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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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The rectal-anal junction (RAJ) is the major colonization site of Shiga toxin-producing Escherichia coli (STEC) O157 in beef cattle, leading to transmission of this foodborne pathogen from farms to food chains. To date, there is limited understanding regarding whether the mucosa-attached microbiome has a profound impact on host-STEC interactions. In this study, the active RAJ mucosa-attached microbiota and its potential role in host immunity-STEC commensal interactions were investigated using RAJ mucosal biopsies collected from calves orally challenged with two STEC O157 strains with or without functional stx2a (stx2a+ or stx2a-). The results revealed that shifts of microbial diversity, topology, and assembly patterns were subjected to stx2a production post-challenge and Paeniclostridium and Gallibacterium were the keystone taxa for both microbial interactions and assembly. Additional mucosal transcriptome profiling showed stx2a-dependent host immune responses (i.e. B- and T-cell signaling and antigen processing and presentation) post-challenge. Further integrated analysis revealed that mucosa-attached beneficial microbes (i.e. Provotella, Faecalibacterium, and Dorea) interacted with host immune genes pre-challenge to maintain host homeostasis; however, opportunistic pathogenic microbes (i.e. Paeniclostridium) could interact with host immune genes after the STEC O157 colonization and interactions were stx2a-dependent. Furthermore, predicted bacterial functions involved in pathogen (O157 and Paeniclostridium) colonization and metabolism were related to host immunity. These findings suggest that during pathogen colonization, host-microbe interactions could shift from beneficial to opportunistic pathogenic bacteria driven and be dependent on the production of particular virulence factors, highlighting the potential regulatory role of mucosa-attached microbiota in affecting pathogen-commensal host interactions in calves with STEC O157 infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

This study developed a novel method for the rapid detection of Escherichia coli ( E. coli ) O157:H7 on a microfluidic platform. First, the concentration of bacteria in a sample was determined with the adenosine triphosphate (ATP) method. Then, the specific detection of E. coli was achieved in a microfluidic chip by the immune-microsphere technique. The influences of the culture time, flow rate and capture time on the detection of the target bacteria were investigated systematically. Generally, with increasing capture time, more bacteria could be captured by the microspheres, which had a positive effect on bacterial detection. Furthermore, the sensitivity and specificity of the method were also tested. The results showed that this method could specifically detect E. coli with a sensitivity as high as 49.1 cfu/μL; the consumption of bacteria was 1 μL, and the reagent was at the microliter level. The testing time can be controlled within one and a half hours, and the cost of testing was approximately RMB 10. The method described in this article is simple and accurate and has great application value in bacterial detection for medical diagnostics.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain is known as one of the major human foodborne pathogens. Lack of effective clinical treatment for human diarrheal diseases confirms the need for vaccine production against enteric bacteria such as E.coli O157:H7. Shiga-like toxin (Stx), EscC, and Intimin are the main important virulent factors of this enteric pathogen. In the present study, a comparative Omics analysis was conducted to identify most invasion EHEC antigenic factors as a potential immunogen. SEI (Stx-EscC-Intimin) trivalent chimeric protein was designed from the exposed and epitope rich part of these virulence factors. Sequence optimization, physicochemical properties, mRNA folding, three-dimensional structure and immunoinformatics data were investigated. The chimeric gene was synthesized with codon bias of E. coli. Recombinant protein was expressed and confirmed by western blot analysis. To evaluate the immunogenicity of the designed protein, the protein was administered to BALB/c mice and the serum IgG was determined by ELISA. Based on the Ramachandran plot, the validation data showed that 90.1 % of residues lie in the favored region. The high antigenicity of the multimeric protein was predicted by the immunoinformatic analysis. Epitope prediction had shown the proper distribution of linear and conformational B-cell epitopes and the competition of T-cell epitopes to bind MHC molecules too. Recombinant ESI Protein with 74.5 kDa was expressed in E. coli. Western blot analysis by anti-Stx antibody, confirmed a single band of chimeric protein. Consequently, the chimeric gene was designed and constructed after assessments. From in silico approach, the protein deduced from this cassette can be an immunogen candidate, and act against toxicity and adherence of EHEC., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Shiga-toxin producing Escherichia coli O157:H7 (O157)-based vaccines can provide a potential intervention strategy to limit foodborne zoonotic transmission of O157. While the peripheral antibody response to O157 vaccination has been characterized, O157-specific cellular immunity at the rectoanal junction (RAJ), a preferred site for O157 colonization, remains poorly described. Vaccine induced mucosal O157-specific antibodies likely provide some protection, cellular immune responses at the RAJ may also play a role in protection. Distinct lymphoid follicles were increased in the RAJ of vaccinated/challenged animals. Additionally, increased numbers of interferon (IFN)γ-producing cells and γδ + T cells were detected in the follicular region of the RAJ of vaccinated/challenged animals. Likewise, adjuvanted-vaccine formulation is critical in immunogenicity of the O157 parenteral vaccine. Local T cell produced IFNγ may impact epithelial cells, subsequently limiting O157 adherence, which was demonstrated using in vitro attachment assays with bovine epithelial cells. Thus, distinct immune changes induced at the mucosa of vaccinated and challenged animals provide insight of mechanisms associated with limiting O157 fecal shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling O157 in ruminants and thus limiting O157 transmission to humans.

