Hybrid nanoflower-based electrochemical lateral flow immunoassay for Escherichia coli O157 detection.

An electrochemical biosensor has been developed for detection of Escherichia coli O157 by integrating lateral flow with screen-printed electrodes. The screen-printed electrodes were attached under the lateral flow detection line, and organic-inorganic nanoflowers prepared from E. coli O157-specific antibodies as an organic component were attached to the lateral flow detection line. In the presence of E. coli O157, an organic-inorganic nanoflower-E. coli O157-antimicrobial peptide-labelled ferrocene sandwich structure is formed on the lateral flow detection line. Differential pulse voltammetry is applied using a smartphone-based device to monitor ferrocene on the detection line. The resulting electrochemical biosensor could specifically detect E. coli O157 with a limit of detection of 25 colony-forming units mL-1. Through substitution of antibodies of organic components in organic-inorganic nanoflowers, biosensors have great potential for the detection of other pathogens in biomedical research and clinical diagnosis.

Differential Outcome between BALB/c and C57BL/6 Mice after Escherichia coli O157:H7 Infection Is Associated with a Dissimilar Tolerance Mechanism.

Enterohemorrhagic Escherichia coli (EHEC) infections can result in a wide range of clinical presentations despite that EHEC strains belong to the O157:H7 serotype, one of the most pathogenic forms. Although pathogen virulence influences disease outcome, we emphasize the concept of host-pathogen interactions, which involve resistance or tolerance mechanisms in the host that determine total host fitness and bacterial virulence. Taking advantage of the genetic differences between mouse strains, we analyzed the clinical progression in C57BL/6 and BALB/c weaned mice infected with an E. coli O157:H7 strain. We carefully analyzed colonization with several bacterial doses, clinical parameters, intestinal histology, and the integrity of the intestinal barrier, as well as local and systemic levels of antibodies to pathogenic factors. We demonstrated that although both strains had comparable susceptibility to Shiga toxin (Stx) and the intestinal bacterial burden was similar, C57BL/6 showed increased intestinal damage, alteration of the integrity of the intestinal barrier, and impaired renal function that resulted in increased mortality. The increased survival rate in the BALB/c strain was associated with an early specific antibody response as part of a tolerance mechanism.

Repeated Oral Vaccination of Cattle with Shiga Toxin-Negative Escherichia coli O157:H7 Reduces Carriage of Wild-Type E. coli O157:H7 after Challenge.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE+E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 109 CFU of a pool of three stx-negative LEE+E. coli O157:H7 strains (vaccine group) or three stx-negative LEE- non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx+ LEE+E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ.IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Identification of novel monoclonal antibodies targeting the outer membrane protein C and lipopolysaccharides for Escherichia coli O157:H7 detection.

AIMS: To identify and evaluate the application of two novel monoclonal antibody (mAb) 2G12 against outer membrane protein (Omp) C and mAb 12B1 targeting the O chain of the lipopolysaccharides (LPS) of Escherichia coli O157:H7 (ECO157). METHODS AND RESULTS: The sensitivity and specificity of these two antibodies were evaluated with eight ECO157 strains and 68 untargeted strains. mAb 2G12 and 12B1 had no detectable binding with any of the non-O157 strains at 6·0 log10 CFU per ml, while its high specificity and affinity remained with all ECO157 strains. When a higher level (8·0 log10 CFU per ml) was tested, 2G12 and 12B1 did not react with 82·35 and 97·06% of the non-O157 strains respectively. Based on the pair of two antibodies, the sandwich enzyme-linked immunosorbent assay detected 100% (8/8) of ECO157 strains and none of the non-ECO157 strains. The detection limit of ECO157 strains in pure culture were 4·2 ± 0·2 log10 CFU per ml. When the developed test was applied to artificially inoculated beef samples, the detection limit was 6·0 log10 CFU per gram without enrichment and 1·0 log10 CFU per gram after 12 h of enrichment. CONCLUSIONS: The two novel antibodies identified in this study served as great candidates for the recovery, and detection of ECO157 from different environmental and food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: ECO157-specific detection was improved by a combination of the novel OmpC mAb and LPS mAb with defined target antigen and good specificity.

