strains can be stimulated to produce sufficient toxin for confident identification. This does not occur with stx1-positive E. coli isolates, however, despite the fact that both Stxs can be identified for several isolates without MMC or CF stimulation. BIOLOGICAL SIGNIFICANCE: Stxs, especially Stx2, are very important causes of severe food-borne disease, even death. This study confirms that receptor analogue-based affinity enrichment of Stxs, after MMC or CF treatment of E. coli, is useful for fast and accurate Stx2 identification through LC-MS/MS.

Selective Detection of Shiga-like Toxin 1 from Complex Samples Using Pigeon Ovalbumin Functionalized Gold Nanoparticles as Affinity Probes.

Escherichia coli O157:H7 is a foodborne pathogen. This bacterial strain can generate Shiga-like toxins (SLTs), which can cause serious sickness and even death. Thus, it is important to develop effective and sensitive methods that can be used to rapidly identify the presence of SLTs from complex samples. Pigeon egg white (PEW) contains abundant glycoproteins, including pigeon ovalbumin (POA) (∼60%). POA possesses Gal-α(1→4)-Gal-ß(1→4)-GlcNAc termini, which can recognize the B subunits in SLT type 1 (SLT-1B). Thus, POA is a suitable probe for trapping SLT-1B. In this work, we used PEW proteins as starting materials to react with aqueous tetrachloroauric acid for generation of PEW-protein-immobilized gold nanoparticles (AuNPs@PEW) via one-pot reactions. We demonstrated that the generated AuNPs@PEW were mainly dominated by POA-immobilized Au NPs. The as-prepared AuNPs@PEW were used as affinity probes to selectively probe SLT-1B from complex cell lysates derived from E. coli O157:H7. The selective trapping step can be completed within ∼90 s under microwave heating (power = 450 W) to enrich sufficient SLT-1B for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis. Furthermore, this approach can be used to detect SLT-1B at a concentration as low as ∼40 pM. The feasibility of using the proposed method to selectively detect SLT-1B from ham contaminated by E. coli O157:H7 was also demonstrated.

Novel monoclonal antibodies against Stx1d and 1e and their use for improving immunoassays.

Shiga toxins (Stxs) are major causative agents for bloody diarrhea and hemolytic uremic syndrome, a life-threatening disease in humans. No effective treatment is available. Early detection of Stxs in clinical samples is critical for disease management. As bacteria evolve, new Stxs are produced; therefore, methods used to identify them need to be improved as well. In this study, new monoclonal antibodies (mAbs) against Stx1d and 1e were developed and used to improve a commercial Stx1 kit. Incorporation of the new mAbs into the Abraxis Stx1 kit not only increased the assay sensitivity to Stx1d, but the assay was conferred the ability to detect Stx1e, a newly identified subtype of Stx1 produced by an atypical Stx-producing bacterial strain, Enterobacter cloacae M12X01451, isolated from a clinical specimen. This toxin was not detectable using existing commercial kits. The signal to noise ratio (s/n) of the new assay was increased 3-fold for Stx1d, and 44-fold for Stx1e at toxin concentration of 10ng/mL. The limit of detection (LOD) was 10pg/mL for Stx1a, and 100pg/mL for Stx1c, 1d and 1e. When used for bacterial strains, the sensitivity of the new assay was improved 2.5- to 60-fold depending on subtypes produced. In summary, high affinity mAbs against Stx1d and 1e were developed and incorporation of these mAbs into the Stx1 kit significantly enhanced the assay sensitivity and broadened the subtype-specificity. This improvement should be useful for reducing product recalls and disease mistreatment due to failures of detecting less common but clinically important subtypes of Stxs.

Prevalence and characteristics of verotoxigenic Escherichia coli strains isolated from pigs and pork products in Umbria and Marche regions of Italy.

In total 1095 samples from 675 pork products, 210 swine colon contents, and 210 swine carcass sponge swabs were collected in Umbria and Marche regions of Italy and examined for the presence of Shiga toxin-producing Escherichia coli (STEC), also known as Verotoxin-producing E. coli (VTEC). After an enrichment step, each sample was analysed by real-time PCR to detect the stx1, stx2, and eae genes. stx-Positive samples were further tested for the "top five" serogroup markers (O157, O26, O103, O111, O145) and cultured onto selective media. The isolates were assigned to stx subtypes and tested for the presence of aaiC and aggR genes. Out of 420 swine samples, 38.6% faecal samples and 13.8% carcass sponge swabs were stx-positive. In total, 33 E. coli STEC isolates were obtained from 30 samples (4 carcasses and 26 colon contents) indicating a culture-positive rate of 7.1%. A higher culture-positive rate was observed in faecal samples (12.4%) than in carcass sponge swabs (1.9%). Out of 675 pork samples, 19 (2.8%) were stx-positive. No STEC strains were isolated from stx-positive pork products. We concluded that STEC isolation from foodstuffs remains difficult, despite the application of ISO/TS 13136:2012. Furthermore, in accordance with the results of studies conducted in other countries, we observed that most of swine STEC strains carried stx2e gene and lacked of virulence genes, such as eae, aaiC and aggR, indicative of potential pathogenic characteristics for humans. Although the majority of STEC isolates did not express virulence factors correlating with severe human diseases, the association between swine STEC strains and human illness requires further investigations.

