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Enterohemorrhagic Escherichia coli (EHEC) are foodborne pathogens causing severe human infections including hemorrhagic colitis and hemolytic uremic syndrome, particularly in children. Ruminants are the main reservoir of EHEC which colonize their intestinal tract through a mechanism involving the bacterial adhesin intimin. Vaccination of cattle has shown efficacy in reducing EHEC O157:H7 shedding in feces. However, most of these vaccines rely on purified proteins and/or adjuvants, making them expensive and not used by breeders. This study introduced the development of a new type of vaccine based on Outer Membrane Vesicles (OMVs) carrying the C-terminal domain of intimin (Int280). A vaccine which combines OMVs carrying luminal Int280 and OMVs displaying surface-exposed Int280 was produced using two addressing systems based on PelB peptide signal and Lpp-OmpA hybrid protein, respectively. Dot blot experiments on OMVs combined with FAS assay with bacteria confirmed the correct localization of the fusion proteins and the functionality of Lpp-OmpA-Int280, respectively. As a proof of concept, the efficiency of the mixed vaccine was tested in a mouse model using the pathogen Citrobacter rodentium which shares a similar intimin-based adhesion mechanism with EHEC. Intraperitoneal vaccination of mice, at two-week intervals with 1 μg of the mixture of OMV-Int280, elicited a strong anti-intimin IgG response. Interestingly, we observed a shortened C. rodentium fecal shedding duration in immunized mice compared to the control unvaccinated group, with significant reduction of C. rodentium colonization from day 14 (q < 0.0001) to day 18 (q = 0.0068). This OMV-Int280 vaccine therefore represents a promising candidate for the control of EHEC intestinal carriage and fecal shedding in ruminants. •Outer membrane vesicles (OMVs) enriched with intimin C-terminal domain were used as vaccine.•Two addressing systems successfully exported Int280 to the lumen or surface of OMVs.•Int280 was addressed to the outer membrane surface of E. coli and was functional.•The OMV-Int280 based vaccine induced a seroconversion against intimin in mice.•OMV-Int280 immunized mice had a shortened duration of C. rodentium fecal shedding.

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. 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.

Background. Escherichia coli O157:H7 is a Shiga toxin-producing E. coli (STEC) associated with numerous foodborne outbreaks in the United States and is an important cause of bacterial gastrointestinal illness. In May 2009, we investigated a multistate outbreak of E. coli O157:H7 infections. Methods. Outbreak-associated cases were identified using serotyping and molecular subtyping procedures. Traceback investigation and product testing were performed. A matched case-control study was conducted to identify exposures associated with illness using age-, sex-, and state-matched controls. Results. Seventy-seven patients with illnesses during the period 16 March-8 July 2009 were identified from 30 states; 35 were hospitalized, 10 developed hemolytic-uremic syndrome, and none died. Sixty-six percent of patients were < 19 years; 71% were female. In the case-control study, 33 of 35 case patients (94%) consumed ready-to-bake commercial prepackaged cookie dough, compared with 4 of 36 controls (11%) (matched odds ratio = 41.3; P < .001); no other reported exposures were significantly associated with illness. Among case patients consuming cookie dough, 94% reported brand A. Three nonoutbreak STEC strains were isolated from brand A cookie dough. The investigation led to a recall of 3.6 million packages of brand A cookie dough and a product reformulation. Conclusions. This is the first reported STEC outbreak associated with consuming ready-to-bake commercial prepackaged cookie dough. Despite instructions to bake brand A cookie dough before eating, case patients consumed the product uncooked. Manufacturers should consider formulating ready-to-bake commercial prepackaged cookie dough to be as safe as a ready-to-eat product. More effective consumer education about the risks of eating unbaked cookie dough is needed.

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.