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., (Copyright © 2021 American Society for Microbiology.)

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 log 10 CFU per ml, while its high specificity and affinity remained with all ECO157 strains. When a higher level (8·0 log 10 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 log 10 CFU per ml. When the developed test was applied to artificially inoculated beef samples, the detection limit was 6·0 log 10 CFU per gram without enrichment and 1·0 log 10 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., (© 2020 The Society for Applied Microbiology.)

Immunochromatographic assay (ICA) is widely applied in various fields. However, severe matrix interference and weak signal output present major challenges in achieving accurate and ultrasensitive detection in ICA. Here, a polydopamine (PDA)-mediated magnetic bimetallic nanozyme (Fe 3 O 4 @PDA@Pd/Pt) with peroxidase-like activity was synthesized and used as a probe in ICA. The magnetic property of Fe 3 O 4 @PDA@Pd/Pt enabled effective magnetic enrichment of targets, thereby reducing the matrix interference in the sample. PDA coating on the magnetic bimetallic nanozyme was employed as a mediator and a stabilizer. It improved the catalytic ability and stability of the magnetic bimetallic nanozyme by providing more coordination sites for Pd/Pt growth and functional groups (-NH and -OH). In addition, the Pd/Pt bimetallic synergistic effect could further enhance the catalytic ability of the nanozyme. A method was developed by integrating Fe 3 O 4 , PDA, and Pd/Pt into Fe 3 O 4 @PDA@Pd/Pt as a probe in ICA. With the proposed method, human chorionic gonadotropin and Escherichia coli O157:H7 were successfully detected to be as low as 0.0094 mIU/mL in human blood serum and 9 × 10 1 CFU/mL in the milk sample, respectively. This method may be readily adapted for accurate and ultrasensitive detection of other biomolecules in various fields.

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 × 10 9 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., (Copyright © 2021 American Society for Microbiology.)

Therapeutic antibodies (Abs) inhibiting bacterial adhesion to host epithelia are an attractive option to reduce the load of Shiga toxin-producing E. coli (STEC) in the intestine of the patient and also in the bovine reservoir, thereby minimizing the risk of STEC contamination in the food chain. Of particular interest are recombinant single-domain Ab fragments called nanobodies (Nbs) derived from the variable domain of camelid heavy chain-only antibodies (VHH). The outer membrane adhesin intimin and the translocated intimin receptor (Tir) are essential for the attachment of STEC to host epithelia. In addition, EspA filaments of the bacterial type III protein secretion system are needed for Tir translocation into the host cell. Given their importance for bacterial adhesion and colonization, we developed Nbs against intimin, Tir and EspA proteins of STEC serotype O157:H7. Here, we report the screening methods used to isolate inhibitory Nbs blocking intimin-Tir protein-protein interaction, actin-pedestal formation, and intimate adhesion of STEC to epithelial cells in vitro. First, we describe how VHH gene repertoires can be produced as Nbs secreted by E. coli using the α-hemolysin (HlyA) protein secretion system. Next, we report the methods for identification of inhibitors of intimin-Tir protein-protein interaction and of STEC intimate adhesion to HeLa cells in culture. These methods can be adapted for the screening of Nbs against different adhesin-receptor complexes to block the adhesion of other pathogens to host cells.

Herein, a label-free and dual-readout immunochromatographic test strip (ITS) for the sensitive detection of Escherichia coli (E. coli) O157:H7 by taking advantages of the strong capture ability of Fe 3 O 4 @CuS nanostructures (NSs) towards bacteria and their ultrahigh photothermal effects (PTEs) was reported. Especially, without the customarily antibody (Ab)-labeled probe, Fe 3 O 4 @CuS NSs could be adsorbed onto the surfaces of bacteria to form Fe 3 O 4 @CuS-bacteria conjugates and then trapped by immobilized Abs on the test line (T-line), forming a characteristic yellow band. After direct immunoreactions, the PTEs of Fe 3 O 4 @CuS NSs endowed T-line to be irradiated by an 808 nm infrared light, obtaining satisfactory sensitivity and accuracy. Under optimal conditions, E. coli O157:H7, as low as 10 3 and 10 2 CFU/mL, could be monitored in colorimetric and photothermal modes. Additionally, E. coli O157:H7 was successfully detected in beef, chicken, milk and honey samples by this proposed platform with a recovery of 80-120%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe 3 O 4 organic-inorganic composites (Ab@Fe 3 O 4 ) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe 3 O 4 were incubated in Cu 2+ phosphate buffer to synthesize the magnetic composite Ab@Fe 3 O 4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu 2+ 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@Fe 3 O 4 -bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H 2 O 2 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 10 1 -10 7  CFU mL -1 and with a detection limit of 14 CFU mL -1 . One-pot process magnetic Fe 3 O 4 organic-inorganic composites (Ab@Fe 3 O 4 , 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.