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection.

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 103 CFU per ml and for H7 positive E. coli is 1 × 104 CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 104 CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.

Immunoassay for foodborne pathogenic bacteria using magnetic composites Ab@Fe3O4, signal composites Ap@PtNp, and thermometer readings.

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe3O4 were incubated in Cu2+ phosphate buffer to synthesize the magnetic composite Ab@Fe3O4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu2+ phosphate buffer to synthesize the signal composites Ap@PtNp with antimicrobial peptides (magainin I), recognizing and labeling E. coli O157:H7. In the presence of E. coli O157:H7, magnetic microcomposites targeted bacteria and signal microcomposites to form the sandwich structure: Ab@Fe3O4-bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H2O2 to generate thermo-signals (temperature rise), which were determined by a thermometer. This point-of-care bioassay detected E. coli O157:H7 in the linear range of 101-107 CFU mL-1 and with a detection limit of 14 CFU mL-1. One-pot process magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4, magnetic microcomposites, MMC) for immunomagnetic separation and nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp, signal microcomposites, SMC) were used as signal amplification and thermometer readings for E. coli O157:H7 detection.

A colorimetric immunoassay for determination of Escherichia coli O157:H7 based on oxidase-like activity of cobalt-based zeolitic imidazolate framework.

Cobalt-based zeolitic imidazolate framework nanosheets (ZIF-67) with oxidase-like catalytic activities as an immunoprobe were employed to enhance the sensitivity of an immunoassay. ZIF-67 was synthesized via the solvothermal method using 2-methylimidazole and cobalt dichloride as substrates. A colorimetric immunoassay for Escherichia coli (E. coli) O157:H7 was designed. Preparation of the immunoprobe involved self-polymerized dopamine being applied for the surface modification of ZIF-67 nanosheets in order to bind to the antibody, which was used to identify E. coli O157:H7. ZIF-67 catalyze the oxidation of 3,3',5,5'-tetramethylbiphenyl (TMB) and produced a color change from colorless to blue. Upon reaction termination, the absorbance was measured at 450 nm. By combining ZIF-67@PDA catalyzed chromogenic reaction with antibody recognition and magnetic separation, the limit of determination is 12 CFU mL-1 and the linear range is 30 to 3.0 × 108 CFU mL-1. The proposed colorimetric immunoassay was successfully utilized to detect E. coli O157:H7 of spiked food samples. Graphical abstract.

Recombinant HcpA-EspA-Tir-Stx2B chimeric protein induces immunity against attachment and toxicity of Escherichia coli O157:H7.

BACKGROUND: It is about 4 decades from the identification of Enterohemorrhagic Escherichia coli (EHEC) as a food-borne pathogen. There are many shreds of evidence that the bacteria are significant sources of intestinal infections and outbreaks even in developed countries. Developing an effective vaccine against O157 and non-O157 serotypes of EHEC is a good strategy to combat the bacteria. Raising antibody against main toxicity, adhering and colonizing apparatus of the bacteria using a multi-antigenic protein can be hopefully applicable. MATERIAL AND METHODS: A synthetic cassette consists of HcpA-EspA-Tir-Stx2B (HETS) subunit proteins were constructed and sub-cloned in pET32a (+). The protein was expressed and purified with Ni-NTA column and the BALB/c mice were immunized by the purified protein. HETS protein efficacy to elicit immune responses, O157 fecal shedding and immunity against Stx toxin were assessed. In addition, the cellular assays were performed to investigate the immune sera capability for neutralizing of Stx toxin and bacterial attachment apparatus. RESULTS: The HETS protein (611 amino acid length) was expressed and validated by Western blotting. Exerted EHEC bacteria ratio in the immunized mice was reduced close to 60% in shedding test. Cellular assays revealed that the sera of the immunized animals were able to neutralize Stx holotoxin in an extent of 70%; also, immunized mice were able to tolerate up to 200 LD50 of the active toxin. Moreover, toxin neutralization assay showed the capability of the immunized sera to block the cell adhesion. CONCLUSION: Regarding a lack of an efficient vaccine against EHEC, the proposed candidate immunogen, which consists of main adhesion and invasion factors, can overcome the lack of a vaccine against the bacteria.