Rapid Detection of Escherichia coli O157 and Shiga Toxins by Lateral Flow Immunoassays.

Shiga toxin-producing Escherichia coli O157:H7 (STEC) cause food-borne illness that may be fatal. STEC strains enumerate two types of potent Shiga toxins (Stx1 and Stx2) that are responsible for causing diseases. It is important to detect the E. coli O157 and Shiga toxins in food to prevent outbreak of diseases. We describe the development of two multi-analyte antibody-based lateral flow immunoassays (LFIA); one for the detection of Stx1 and Stx2 and one for the detection of E. coli O157 that may be used simultaneously to detect pathogenic E. coli O157:H7. The LFIA strips were developed by conjugating nano colloidal gold particles with monoclonal antibodies against Stx1 and Stx2 and anti-lipid A antibodies to capture Shiga toxins and O157 antigen, respectively. Our results indicate that the LFIA for Stx is highly specific and detected Stx1 and Stx2 within three hours of induction of STEC with ciprofloxacin at 37 °C. The limit of detection for E. coli O157 LFIA was found to be 105 CFU/mL in ground beef spiked with the pathogen. The LFIAs are rapid, accurate and easy to use and do not require sophisticated equipment or trained personnel. Following the assay, colored bands on the membrane develop for end-point detection. The LFIAs may be used for screening STEC in food and the environment.

Stable Flies (Stomoxys calcitrans L.) from Confined Beef Cattle Do Not Carry Shiga-Toxigenic Escherichia coli (STEC) in the Digestive Tract.

BACKGROUND AND OBJECTIVE: Stable flies (Stomoxys calcitrans L.) are very common around confined and pastured cattle, and due to their painful bites they are very important animal pests. Cattle are asymptomatic reservoirs of foodborne pathogens, Escherichia coli O157:H7 and other Shiga-toxigenic E. coli serotypes (STEC). In the present study, the potential of stable flies to carry STEC in a beef cattle feedlot was assessed. METHODS: Stable flies (n = 180) were collected over 3 summer months and processed individually for STEC-8 that included the serotype O157 and seven non-O157 serotypes (O26, O45, O103, O104, O111, O121, and O145). Isolation and detection of STEC was based on direct plating as well as the enrichment/immunomagnetic separation approach. Modified Posse agar (mP) was used for culturing non-O157 serotypes and sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) for E. coli O157. Multiplex polymerase chain reactions were used for differentiation of individual serotypes and detection of virulence genes (stx1, stx2, eae, and ehxA). RESULTS AND CONCLUSIONS: Of 180 stable flies, 67 (37.2%) carried enterics on mP (mean: 3.6 ± 1.05 × 10(6) colony-forming units [CFU]/fly) and 55/180 (30.5%) were positive for bacteria on CT-SMAC (mean: 1.2 ± 1.08 × 10(4) CFU/fly). However, stable flies positive for E. coli serotypes of interest were very rare (prevalence: 1.1%). The three serotype-positive isolates, two E. coli O26 and one E. coli O45, were recovered from two flies and neither of them harbored the virulence genes. We conclude that stable flies likely do not play a role as a biological vector and/or reservoir of STEC-8 in cattle feedlots.

Mass Spectrometry-Based Method of Detecting and Distinguishing Type 1 and Type 2 Shiga-Like Toxins in Human Serum.