To overcome the drawbacks of antibody labeling dependence and single-readout system in the conventional lateral flow immunoassays (LFIAs) as well as the non-targeted combination of new capture agents reported recently for pathogen detection, in this work, a multi-readout and label-free LFIA was proposed for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7) based on a nanozyme-bacteria-antibody sandwich pattern. A type of functional nanozyme-mannose modified Prussian blue (man-PB), was introduced as the recognition agent as well as signal indicator. Apart from original signal intensity on the T-line, the peroxidase-like catalytic activity-driven generation of colorimetric signal could be used as another format of quantitation. Importantly, such LFIA could exhibit excellent performance for target pathogens detection separately with a quantitative range of 10 2 -10 8 cfu·mL -1 and a low detection limit of 10 2 cfu·mL -1 based on different readout formats, indicating the application potential of the proposed LFIA in real samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 × 10 3 CFU per ml and for H7 positive E. coli is 1 × 10 4 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 × 10 4 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., (© 2020 The Society for Applied Microbiology.)

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 × 10 8  CFU mL -1 . The proposed colorimetric immunoassay was successfully utilized to detect E. coli O157:H7 of spiked food samples. Graphical abstract.

Escherichia coli O157:H7 and Shigella flexneri are the predominant diarrhoeal pathogens and those strains producing Shiga toxins cause life-threatening sequelae including hemolytic uremic syndrome (HUS) upon their entry into the host. Intimate adherence of E. coli O157 and invasion of S. flexneri in the host intestinal epithelial cells is mainly mediated by Intimin and IpaB proteins, respectively. In this study, we have synthesized chimera of immunodominant regions of Intimin (eae) and IpaB (ipaB) designated as EI and expressed it in Lactococcus lactis (LL-EI) to develop a combinatorial oral vaccine candidate. Immune parameters and protective efficacy of orally administered LL-EI were assessed in the murine model. Significant EI-specific serum IgG, IgA, and fecal IgA antibody titer were observed in the LL-EI group. Considerable increase in EI-specific splenocyte proliferation and a concurrent upregulation of both Th1 and Th2 cytokines was observed in LL-EI immunized mice. Flow cytometry analysis also revealed a significant increase in CD4 and CD8 cell counts in LL-EI immunized group compared to PBS, LL control group.In vitro studies using LL-EI immunized mice sera showed substantial protection against bacterial adhesion and invasion caused by E. coli O157 and Shigella flexneri¸ respectively. LL-EI immunized group challenged with E. coli O157 ceased fecal shedding within 6 days, and mice challenged with S. flexneri showed 93% survival with minimal bacterial load in the lungs. Our results indicate that LL-EI immunization elicits systemic, mucosal and cell-mediated immune responses, and can be a promising candidate for oral vaccine development against these pathogens., Competing Interests: Declaration of Competing Interest Authors authenticate no conflict of interest among them., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p <0.05) association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p < 0.05). The Firmicutes:Bacteroidetes ratio (p > 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7., Competing Interests: We did not have any commercial affiliations with Thermo Fisher and Elanco International as indicated above in the Updated Funding Statement, but we do adhere to PLOS ONE policies on sharing data and materials.

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD 50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.

Enterohemorrhagic E. coli (EHEC) is a major cause of large outbreaks worldwide associated with hemorrhagic colitis and hemolytic uremic syndrome. While vaccine development is warranted, a licensed vaccine, specific for human use, against EHEC is not yet available. In this study, the reverse vaccinology approach combined with genomic, transcriptional and molecular epidemiology data was applied on the EHEC O157:H7 genome to select new potential vaccine candidates. Twenty-four potential protein antigens were identified and one of them (MC001) was successfully expressed onto Generalized Modules for Membrane Antigens (GMMA) delivery system. GMMA expressing this vaccine candidate was immunogenic, raising a specific antibody response. Immunization with the MC001 candidate was able to reduce the bacterial load of EHEC O157:H7 strain in feces, colon and caecum tissues after murine infection. MC001 is homologue to lipid A deacylase enzyme (LpxR), and to our knowledge, this is the first study describing it as a potential vaccine candidate. Gene distribution and sequence variability analysis showed that MC001 is present and conserved in EHEC and in enteropathogenic E. coli (EPEC) strains. Given the high genetic variability among and within E. coli pathotypes, the identification of such conserved antigen suggests that its inclusion in a vaccine might represent a solution against major intestinal pathogenic strains.

Lipocalin 2 (Lcn2) is an essential component of the antimicrobial innate immune system. It attenuates bacterial growth by binding and sequestering the iron-scavenging siderophores to prevent bacterial iron acquisition. Whereas, the ability of Lcn2 to sequester iron is well-described, the role of Lcn2 in regulating immune cells during bacterial infection remains unclear. In this study, we showed that upon infection with Escherichia coli (O157:H7), Lcn2-deficient ( Lcn2 -/- ) mice carried more bacteria in blood and liver, and the acute-phase sera lost their antibacterial activity in vitro . Neutrophils from Lcn2 -/- mice were defective in homeostasis and morphological development. E. coli O157:H7 infection of Lcn2 -/- mice resulted in a reduced neutrophil migration capacity, with 30% reduction of extravasated neutrophils, and impaired chemotaxis, as shown by a reduction in the secretion of chemoattractants, such as tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2, which are instrumental in eliciting a neutrophil response. We also found that some secreted cytokines [interleukin (IL)-6, IL-1β, and TNF-α] were decreased. Transcripts of inflammatory cytokines (IL-6, IL-1β, TNF-α, and IL-10), chemokines (MIP-2 and MCP-1), and iNOS production were all strongly repressed in Lcn2 -/- macrophages. Furthermore, Lcn2 could induce the production of chemokines and promote the migration and phagocytosis of macrophages. Thus, Lcn2 deficiency could impair the migration and chemotaxis ability of neutrophils and disturb the normal secretion of inflammatory cytokines of macrophages. Therefore, the heightened sensitivity of Lcn2 -/- mice to E. coli O157:H7 is not only due to the antibacterial function of Lcn2 but also a consequence of impaired functions of immune cells, including neutrophils and macrophages., (Copyright © 2019 Wang, Li, Tang, Liang, Wang and Du.)