Butyrate has been used to treat different inflammatory disease with positive outcomes, the mechanisms by which butyrate exerts its anti-inflammatory effects remain largely undefined. Here we proposed a new mechanism that butyrate manipulate endogenous host defense peptides (HDPs) which contributes to the elimination of Escherichia coli O157:H7, and thus affects the alleviation of inflammation. An experiment in piglets treated with butyrate (0.2% of diets) 2 days before E. coli O157:H7 challenge was designed to investigate porcine HDP expression, inflammation and E. coli O157:H7 load in feces. The mechanisms underlying butyrate-induced HDP gene expression and the antibacterial activity and bacterial clearance of macrophage 3D4/2 cells in vitro were examined. Butyrate treatment ( i ) alleviated the clinical symptoms of E. coli O157:H7-induced hemolytic uremic syndrome (HUS) and the severity of intestinal inflammation; ( ii ) reduced the E. coli O157:H7 load in feces; ( iii ) significantly upregulated multiple, but not all, HDPs in vitro and in vivo via histone deacetylase (HDAC) inhibition; and ( iv ) enhanced the antibacterial activity and bacterial clearance of 3D4/2 cells. Our findings indicate that butyrate enhances disease resistance, promotes the clearance of E. coli O157:H7, and alleviates the clinical symptoms of HUS and inflammation, partially, by affecting HDP expression via HDAC inhibition.

Escherichia coli O157:H7 is a predominant foodborne pathogen with severe pathogenicity, leading to increasing attention given to rapid and sensitive detection. Herein, we propose an impedance biosensor using new kinds of screen-printed interdigitated microelectrodes (SPIMs) and wheat germ agglutinin (WGA) for signal amplification to detect E. coli O157:H7 with high sensitivity and time-efficiency. The SPIMs integrate the high sensitivity and short response time of the interdigitated electrodes and the low cost of the screen-printed electrodes. Self-assembling of bi-functional 3-dithiobis-(sulfosuccinimidyl-propionate) (DTSP) on the SPIMs was investigated and was proved to be able to improve adsorption quantity and stability of biomaterials. WGA was further adopted to enhance the signal taking advantage of the abundant lectin-binding sites on the bacteria surface. The immunosensor exhibited a detection limit of 102 cfu·mL−1, with a linear detection range from 102 to 107 cfu·mL−1 (r2 = 0.98). The total detection time was less than 1 h, showing its comparable sensitivity and rapid response. Furthermore, the low cost of one SPIM significantly reduced the detection cost of the biosensor. The biosensor may have great promise in food safety analysis and lead to a portable biosensing system for routine monitoring of foodborne pathogens.

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.

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. Graphical Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen that can cause gastrointestinal disease with potentially fatal consequences as a result of systemic Shiga toxin activity. Cattle are the main reservoir host of EHEC O157 and interventions need to be developed that prevent cattle colonization or limit shedding of the organism from this host. EHEC O157 predominately colonizes the bovine terminal rectum and requires a type III secretion system (T3SS) for adherence and persistence at this site. A vaccine based on concentrated bacterial supernatant that contains T3S proteins has shown some efficacy. Here we have demonstrated that vaccination with a combination of antigens associated with T3S-mediated adherence; the translocon filament protein, EspA, the extracellular region of the outer membrane adhesin, intimin, and the translocated intimin receptor (Tir) significantly reduced shedding of EHEC O157 from experimentally infected animals. Furthermore, this protection may be augmented by addition of H7 flagellin to the vaccine preparation that has been previously demonstrated to be partially protective in cattle. Protection correlates with systemic and mucosal antibody responses to the defined antigens and validates the targeting of these colonization factors.

Escherichia coli ( E. coli ) O157:H7 is an important food-borne pathogen that can cause hemorrhagic diarrhea and enteritis in humans and animals. Realizing the rapid quantitation of E. coli O157:H7 is of great significance for the guarantee of food safety and disease control. In this study, an electrochemical immunosensing technique based on a functionalized composite of Cu-metal organic framework (Cu-MOF) and poly (3,4-ethylenedioxythiophene)-poly (styrenesulfonate) (PEDOT:PSS) is developed, achieving rapid and sensitive quantitation of E. coli O157:H7 in food and clinical feces samples. The organic functionalization of Cu-MOF significantly improves the interface conductivity to facilitate electron transfer and provides the sulfonic groups (–SO 3 H) to conjugate bio-recognizing elements for target determination. The immunosensor delivers a linear detection range of 3 × 10 2  ~ 3 × 10 8  cfu/mL, a low limit of detection (LOD) of 7.4 cfu/mL, and a short analysis time of 40 min. In addition, it does not show any cross-reactivity with other common pathogens and exhibits high repeatability with relative standard deviations (RSDs) all lower than 2.09%, providing a promising approach for warranting  food safety and control of E. coli O157:H7 disease. Graphical Abstract