Shiga-like toxins (verotoxins) are responsible for the virulence associated with a variety of foodborne bacterial pathogens. Direct detection of toxins requires a specific and sensitive technique. In this study, we describe a mass spectrometry-based method of analyzing the tryptic decapeptides derived from the non-toxic B subunits. A gene encoding a single protein that yields a set of relevant peptides upon digestion with trypsin was designed. The (15)N-labeled protein was prepared by growing the expressing bacteria in minimal medium supplemented with (15)NH4Cl. Trypsin digestion of the (15)N-labeled protein yields a set of (15)N-labeled peptides for use as internal standards to identify and quantify Shiga or Shiga-like toxins. We determined that this approach can be used to detect, quantify and distinguish among the known Shiga toxins (Stx) and Shiga-like toxins (Stx1 and Stx2) in the low attomole range (per injection) in complex media, including human serum. Furthermore, Stx1a could be detected and distinguished from the newly identified Stx1e in complex media. As new Shiga-like toxins are identified, this approach can be readily modified to detect them. Since intact toxins are digested with trypsin prior to analysis, the handling of intact Shiga toxins is minimized. The analysis can be accomplished within 5 h.

Magnetic Nanoparticle-Based Platform for Characterization of Shiga-like Toxin 1 from Complex Samples.

Foodborne illness outbreaks resulting from contamination of Escherichia coli O157:H7 remain a serious concern in food safety. E. coli O157:H7 can cause bloody diarrhea, hemolytic uremic syndrome, or even death. The pathogenicity of E. coli O157:H7 is mainly caused by the expression of Shiga-like toxins (SLTs), i.e., SLT-1 and SLT-2. SLTs are pentamers composed of a single A and five B subunits. In this study, we propose a magnetic nanoparticle (MNP)-based platform to rapidly identify SLT-1 from the complex cell lysate of E. coli O157:H7. The core of the MNPs is made of iron oxide, whereas the surface of the core is coated with a thin layer of alumina (Fe3O4@Al2O3 MNPs). The Fe3O4@Al2O3 MNPs are functionalized with pigeon ovalbumin (POA), which contains Gal-α(1→4)-Gal-ß(1→4)-GlcNAc termini that can bind SLT-1B selectively. Furthermore, POA is a phosphate protein. Thus, it can be easily immobilized on the surface of the Fe3O4@Al2O3 MNPs through aluminum phosphate chelation under microwave heating within 1.5 min. The generated POA-Fe3O4@Al2O3 MNPs are capable of effectively enriching SLT-1B from complex cell lysates simply by pipetting 20 µL of the sample in and out of the tip in a vial for ∼1 min. To release SLT-1 from the MNPs, Gal-α(1→4)-Gal disaccharides were used for displacement. The released target species are sufficient to be identified by matrix-assisted laser desorption/ionization mass spectrometry. Although the sample volume used in this approach is small (20 µL) and the enrichment time is short (1 min), the selectivity of this approach toward SLT-1B is quite good. We have demonstrated the effectiveness of this approach for rapid determination of the presence of SLT-1 from complex cell lysates and ham/juice samples based on the detection of SLT-1B.

A Four-Plex Real-Time PCR Assay, Based on rfbE, stx1, stx2, and eae Genes, for the Detection and Quantification of Shiga Toxin-Producing Escherichia coli O157 in Cattle Feces.

Several real-time polymerase chain reaction (PCR) assays have been developed to detect and quantify Shiga toxin-producing Escherichia coli (STEC) O157:H7, but none have targeted the O-antigen specific gene (rfbEO157) in combination with the three major virulence genes, stx1, stx2, and eae. Our objectives were to develop and validate a four-plex, quantitative PCR (mqPCR) assay targeting rfbE(O157), stx1, stx2, and eae for the detection and quantification of STEC O157 in cattle feces, and compare the applicability of the assay to detect STEC O157 to a culture method and conventional PCR (cPCR) targeting the same four genes. Specificity of the mqPCR assay to differentially detect the four genes was confirmed with strains of O157 and non-O157 STEC with different profiles of target genes. In cattle feces spiked with pure cultures, detection limits were 2.8×10(4) and 2.8×10(0) colony-forming units/g before and after enrichment, respectively. Detection of STEC O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. The mqPCR detected 48.9% (136/278) of samples as positive for E. coli O157. Of the 100 samples that were randomly picked from 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar's chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect STEC O157 in cattle feces. However, the use of real-time PCR as a screening method to identify positive samples and then subjecting only positive samples to a culture method may underestimate the presence of STEC O157 in fecal samples.

Multiplexed Microsphere Suspension Array-Based Immunoassays.

ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use.

Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

A high-throughput, precipitating colorimetric sandwich ELISA microarray for Shiga toxins.

Shiga toxins 1 and 2 (Stx1 and Stx2) from Shiga toxin-producing E. coli (STEC) bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies) and pooled horseradish peroxidase (HRP)-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB), the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1-2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic) and/or B-PER (a cell-disrupting, protein extraction reagent). Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

Safe and effective means of detecting and quantitating Shiga-like toxins in attomole amounts.