Considering the health risks of E. coli O157:H7 presence in food and water, an affordable and highly sensitive detection method is crucial. Herein, we report the first use of a single antibody-based fluorescent lateral flow immunoassay (FLFIA) depending on non-radiative energy transfer between graphene oxide and quantum dots for determination of E. coli O157:H7 in beef and river water. FLFIA showed a high sensitivity rate thousand-fold better than the conventional lateral flow (LF). In inoculated minced beef and river water samples, the limits of detection were 178 and 133 CFU g -1 or mL -1 , respectively. Besides, it presented a high selectivity in the presence of other possible interfering bacteria. The single antibody approach reduced the assay cost to 60% less than the conventional LF. Alongside, the results could be read by portable LF readers or smartphones. These advantages offer FLFIA as a promising technology for pathogen detection in food and water., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Enterohemorrhagic Escherichia coli (EHEC) are known as the gastrointestinal pathogens and major causes of enterohemorrhagic colitis since decades ago. There is no efficient approved vaccine against EHEC O157 and non-O157. In the present study, a recombinant candidate vaccine against enterohemorrhagic E. coli (EHEC) O157:H7 entrapped in the sodium alginate and PLGA nanoparticles and the efficiency of the immunization of these formulations were investigated. nanoparticles due to their properties like controlled cargoes release, adjuvanticity, cargo protection, increased bioavailability, etc have been noticed for drug delivery. A chimeric protein composed of HcpA, EspA, Tir and Stx2B antigens was designed, recombinantly expressed, purified and entrapped in nanoparticles. BALB/c mice were administrated with nano-formulated and free proteins. IgG titer, EHEC fecal shedding and the ability of the immune sera to neutralize Stx toxin and inhibit the bacterial attachment to Caco-2 cells were analyzed. Fecal shedding analysis demonstrated that the colonization of the bacteria in the intestine of the mice was reduced significantly (P > 0.01). Immune mice were able to tolerate up to 200 LD 50 of the active Stx toxin. About 80% of the bacterial binding capacity to Caco-2 cells was declined, especially in groups immunized with nano-formulations. Considering the importance of EHEC, especially O157 serotype, on public health and the other hand, the lack of an efficient vaccine in this regard, delivery of HETS candidate vaccine with NPs can be applied to prevent the infection by the pathogen., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin-inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL-Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD 50 of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL-Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae., (© 2019 APMIS. Published by John Wiley & Sons Ltd.)

Here we exploit the natural properties of a synthetic nanoparticle (NP) scaffold as a subunit vaccine against enterohemorrhagic Escherichia coli (EHEC). Two EHEC-specific immunogenic antigens, namely, LomW and EscC, either alone or in combination, were covalently linked on the surface of gold nanoparticles (AuNPs) and used to immunize mice prior to challenge with EHEC O157:H7 strain 86-24. LomW is a putative outer membrane protein encoded in bacteriophage BP-933W, while EscC is a structural type III secretion system protein which forms a ring in the outer membrane. The resulting AuNP preparations, AuNP-LomW and AuNP-EscC, showed that the nanoparticles were able to incorporate the antigens, forming stable formulations that retained robust immunogenicity in vivo after subcutaneous immunization. When administered subcutaneously, AuNP-LomW or AuNP-EscC or a combination containing equivalent amounts of both candidates resulted in higher IgG titers in serum and secretory IgA titers in feces. The serum IgG titers correlated with a significant reduction in EHEC intestinal colonization after 3 days postinoculation. In addition, we showed that serum from antigen-coated AuNP-immunized mice resulted in a reduction of adherence to human intestinal epithelial cells for EHEC, as well as for two other E. coli pathotypes (enteropathogenic E. coli [EPEC], encoding EscC, and enteroaggregative E. coli [EAEC], encoding LomW). Further, the serum had antigen-specific bactericidal properties, engaging the classical complement pathway. Overall, our results demonstrate the immunogenicity and stability of a novel nanovaccine against EHEC. These results also strengthen the prospect of development of a synthetic nanoparticle vaccine conjugated to E. coli antigens as a promising platform against other enteric pathogens. IMPORTANCE Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis, which can progress to hemolytic uremic syndrome. These complications represent a serious global public health problem that requires laborious public health interventions and safety control measures to combat recurrent outbreaks worldwide. Today, there are no effective interventions for the control of EHEC infections, and, in fact, the use of antibiotics is counterindicated for EHEC disease. Therefore, a viable alternative for the prevention of human infections is the development of vaccines; however, no such vaccines are approved for human use. In this study, we developed a novel gold nanoparticle platform which acts as a scaffold for the delivery of various antigens, representing a nanovaccine technology which can be applied to several disease models., (Copyright © 2019 Sanchez-Villamil et al.)