Rapid detection of Escherichia coli O157:H7 by a fluorescent microsphere-based immunochromatographic assay and immunomagnetic separation.

To ensure food safety and avoid infections by Escherichia coli O157:H7 (E. coli O157:H7), we developed a novel fluorescent microsphere (FM)-based immunochromatography assay (FM-ICA). FMs were conjugated to anti-E. coli O157:H7 monoclonal antibody (MAb) as an ICA probe, Immunomagnetic beads (IM-beads) were prepared by conjugating functionalized magnetic microspheres with the antibody for enrichment and separation of pathogenic bacteria from complex food matrices. Under selected conditions, a standard curve of FM-ICA measurement of E. coli O157:H7 was developed with a linear range from 3 × 105 to 6 × 107 colony-forming units (CFU)/mL in PBS buffer. The recoveries of intra- and inter assay ranged from 101.64% to 107.03% and 95.62%-110.2% respectively, with CV below 10%. The FM-ICA showed good sensitivity, accuracy and precision. When IM-beads separation plus FM-ICA (IMS-FICA) were used to assay raw food samples, sensitivity was 3 × 103 CFU/mL, a 33-fold improvement compared with FM-ICA only. Moreover, this method had high specificity for E. coli O157:H7 and can be used to assay E. coli O157:H7 in beef, milk and water samples. This assay can be completed within 2 h and has great potential for on-site quantitation of E. coli O157:H7 with simplicity, rapidity, sensitivity, and cost-effectiveness.

Activation of lpxR gene through enterohaemorrhagic Escherichia coli virulence regulators mediates lipid A modification to attenuate innate immune response.

During the course of infection, pathogens must overcome a variety of host defence systems. Modulation of lipid A, which is a strong stimulant for host immune systems, is one of the strategies used by microorganisms to evade the host response. The lpxR gene, which encodes a lipid A 3'-O-deacylase, is commonly found in several pathogens and has been shown to reduce the inflammatory response. Here, we demonstrated that the lpxR gene of enterohaemorrhagic Escherichia coli (EHEC) was positively regulated by two virulence regulators, Pch and Ler, and that this regulation was coordinated with the locus of enterocyte effacement genes, which encode major virulence factors for colonisation. The lpxR promoter was repressed by the binding of H-NS, but the competitive binding of both regulators resulted in transcription activation. Next, we showed that lipid A from the lpxR mutant was more stimulatory of the inflammatory response in macrophage-like cells than lipid A from wild-type EHEC. Furthermore, phagocytic activity and phagosome maturation in host cells infected with the lpxR mutant were increased in a p38 mitogen-activated protein kinase-dependent manner in comparison with wild-type EHEC infection. Finally, we demonstrated that the pch mutant, which is deficient in activation of the locus of enterocyte effacement genes, was phagocytised more efficiently than the wild type. Thus, EHEC modulates lipid A to dampen the host immune response when activating virulence genes for colonisation.

Intranasal co-administration of recombinant active fragment of Zonula occludens toxin and truncated recombinant EspB triggers potent systemic, mucosal immune responses and reduces span of E. coli O157:H7 fecal shedding in BALB/c mice.