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.

Single-step CE for miniaturized and easy-to-use system.

We developed a novel single-step capillary electrophoresis (SSCE) scheme for miniaturized and easy to use system by using a microchannel chip, which was made from the hydrophilic material polymethyl methacrylate (PMMA), equipped with a capillary stop valve. Taking the surface tension property of liquids into consideration, the capillary effect was used to introduce liquids and control capillary stop valves in a partial barrier structure in the wall of the microchannel. Through the combined action of stop valves and air vents, both sample plug formation for electrophoresis and sample injection into a separation channel were successfully performed in a single step. To optimize SSCE, different stop valve structures were evaluated using actual microchannel chips and the finite element method with the level set method. A partial barrier structure at the bottom of the channel functioned efficiently as a stop valve. The stability of stop valve was confirmed by a shock test, which was performed by dropping the microchannel chip to a floor. Sample plug deformation could be reduced by minimizing the size of the side partial barrier. By dissolving hydroxyl ethyl cellulose and using it as the sample solution, the EOF and adsorption of the sample into the PMMA microchannel were successfully reduced. Using this method, a 100-bp DNA ladder was concentrated; good separation was observed within 1 min. At a separation length of 5 mm, the signal was approximately 20-fold higher than a signal of original sample solution by field-amplified sample stacking effect. All operations, including liquid introduction and sample separation, can be completed within 2 min by using the SSCE scheme.

Comparison of the presence of Shiga toxin 1 in food matrices as determined by an enzyme-linked immunosorbent assay and a biological activity assay.

This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q(10), and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R(2) = 0.994) with those determined with the BAA.

Public health approach to detection of non-O157 Shiga toxin-producing Escherichia coli: summary of two outbreaks and laboratory procedures.

Routine laboratory testing may not detect non-O157 Shiga toxin-producing Escherichia coli (STEC) reliably. Active clinical, epidemiological, environmental health, and laboratory collaboration probably influence successful detection and study of non-O157 STEC infection. We summarized two outbreak investigations in which such coordinated efforts identified non-O157 STEC disease and led to effective control measures. Outbreak 1 involved illness associated with consuming unpasteurized apple cider from a local orchard. Public health personnel were notified by a local hospital; stool specimens from ill persons contained O111 STEC. Outbreak 2 involved bloody diarrhoea at a correctional facility. Public health personnel were notified by the facility infection control officer; O45 STEC was the implicated agent. These reports highlight the ability of non-O157 STEC to cause outbreaks and demonstrate that a coordinated effort by clinicians, infection-control practitioners, clinical diagnostic laboratorians, and public health personnel can lead to effective identification, investigation, and prevention of non-O157 STEC disease.

Investigation of a spatiotemporal cluster of verotoxin-producing Escherichia coli O157 infections in eastern England in 2007.

An outbreak of verotoxin-producing Escherichia coli O157 (VTEC O157) infections linked to an open farm occurred in eastern England in April and May 2007. This paper describes the investigation and highlights the importance of multidisciplinary collaboration for successful control of such outbreaks. There was a temporal cluster of 12 confirmed symptomatic cases of VTEC O157 and one asymptomatic carrier, from five families. The investigation revealed that four of these cases formed part of an outbreak involving two families who visited an open farm. The phenotypic and genotypic characteristics of the isolates from the two families and the putative farm animal contacts were indistinguishable, indicating that the animals were the source of the primary infections. No epidemiological link could be established between the remaining three families affected and the open farm or people having visited the farm. Control measures included improved hand washing facilities on the farm, information for visitors and staff, restricted access and suspended petting and feeding of animals, and thorough cleaning and disinfection of affected areas.

Local bacteriophage isolates showed anti- Escherichia coli O157:H7 potency in an experimental ligated rabbit ileal loop model.

Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity (P < 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E. coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages.

Use of a Vero cell-based fluorescent assay to assess relative toxicities of Shiga toxin 2 subtypes from Escherichia coli.

Shiga toxin-producing Escherichia coli is a leading cause of human gastroenteritis from food and waterborne sources worldwide. Shiga toxins 1 and 2 are important virulence factors linked to severe human illness. In particular, Shiga toxin 2 is composed of a diverse and heterogeneous group of subtypes with differential cytotoxicities in mammalian cells. In this chapter, we describe the use of the Vero-d2EGFP fluorescent assay to examine the relative toxicities of Stx2 and Stx2 subtypes expressed by strains of Shiga toxin-producing E. coli.