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.

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.

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 LD 50 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The most bacterial cause of infectious diseases associated with diarrhea are enterotoxigenic and enterohemorrhagic Escherichia coli (ETEC and EHEC, respectively). These strains use colonization factors for the attachment to the human intestinal mucosa, followed by enterotoxins production that could induce more host damage. The Heat-labile enterotoxin (LT) and colonization factors (CFs) are momentous factors for the pathogenesis of ETEC. Also, Intimin and Shiga like toxin (STX) are the main pathogenic factors expressed by EHEC. Because of mucosal surfaces are the major entry site for these pathogens, oral immunization with providing the protective secretary IgA antibody (sIgA) responses in the mucosa, could prevent the bacterial adherence to the intestine. In this study oral immunogenicity of a synthetic recombinant protein containing StxB, Intimin, CfaB and LtB (SICL) was investigated. For specific expression in canola seeds, the optimized gene was cloned in to plant expression vector containing the Fatty Acid Elongase (FAE) promoter. The evaluation of the expression level in canola seeds was approximately 0.4% of total soluble protein (TSP). Following to oral immunization of mice, serum IgG and fecal IgA antibody responses induced. Caco-2 cell binding assay with ETEC shows that the sera from immunized mice could neutralize the attachment properties of toxigenic E. coli. The reduction of bacterial shedding after the challenge of immunized mice with E. coli O157:H7 was significant. The sera from immunized mice in the rabbit ileal loop experiment exhibited a significant decrease in the fluid accumulation compared to the control. The results indicate efficacy of the recombinant chimeric protein SICL in transgenic canola seed as an effective immunogen, which elicits both systemic and mucosal immune responses as well as protection against EHEC and ETEC adherence and toxicity., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

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.

One of highly effective methods for prevention and control of Entrohemorragic Esherichia coli (EHEC) infections is to use vaccination against extremely immunogenic part of attachment factors. In this study rEIT (EspA, Intimin, Tir) was produced in bacteria and then encapsulated with chitosan nanoparticle as a candidate nanovaccine. A chimeric trivalent recombinant protein which was previously found to provide reasonable immunogenicity against E.coli O157:H7 was used as a base. Mice immunized orally with chitosan based nanoparticle containing rEIT antigen. The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. In challenging tests different groups of immunized mice were infected orally with E.coli O157:H7. The results showed that the recombinant nanovaccine candidate could induce the strong humoral and mucosal immune responses and protect the mice from live EHEC O157:H7 challenge. Higher titers of serum anti rEIT IgG were achieved after the last immunization in all of the groups. Comparison of the amount of IgA titers in serum and feces showed higher values for the latter. In vitro study of binding inhibition assay on Caco-2 cell monolayers by pre-incubated antisera with EHEC bacteria, showed that immunized mice antibody could reduce adhesion properties of E. coli O157:H7. In a challenging study with EHEC bacteria, reduction in number of colonies was observed in all of the immunized groups for over two weeks. Results from the present study prove that nanovaccine candidate with rEIT can reduce signs and symptoms of EHEC infections. This novel approach can be a new strategy for inducing immunity against E. coli O157:H7. This study suggests the use of oral -injection combined vaccination routes comparing to other methods available in order to achieve higher humoral and mucosal immunogenicity levels., (Copyright © 2019. Published by Elsevier B.V.)

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.

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 (H 2 O 2 ) to generate O 2 . 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.

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 × 10 5 to 6 × 10 7  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 × 10 3  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., (Copyright © 2018. Published by Elsevier Inc.)

A universal and reusable hapten-antibody-mediated portable optofluidic immunosensing platform (OIP) was developed for rapid on-site detection of pathogens. By using Escherichia coli O157:H7 (E. coli O157:H7) and bisphenol A-Bovine serum albumin (BPA-BSA)/anti-BPA antibody as a model pathogen and a mediated hapten-antibody, respectively, a novel immunoassay mechanism was proposed to detect pathogens. The BPA-BSA-modified immunosensor and E. coli O157:H7 were initially saturated with anti-BPA antibodies (mouse IgG) and anti-E. coli O157:H7 antibodies (mouse IgG), respectively. Then, the fluorescence-labeled secondary antibodies (goat anti-mouse IgG antibody) were incubated with E. coli O157:H7 with their antibodies. Next, the mixture was introduced into the immunosensor surface bound to the anti-BPA antibodies. A high concentration of E. coli O157:H7 in the sample reduced the number of fluorescence-labeled secondary antibodies bound to the immunosensor surface, thus resulting in the detection of low fluorescence signals. Under optimized conditions, the hapten-antibody-mediated OIP system exhibited a detection limit of 8 cfu/mL E. coli O157:H7 after concentrating 100 times by using centrifugation, and a test cycle, including prereaction, detection, and regeneration, was less than 1 h. The robustness of the hapten-carrier protein-modified immunosensor surface allowed multiple pathogen immunoassays. The proposed strategy demonstrated good recovery, precision, and accuracy through the evaluation of the spiked water samples. We expect that the new platform can be readily used for the detection of other pathogens in a variety of application fields ranging from environmental monitoring and food safety to medical diagnosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

According to European Union (EU) regulations, the serological tests for the eradication of bovine and ovine brucellosis are the Rose Bengal Test, Complement Fixation Test, and i-ELISA. These methods, also recommended by the World Organization for Animal Health (OIE) for international trades, have limitations related to the use of suspensions of smooth Brucellae or LPS extracts. Limitations include false-positive serological reactions to brucellosis, which in turn impedes accurate diagnosis in some herds. False positive reactions should be considered carefully during the final stages of an eradication programme and for surveillance purposes in brucellosis-free areas. In this study, we produced specific sera through the experimental infection of sheep with Y. enterocolitica O:9 and E. coli O157:H7. These are the most important cross-reactive bacteria with Brucella. We then evaluated the antibody response of groups of sheep that had been immunised towards homologous antigens and official antigens for brucellosis, in order to identify a differential diagnostic protocol to distinguish cross-reaction in Brucella-infected animals.

Escherichia coli O157:H7 is a zoonotic pathogen of global importance and the serotype of Shiga toxin-producing E.coli (STEC) most frequently associated with Hemolytic Uremic Syndrome (HUS) in humans. The main STEC reservoir is cattle. Vaccination of calves with the carboxy-terminal fraction of Intimin γ (IntC280) and EspB can reduce E.coli O157:H7 fecal shedding after experimental challenge. Shiga toxin (Stx) exerts local immunosuppressive effects in the bovine intestine and Stx2B fused to Brucella lumazine synthase (BLS-Stx2B) induces Stx2-neutralizing antibodies. To determine if an immune response against Stx could improve a vaccine's effect on fecal shedding, groups of calves were immunized with EspB + IntC280, with EspB + IntC280 + BLS-Stx2B, or kept as controls. At 24 days post vaccination calves were challenged with E.coli O157:H7. Shedding of E.coli O157:H7 was assessed in recto-anal mucosal swabs by direct plating and enrichment followed by immunomagnetic separation and multiplex PCR. Calves were euthanized 15 days after the challenge and intestinal segments were obtained to assess mucosal antibodies. Vaccination induced a significant increase of IntC280 and EspB specific antibodies in serum and intestinal mucosa in both vaccinated groups. Antibodies against Stx2B were detected in serum and intestinal mucosa of animals vaccinated with 3 antigens. Sera and intestinal homogenates were able to neutralize Stx2 verocytotoxicity compared to the control and the 2-antigens vaccinated group. Both vaccines reduced E.coli O157:H7 shedding compared to the control group. The addition of Stx2B to the vaccine formulation did not result in a superior level of protection compared to the one conferred by IntC280 and EspB alone. It remains to be determined if the inclusion of Stx2B in the vaccine alters E.coli O157:H7 shedding patterns in the long term and after recurrent low dose exposure as occurring in cattle herds., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Escherichia coli O157:H7 (O157) can cause from a mild diarrheal illness to hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are the primary reservoir for O157 and fecal shedding of O157 by these animals is a major risk factor in contamination of cattle hides and carcasses at slaughter. Vaccination is an important strategy to reduce fecal shedding of O157 in cattle. In this study, we evaluated the immunogenicity and efficacy of an inactivated vaccine strain of O157 formulated with an adjuvant. This vaccine strain was deleted of the hha gene enabling high level expression of the locus of enterocyte effacement (LEE) encoded proteins required for O157 colonization in cattle. The inactivated vaccine strain emulsified with the adjuvant or suspended in the phosphate-buffered saline (PBS) was injected in the neck muscles of two groups of weaned calves followed by a booster three weeks later with the corresponding formulation. Animals in groups 3 and 4 were injected similarly with the adjuvant and PBS, respectively. All animals were orally inoculated three weeks post-booster vaccination with a live culture of O157. The animals vaccinated with the adjuvanted vaccine showed higher serum antibody titers to the vaccine strain and shed O157 for a shorter duration and at lower numbers compared to the animals vaccinated with the non-adjuvanted vaccine, adjuvant-only, or PBS. Western blotting of the vaccine strain lysates showed higher immunoreactivity of serum IgG in vaccinated animals to several O157-specific proteins and lipopolysaccharides (LPS). The vaccination induced IgG showed specificity to LEE-encoded proteins and outer membrane LPS as LEE and waaL deletion mutants, unable to produce LEE proteins and synthesize high molecular weight LPS, respectively, yielded significantly lower antibody titers compared to the parent vaccine strain. The positive reactivity of the immune serum was also observed for purified LEE-encoded proteins EspA and EspB. In conclusion, the results of this animal study showed that a two-dose regimen of an adjuvanted vaccine is capable of inducing O157-specific immune response that directly or indirectly reduced fecal shedding of O157 in cattle., (Published by Elsevier B.V.)

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene (PR1; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly (P < 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly (P < 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly (P < 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Individuals often respond differently to the same vaccine; some of this variation may be caused by genetic differences among animals. Our objective was to estimate heritability and identify genomic regions associated with humoral response to an Escherichia coli O157:H7 vaccine in beef cattle. Crossbred beef cattle (n = 651) were vaccinated with a commercially available E. coli O157:H7 vaccine. Serum was collected at time of initial vaccination (d 0), booster (d 21), and d 56 after initial vaccination. Total antibodies specific to siderophore receptor and porin proteins in the vaccine were quantified by enzyme-linked immunosorbent assay. Genomic DNA was isolated from whole blood and genotyped with the bovine GeneSeek Genomic Profiler-High Density 78K or 26K Single Nucleotide Polymorphism BeadChip and imputed to 777,000 SNP genotypes. Heritability was estimated by restricted maximum likelihood (REML) using both 1) pedigree and 2) genomic relationships among individuals. Fixed effects were contemporary group, calf age, sex, principal components from SNP genotype data, and pedigree-derived heterozygosity effects. Additive and dominance effects of SNPs were estimated individually while accounting for contemporary group, sex, and the top 20 principal components calculated from the genomic relationship matrix. Heritability of initial response to vaccination (d 21 -d 0) was 0.10 ± 0.175 using pedigree relationships and 0.14 ± 0.149 using genomic relationships, but neither estimate was statistically different from zero. Heritability of booster (d 56 -d 21) and overall (d 56 -d 0) responses were low and not statistically significant from zero. There were no clusters of linked SNP associated with vaccine response, but eight regionally isolated SNPs were significantly associated with initial or overall response to vaccination. Regional genetic variation for initial response to an E. coli O157:H7 vaccine was observed, although overall heritability of this response was not statistically significant from zero.

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., (Published by Elsevier Ltd.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Here, we report a simple and non-emulsion based approach to prepare starch-based magnetic polymer beads with well-defined size, shape and dispersibility in aqueous environment through co-precipitation of enzymatically synthesized short chain amylose (SCA) and dextran-coated iron oxide nanoparticles (Dex@IONPs). The size and morphology of magnetic polymer beads (MPBs) were controlled by employing Dex@IONPs as a seeding agent. The resulting superparamagnetic amylose microbeads (SAMBs) were readily functionalized with antibody through one step reaction using a linker protein, which showed great capture efficiency (>90%) and specificity for target bacteria present in complicated food matrix. The excellent magnetic sensitivity also enabled the SAMBs readily assembled into ordered 1D arrays by external magnetic field whose structure could permanently be fixed by SCA-mediated precipitation process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Colloidal gold immunochromatographic assay (ICA) has poor sensitivity when used for Escherichia coli O157:H7 (E. coli O157:H7) detection. Eu (III)-doped polystyrene nanoparticle (EuNP) has a large range of stokes shift, long decay time, and wide excitation spectrum and narrow emission spectra. EuNP has been used as novel probe in ICA to improve sensitivity. In this study, carboxyl-modified EuNPs were prepared with different linkers. ICA based on EuNP, EuNP-6 carbon chain (CC) complex, EuNP-200CC complex, EuNP-1000CC complex, and EuNP-streptavidin (EuNP-SA) complex were systematically compared for the detection of E. coli O157:H7. Under optimized working conditions, the limits of detection (LOD) of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 9.54 × 10 2 , 1.59 × 10 2 , 3.18 × 10 2 , 2.98 × 10 2 , and 1.08 × 10 2  colony-forming units (CFU) mL -1 , respectively. The linear ranges of EuNP-ICA, EuNP-6CC-ICA, EuNP-200CC-ICA, EuNP-1000CC-ICA, and EuNP-SA-ICA were 6.36 × 10 2 -1.59 × 10 5 , 3.18 × 10 2 -1.59 × 10 5 , 6.36 × 10 2 -1.59 × 10 5 , 6.36 × 10 2 -1.59 × 10 5 , and 8.0 × 10 1 -1.59 × 10 5  CFU mL -1 , respectively. EuNP-SA-ICA exhibited the highest sensitivity and the widest linear range with good specificity, accuracy, and precision. It could be a promising analytical method for detecting E. coli O157:H7 in food samples. EuNP-SA-ICA may be a good model for detecting low concentrations of other food-borne pathogens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In the present study, the application of mesoporous silica nanoparticles (MSNPs) loaded with recombinant EspA protein, an immunogen of enterohaemorrhagic E. coli, was investigated in the case of BALB/c mice immunization against the bacterium. MSNPs of 96.9 ± 15.9 nm in diameter were synthesized using template removing method. The immunization of mice was carried out orally and subcutaneously. Significant immune responses to the antigen were observed for the immunized mice when rEspA-loaded MSNPs were administered in both routes in comparison to that of the antigen formulated using a well-known adjuvant, i.e. Freund's. According to the titretitre of serum IL-4, the most potent humoral responses were observed when the mice were immunized subcutaneously with antigen-loaded MSNPs (244, 36 and 14 ng/dL of IL-4 in the serum of mice immunized subcutaneously or orally by antigen-loaded MSNPs, and subcutaneously by Freund's adjuvant formulated-antigen, respectively). However, the difference in serum IgG and serum IgA was not significant in mice subcutaneously immunized with antigen-loaded MSNPs and mice immunized with Freund's adjuvant formulated-antigen. Finally, the immunized mice were challenged orally by enterohaemorrhagic E. coli cells. The amount of bacterial shedding was significantly reduced in faecesfaeces of the animals immunized by antigen-loaded MSNPs in both subcutaneous and oral routes.

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., (© 2017 John Wiley & Sons Ltd.)

Escherichia coli (E. coli) O157:H7 is an important food‑borne pathogen with a low infective threshold and high resistance to treatment. There are currently a number of detection methods available, however, the majority are time‑consuming, complex and expensive, thus it is hard for these methods to be applied in routine detection. Therefore, there is urgent requirement to develop more sensitive, rapid and specific detective techniques. In the present study, an immuno‑biosensor based on the interference of load to the F0F1‑ATPase rotation, indicated by the fluorescence fluctuation, was constructed to detect O157:H7. The results demonstrated a good linear relationship (R2=0.9818) between antigen concentration (range, 102 cfu to 104 cfu) and the fluorescence intensity. The detection signals of the samples containing 102 cfu/well and 104 cfu/well E. coli O157:H7 were significantly stronger than the signal produced by the control sample (P<0.01). Due to its higher sensibility and simplicity when compared with the current methods applied, the results of the present study indicate a promising future for the application of this technique in detecting food source pathogens.

In this report, we developed a rapid immunoassay system, designated the bioluminescent interference gathering optical (BINGO) assay, which required no time-consuming washing steps for removal of unbound antibodies. This system employed a luciferase (Luc)-conjugated antibody (LucAb) and a dye that absorbed light from the LucAb. The antigen-associated LucAb was localized by transfer of an antigen to the detector-side of a chamber where a detector photomultiplier tube (PMT) was installed. In contrast, the free LucAb was distributed throughout the solution, and the light emitted by the free LucAb was absorbed by the dye. Therefore, only light from LucAb associated with antigen could be detected by the PMT. The new system could be used to rapidly detect the amount of antigen-antibody-Luc complex by collecting steps, such as centrifugation or magnetic collection of antibody-coated magnetic beads. Proof-of-principle experiments were performed using a model system with streptavidin beads and biotinylated Luc. The feasibility of the system was demonstrated using magnetic beads coated with anti-Escherichia coli O157 antibody, enabling detection of 4 × 10 3  cells in only 15 min. Thus, this system may have applications in a variety of biomedical fields., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In recent decades, diseases caused by pathogenic Escherichia coli (E. coli), enterohaemorrhagic E. coli (EHEC) O26 have been increasingly reported worldwide, which are as severe as those caused by EHEC strain O157:H7 and require effective intervention strategies. Herein, we report the application of clay nanoparticles, i.e. hectorites as effective nano-adjuvants for vaccination against EHEC O26 colonization. We show that medium size HEC (hectorite, around 73~77 nm diameter) is able to induce efficient humoral and cellular immune responses against EHEC antigen - intimin β (IB), which are significantly higher than those triggered by commercially used adjuvants - QuilA and Alum. We also demonstrate that mice immunized with IB adjuvanted with HEC nanoparticles elicit sufficient secretion of mucosal IgA, capable of providing effective protection against EHEC O26 binding to ruminant and human cells. In addition, we demonstrate for the first time that hectorites are able to initiate maturation of RAW 264.7 macrophages, inducing expression of co-stimulatory cytokines at a low nanoparticle concentration (10 μg/mL). Together these data strongly suggest that hectorite with optimized size is a highly efficient vaccine nano-adjuvant.

Shiga toxin-producing Escherichia coli (STEC) is the major etiologic agent of hemolytic-uremic syndrome (HUS). The high rate of HUS emphasizes the urgency for the implementation of primary prevention strategies to reduce its public health impact. Argentina shows the highest rate of HUS worldwide, being E. coli O157 the predominant STEC-associated HUS serogroup (>70%), followed by E. coli O145 (>9%). To specifically detect these serogroups we aimed at developing highly specific monoclonal antibodies (mAbs) against the O-polysaccharide (O-PS) section of the lipopolysaccharide (LPS) of the dominant STEC-associated HUS serogroups in Argentina. The development of hybridomas secreting mAbs against O157 or O145 was carried out through a combined immunization strategy, involving adjuvated-bacterial immunizations followed by immunizations with recombinant O-PS-protein conjugates. We selected hybridoma clones that specifically recognized the engineered O-PS-protein conjugates of O157 or O145 serogroups. Indirect ELISA of heat-killed bacteria showed specific binding to O157 or O145 serogroups, respectively, while no cross-reactivity with other epidemiological important STEC strains, Brucella abortus, Salmonella group N or Yersinia enterocolitica O9 was observed. Western blot analysis showed specific recognition of the sought O-PS section of the LPS by all mAbs. Finally, the ability of the developed mAbs to bind the surface of whole bacteria cells was confirmed by flow cytometry, confocal microscopy and agglutination assays, indicating that these mAbs present an exceptional degree of specificity and relative affinity in the detection and identification of E. coli O157 and O145 serogroups. These mAbs may be of significant value for clinical diagnosis and food quality control applications. Thus, engineered O-PS specific moieties contained in the recombinant glycoconjugates used for combined immunization and hybridoma selection are an invaluable resource for the development of highly specific mAbs.