Escherichia coli O157:H7 with its traits such as intestinal colonization and fecal-oral route of transmission demands mucosal vaccine development. E. coli secreted protein B (EspB) is one of the key type III secretory system (TTSS) targets for mucosal candidate vaccine due to its indispensable role in the pathogenesis of E. coli O157:H7. However, mucosally administered recombinant proteins have low immunogenicity which could be overcome by the use of mucosal adjuvants. The quest for safe, potent mucosal adjuvant has recognized ΔG fragment of Zonula occludens toxin of Vibrio cholerae with such properties. ΔG enhances mucosal permeability via the paracellular route by altering epithelial tight junction structure in a reversible, ephemeral and non-toxic manner. Therefore, we tested whether recombinant ΔG intranasally co-administered with truncated EspB (EspB + ΔG) could serve as an effective mucosal adjuvant. Results showed that EspB + ΔG group induced higher systemic IgG and mucosal IgA than EspB alone. Moreover, EspB alone developed Th2 type response with IgG1/IgG2a ratio (1.64) and IL-4, IL-10 cytokines whereas that of EspB + ΔG group generated mixed Th1/Th2 type immune response evident from IgG1/IgG2a ratio (1.17) as well as IL-4, IL-10 and IFN-γ cytokine levels compared to control. Sera of EspB + ΔG group inhibited TTSS mediated haemolysis of murine RBCs more effectively compared to EspB, control group and sera of both EspB + ΔG, EspB group resulted in similar levels of efficacious reduction in E. coli O157:H7 adherence to Caco-2 cells compared to control. Moreover, vaccination with EspB + ΔG resulted in significant reduction in E. coli O157:H7 fecal shedding compared to EspB and control group in experimentally challenged streptomycin-treated mice. These results demonstrate mucosal adjuvanticity of ΔG co-administered with EspB in enhancing overall immunogenicity to reduce E. coli O157:H7 shedding.

An immunofiltration strip method based on the photothermal effect of gold nanoparticles for the detection of Escherichia coli O157:H7.

An immunofiltration strip is a promising rapid and powerful tool for the routine monitoring of foodborne pathogenic bacteria. However, the strip assay is generally less sensitive than other immunological techniques. A novel immunofiltration strip method based on the photothermal effect of gold nanoparticles (GNPs) was developed for the sensitive, rapid, simple handheld and low-cost detection of Escherichia coli O157:H7. The photothermal effect of GNPs was used to achieve the amplification of signals to improve the sensitivity of the strip method. The thermal contrasts caused by the photothermal effect were proportional to the bacteria concentrations and yield an almost logarithmic relation. Under optimal conditions, the detection limit was 1.95 × 104 CFU mL-1, and the sensitivity was improved about ten times compared to that of the conventional visual strip method. In brief, the photothermal based immunofiltration strip could be used for the rapid and sensitive detection of other pathogens as a great potential food quality control technique.

Detection of Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium based on cell elongation induced by beta-lactam antibiotics.

Pathogenic bacteria such as Shiga toxigenic Escherichia coli and Salmonella can cause severe food-borne diseases. Rapid and sensitive detection of these foodborne pathogens is essential to ensure food safety. In this study, a novel method based on cell elongation induced by beta-lactam antibiotics for direct microscopic counting of Gram-negative bacteria was established. Combined with the sample preparation steps of membrane filtration and magnetic separation, the detection of E. coli O157:H7 and Salmonella enterica serotype Typhimurium was achieved by direct optical microscopic counting of the number of elongated bacteria. The limit of detection of E. coli O157:H7 and S. typhimurium could reach 20 CFU mL-1. The recovery tests for E. coli O157:H7 and S. typhimurium in water and milk samples showed acceptable recovery values between 93.6% and 106.2%. This method is sensitive, cost effective, and rapid (<2 h) and shows great potential for the detection of Gram-negative pathogens in various environmental and food samples.

Glyco-iELISA: a highly sensitive and unambiguous serological method to diagnose STEC-HUS caused by serotype O157.

BACKGROUND: Providing proof of presence of Shiga toxin-producing E. coli (STEC) infection forms the basis for differentiating STEC-hemolytic uremic syndrome (HUS) and atypical HUS. As the gold standard to diagnose STEC-HUS has limitations, using ELISA to detect serum antibodies against STEC lipopolysaccharides (LPS) has proven additional value. Yet, conventional LPS-ELISA has drawbacks, most importantly presence of cross-reactivity due to the conserved lipid A part of LPS. The newly described glyco-iELISA tackles this issue by using modified LPS that eliminates the lipid A part. Here, the incremental value of glyco-iELISA compared to LPS-ELISA is assessed. METHODS: A retrospective study was performed including all pediatric patients (n = 51) presenting with a clinical pattern of STEC-HUS between 1990 and 2014 in our hospital. Subsequently, the diagnostic value of glyco-iELISA was evaluated in a retrospective nationwide study (n = 264) of patients with thrombotic microangiopathy (TMA). LPS- and glyco-iELISA were performed to detect IgM against STEC serotype O157. Both serological tests were compared with each other and with fecal diagnostics. RESULTS: Glyco-iELISA is highly sensitive and has no cross-reactivity. In the single-center cohort, fecal diagnostics, LPS-ELISA, and glyco-iELISA identified STEC O157 infection in 43%, 65%, and 78% of patients, respectively. Combining glyco-iELISA with fecal diagnostics, STEC infection due to O157 was detected in 89% of patients. In the nationwide cohort, 19 additional patients (8%) were diagnosed with STEC-HUS by glyco-iELISA. CONCLUSION: This study shows that using glyco-iELISA to detect IgM against STEC serotype O157 has clear benefit compared to conventional LPS-ELISA, contributing to optimal diagnostics in STEC-HUS.

Disposable syringe-based visual immunotest for pathogenic bacteria based on the catalase mimicking activity of platinum nanoparticle-concanavalin A hybrid nanoflowers.

Disposable syringes were used in a novel point-of-care visual test for detecting pathogenic bacteria (Escherichia coli O157:H7 and Salmonella typhimurium). Hybrid nanoflowers composed of platinum nanoparticles and concanavalin A (Pt-nanoflowers) were prepared through a one-pot reaction and were found to be viable catalase mimics. They catalyze the decomposition of hydrogen peroxide (H2O2) to generate O2. When used as labels in immunoassays, they integrate both the functions of biological recognition and signal amplification. The disposable syringe pressure readout was combined with Pt-nanoflower signal conversion and successfully applied to a visual bacteria detection scheme. Both Escherichia coli O157:H7 and Salmonella typhimurium can be quantified with detection limits of as low as 15 and 7 CFU·mL-1, respectively. Graphical abstract One-pot synthetic platinum nanoparticle (PtNP)-concanavalin A hybrid nanoflowers (Pt-nanoflowers), have been used as ideal signal labels for immunoassays and integrating both essential functions of biological recognition and signal amplification. Disposable syringes were used as a readout to detect pathogenic bacteria.

Escherichia coli O157:H7 virulence factors differentially impact cattle and bison macrophage killing capacity.

Enterohemorrhagic Escherichia coli O157:H7 colonizes the gastrointestinal tract of ruminants, including cattle and bison, which are reservoirs of these zoonotic disease-causing bacteria. Healthy animals colonized by E. coli O157:H7 do not experience clinical symptoms of the disease induced by E. coli O157:H7 infections in humans; however, a variety of host immunological factors may play a role in the amount and frequency of fecal shedding of E. coli O157:H7 by ruminant reservoirs. How gastrointestinal colonization by E. coli O157:H7 impacts these host animal immunological factors is unknown. Here, various isogenic mutant strains of a foodborne isolate of E. coli O157:H7 were used to evaluate bacterial killing capacity of macrophages of cattle and bison, the two ruminant species. Cattle macrophages demonstrated an enhanced ability to phagocytose and kill E. coli O157:H7 compared to bison macrophages, and killing ability was impacted by E. coli O157:H7 virulence gene expression. These findings suggest that the macrophage responses to E. coli O157:H7 might play a role in the variations observed in E. coli O157:H7 fecal shedding by ruminants in nature.

Influence of surface polysaccharides of Escherichia coli O157:H7 on plant defense response and survival of the human enteric pathogen on Arabidopsis thaliana and lettuce (Lactuca sativa).

This study aimed to determine the influence of bacterial surface polysaccharides (cellulose, colanic acid, and lipopolysaccharide; LPS) on the colonization or survival of Escherichia coli O157:H7 on plants and the plant defense response. Survival of E. coli O157:H7 were evaluated on Arabidopsis thaliana and romaine lettuce as a model plant and an edible crop (leafy vegetable), respectively. The population of the wild-type strain of E. coli O157:H7 on Arabidopsis plants and lettuce was significantly (P < 0.05) greater compared with the colanic acid-deficient and LPS-truncated mutants on day 1 and day 5 post-inoculation. This result indicates that colanic acid and LPS structures may contribute to the ability of bacterial survival or persistence on plants. The wild-type strain of E. coli O157:H7 produced approximately twice the amount (P < 0.05) of capsular polysaccharide (CPS) than the colanic acid and LPS-truncated mutants. The significantly lower production of CPS was associated with significantly greater (2-fold) expression of pathogenesis-related gene (PR1) compared with the wild-type and cellulose-deficient mutant (P < 0.05). Collectively, the results of this study may suggest that specific surface polysaccharides of E. coli O157:H7 differentially induce the plant defense response, consequently affecting the survival of the human pathogen on plants. The survival and persistence of E. coli O157:H7 was similar on Arabidopsis and lettuce regardless of day post-inoculation.

Intranasal immunization with novel EspA-Tir-M fusion protein induces protective immunity against enterohemorrhagic Escherichia coli O157:H7 challenge in mice.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome in humans. Due to the risks associated with antibiotic treatment against EHEC O157:H7 infection, vaccines represent a promising method for prevention of EHEC O157:H7 infection. Therefore, we constructed the novel bivalent antigen EspA-Tir-M as a candidate EHEC O157:H7 subunit vaccine. We then evaluated the immunogenicity of this novel EHEC O157:H7 subunit vaccine. Immune responses to the fusion protein administered by intranasal and subcutaneous routes were compared in mice. Results showed higher levels of specific mucosal and systemic antibody responses induced by intranasal as compared to subcutaneous immunization. Intranasal immunization enhanced the concentration of interleukin-4, interleukin-10, and interferon-γ, while subcutaneous immunization enhanced only the latter two. In addition, intranasal immunization protected against EHEC O157:H7 colonization and infection in mice at a rate of 90%.Histopathological analysis revealed that vaccination reduced colon damage, especially when administered intranasally. In contrast, subcutaneous immunization elicited a weak immune response and exhibited a low protection rate. These findings demonstrate that intranasal immunization with the fusion protein induces both humoral and cellular immune (Th1/Th2) responses in mice. The novel EspA-Tir-M novel fusion protein therefore represents a promising subunit vaccine against EHEC O157:H7 infection.

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Nano-encapsulation of chicken immunoglobulin (IgY) in sodium alginate nanoparticles: In vitro characterization.

Controlled delivery of therapeutic agents by alginate nanoparticles became an attractive issue in the gastric organ. Some therapeutic agents such as proteins could not tolerate in severe condition in the gastrointestinal tract. In the present study, four concentrations of a specific IgY as a prophylactic agent against E. coli O157: H7 was entrapped in 0.2% w/v sodium alginate nanoparticles by ionic gelation method. Depending on the IgY concentration entrapment efficacy was 28.31-99.84%. The physicochemical and structural characteristics of free and IgY-loaded Alg NPs revealed that the individual particles exhibited a spherical shape with a diameter of 45-85 nm, and a negatively charged surface with a zeta potential value of 26-36 mV. In vitro release study showed a high significant difference of released amounts of IgY at 10% and 99.84% in simulated gastric fluid (pH 1.2) and simulated intestine fluid (pH 6.8), respectively. Also, the quality and activity of released IgY from Alg NPs not changed. The cytotoxicity of different concentrations of Alg NPs on the Vero cells was measured. Our results indicated that Alg NPs prepared from 0.2%w/v stock solution could be appropriate candidates for efficient and safe delivery of IgY through the gastrointestinal tract.