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.

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.

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 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.

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.

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.

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.

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.

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.

An integrated microfluidic platform for sensitive and rapid detection of biological toxins.

Towards designing a portable diagnostic device for detecting biological toxins in bodily fluids, we have developed microfluidic chip-based immunoassays that are rapid (< 20 minutes), require minimal sample volume (<10 microL) and have appreciable sensitivity and dynamic range (microM-pM). The microfluidic chip is being integrated with miniaturized electronics, optical elements, fluid-handling components, and data acquisition software to develop a portable, self-contained device. The device is intended for rapid, point-of-care (and, in future, point-of-incident) testing in case of an accidental or intentional exposure/intoxication to biotoxins. Detection of toxins and potential host-response markers is performed using microfluidic electrophoretic immunoassays integrated with sample preconcentration and mixing of analytes with fluorescently labeled antibodies. Preconcentration is enabled by photopolymerizing a thin, nanoporous membrane with a MW cut-off of approximately 10 kDa in the sample loading region of the chip. Polymeric gels with larger pores are located adjacent to the size exclusion membrane to perform electrophoretic separation of antibody-analyte complex and excess antibody. Measurement of the ratio of bound and unbound immune-complex using sensitive laser-induced fluorescence detection provides quantitation of analyte in the sample. We have demonstrated electrophoretic immunoassays for the biotoxins ricin, Shiga toxin I, and Staphylococcal enterotoxin B (SEB). With off-chip mixing and no sample preconcentration, the limits of detection (LOD) were 300 pM for SEB, 500 pM for Shiga toxin I, and 20 nM for ricin. With a 10 min on-chip preconcentration, the LOD for SEB is <10 pM. The portable device being developed is readily applicable to detection of proteinaceous biomarkers of many other diseases and is intended to represent the next-generation diagnostic devices capable of rapid and quantitative measurements of multiple analytes simultaneously.

Globotriose-functionalized gold nanoparticles as multivalent probes for Shiga-like toxin.

Compared to monovalent carbohydrates, multivalent carbohydrate ligands exhibit significantly enhanced binding affinities to their interacting proteins. Here, we report globotriose (P(k) ligand)-functionalized gold nanoparticle (AuNP) probes for the investigation of multivalent interactions with the B(5) subunit of Shiga-like toxin I (B-Slt). Six P(k)-ligand-encapsulated AuNPs (P(k)-AuNPs) of varying particle size and linker length were synthesized and evaluated for their potential as multivalent affinity probes by using a surface plasmon resonance competition assay. The affinity of these probes for the interacting proteins was greatly affected by nanoparticle size, linker length, and ligand density on nanoparticle surface. For example, the 20-nm 20-P(k)-l-AuNP, which had a relatively long linker showed a >10(8)-fold increase in affinity compared with the mono P(k) ligand. This intrinsic high-affinity AuNP probe specifically captured the recombinant B-Slt from Escherichia coli lysate, and the resulting purity of the B-Slt was >95 %. We also developed a robust P(k)-AuNP-based detection method for Slt-I by combining the technique with silver enhancement.

[Molecular detection of shiga toxin-producing (stx1) Escherichia coli and rotavirus in stools of children with diarrhea]. / Detección molecular de Escherichia coli productor de shiga toxina (Stx1) y rotavirus en heces de niños con diarrea.

The presence of E. coli producer of shiga toxin and rotavirus was investigated in 90 stool samples from children less than 3 years old with diarrhea. Three aliquots were separated from each sample: the first one underwent previous enrichment for E. coli O157, the second one was plated on agar MacConkey-Sorbitol and Red Eosine with MUG, and the last one was frozen at -70 degrees C for the later analysis of rotavirus. The search of the antigen O157 of E. coli was carried out by immunochromatography in vitro of Coris Bioconcept (Belgium). The presence of the antigen O157 (rbf O157) and the genes that code for the shiga toxin (stx1 and stx2) were determined by PCR. Rotavirus were detected by electrophoresis in polyacrylamide gels. The 90 samples analyzed by immunochromatography were negative for the antigen O157. The isolates were STEC strains non O157 and contained the gene sx1, showing a 10% of positivity. The electrophoresis for the viral RNA detected rotavirus in 21 (23.33%) samples. This result confirms the rotavirus prevalence and suggests, that the circulation of STEC strains non O157 is an indication of the involvement of these strains in the ethiology of acute diarrheas.

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction.

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5 degrees C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (gamma-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA+, gamma-tir+, stx2+, rfbE+, fliC+, hlyA+) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable gamma-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.

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.

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.

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.

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.

Stability of the homopentameric B subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry.

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B(5) homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60 degrees C and subunit concentrations ranging from 5 to 85 microM, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 microM, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B(5) ions of Stx2 at the +12 and +13 charge states proceeds, at reaction temperatures of 120 to 180 degrees C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B(5) ions at the +12 and +13 charge states were compared with values reported for the corresponding B(5) ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E(a)) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E(a) values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins.