Impact of Western Diet on Enterohemorrhagic Escherichia coli Colonization in the Human In Vitro Mucosal Artificial Colon as Mediated by Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) is a major food-borne pathogen that causes human disease ranging from diarrhea to life-threatening complications. Accumulating evidence demonstrates that the Western diet enhances the susceptibility to enteric infection in mice, but the effect of diet on EHEC colonization and the role of human gut microbiota remains unknown. Our research aimed to investigate the effects of a Standard versus a Western diet on EHEC colonization in the human in vitro Mucosal ARtificial COLon (M-ARCOL) and the associated changes in the gut microbiota composition and activities. After donor selection using simplified fecal batch experiments, two M-ARCOL bioreactors were inoculated with a human fecal sample (n = 4) and were run in parallel, one receiving a Standard diet, the other a Western diet and infected with EHEC O157:H7 strain EDL933. EHEC colonization was dependent on the donor and diet in the luminal samples, but was maintained in the mucosal compartment without elimination, suggesting a favorable niche for the pathogen, and may act as a reservoir. The Western diet also impacted the bacterial short-chain fatty acid and bile acid profiles, with a possible link between high butyrate concentrations and prolonged EHEC colonization. The work demonstrates the application of a complex in vitro model to provide insights into diet, microbiota, and pathogen interactions in the human gut.

Characterization of Shiga Toxin-producing Escherichia coli Isolated from Cattle Around Ulaanbaatar City, Mongolia.

Shiga toxin-producing Escherichia coli (STEC) are associated with severe infections including hemorrhagic colitis and hemolytic uremic syndrome in humans. Ruminants are known as reservoirs of STEC; however, no data are available on STEC in ruminants in Mongolia, where more than 5 million cattle and 25 million sheep are raised. To disclose the existence and characteristics of STEC in Mongolia, in this study, we isolated and characterized STEC from cattle in Mongolia. We collected 350 rectal swabs of cattle from 30 farms near Ulaanbaatar city and isolated 45 STEC from 21 farms. Rectal swabs were precultured with modified Escherichia coli broth and then inoculated to Cefixime-Tellurite Sorbitol MacConkey agar plate and/or CHROMagar STEC agar plate for the isolation of STEC. The isolation ratios in each farm were from 0% to 40%. Multiplex PCR for the estimation of O- and H-serotypes identified 12 O-genotypes (Og-types) and 11 H-genotypes (Hg-types) from 45 isolates; however, Og-types of 19 isolates could not be determined. Stx gene subtyping by PCR identified 2 stx1 subtypes (1a and 1c) and 4 stx2 subtypes (2a, 2c, 2d, and 2g). Forty-five isolates were divided into 21 different groups based on the Og- and Hg-types, stx gene subtypes and the existence of virulence factors, ehxA, eae, and saa, which includes several major serotypes associated with human illness such as O26:H11 and O157:H7. The most dominant isolate, OgUT:H19 [stx1a (+), stx2a (+), ehxA (+) and saa (+)], was isolated from eight farms. This is the first report on the characterization of STEC in cattle in Mongolia, and the results suggest the importance of further monitoring of STEC contamination in the food chains as well as STEC infection in humans.

Differential Colonization and Mucus Ultrastructure Visualization in Bovine Ileal and Rectal Organoid-Derived Monolayers Exposed to Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.

Bifidobacterium longum K5 Prevents Enterohaemorrhagic Escherichia coli O157:H7 Infection in Mice through the Modulation of the Gut Microbiota.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a commonly encountered foodborne pathogen that can cause hemorrhagic enteritis and lead to hemolytic uremic syndrome (HUS) in severe cases. Bifidobacterium is a beneficial bacterium that naturally exists in the human gut and plays a vital role in maintaining a healthy balance in the gut microbiota. This study investigated the protective effects of B. longum K5 in a mouse model of EHEC O157:H7 infection. The results indicated that pretreatment with B. longum K5 mitigated the clinical symptoms of EHEC O157:H7 infection and attenuated the increase in myeloperoxidase (MPO) activity in the colon of the mice. In comparison to the model group, elevated serum D-lactic acid concentrations and diamine oxidase (DAO) levels were prevented in the K5-EHEC group of mice. The reduced mRNA expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) and mucin MUC2, as well as the elevated expression of virulence factors Stx1A and Stx2A, was alleviated in the colon of both the K5-PBS and K5-EHEC groups. Additionally, the increase in the inflammatory cytokine levels of TNF-α and IL-1ß was inhibited and the production of IL-4 and IL-10 was promoted in the K5-EHEC group compared with the model group. B. longum K5 significantly prevented the reduction in the abundance and diversity of mouse gut microorganisms induced by EHEC O157:H7 infection, including blocking the decrease in the relative abundance of Roseburia, Lactobacillus, and Oscillibacter. Meanwhile, the intervention with B. longum K5 promoted the production of acetic acid and butyric acid in the gut. This study provides insights into the use of B. longum K5 for developing probiotic formulations to prevent intestinal diseases caused by pathogenic bacterial infections.

Ellagitannin content and anti-enterohemorrhagic Escherichia coli activity of aqueous extracts derived from commercial pomegranate products.

This study compared the efficacy of aqueous extracts of commercially available pomegranate peel products and a juice powder in inhibiting the growth of two enterohemorrhagic Escherichia coli strains. Cell suspension of each E. coli strain (5 Log CFU/ml) was added into tryptic soy broth amended with 9 or 23% of each extract prepared with two different methods. After treatment for 5, 10, and 24 h at 25 °C, surviving E. coli cells were enumerated on tryptic soy agar to determine cell population reduction compared to the controls. The concentrations of six different ellagitannins and titratable activity in each treatment system were determined and correlated to E. coli cell population reduction. The extracts from three powdered pomegranate peels caused a significantly greater (p ≤ 0.05) reduction in E. coli population than the extract from the whole peel and juice powder. The higher dose of extracts resulted in a greater cell population reduction than the lower dose. The level of E. coli population reduction correlated positively with the total ellagitannins content (R2 0.67-0.98) and the titratable acidity (R2 0.69-0.98) in the treatment systems. The study suggests that pomegranate peels are promising natural additives or preservatives to control pathogens like EHEC.

The fate of enterohemorrhagic Escherichia coli on alfalfa and fenugreek seeds and sprouts as affected by ascaroside #18 treatments.

Alfalfa and fenugreek sprouts are healthy foods, but they are occasionally contaminated with bacterial pathogens and serve as vehicles for transmitting foodborne illnesses. This study examined the efficacy of ascaroside (ascr)#18 treatment for the control of enterohemorrhagic E. coli (EHEC) growth on sprouts. Commercial alfalfa and fenugreek seeds were decontaminated with 20,000 ppm of NaClO, and residual chlorine was neutralized with Dey-Engley broth. Decontaminated seeds were treated with 1 mM or 1 µM ascr#18, a plant immunity modulator, before being dried and mixed with sandy soil inoculated with E. coli F4546 or BAA-2326 at 104-105 CFU/g. The inoculated seeds were sprouted on 1% water agar at 25ºC for 7 days in the dark. Seed or sprout samples were collected on days 0, 1, 3, 5, and 7 for enumeration of bacterial populations. Data was fit into the general linear model and analyzed using Fisher's least significant different test of the statistical analysis software. Treatment with ascr#18 significantly (P ≤ 0.05) reduced the cell population of EHEC on sprouts. The mean EHEC populations in the 1 mM or 1 µM treatment groups were 3.31 or 1.56 log CFU/g lower compared to the control groups. Besides treatment, sprout seed type and sprouting time were also significant independent variables influencing the growth of EHEC, according to the results of type III error analysis. However, EHEC strain type was not a significant independent variable. The study suggests that ascr#18 could be potentially used to control EHEC contamination and improve the microbial safety of sprouts.

Genomic island-encoded regulatory proteins in enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important zoonotic pathogen that is a major cause of foodborne diseases in most developed and developing countries and can cause uncomplicated diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome. O islands (OIs), which are unique genomic islands in EHEC O157:H7, are composed of 177 isolated genomic features and harbour 26% of the total genes that are absent in the non-pathogenic E. coli K-12 genome. In the last twenty years, many OI-encoded proteins have been characterized, including proteins regulating virulence, motility, and acid resistance. Given the critical role of regulatory proteins in the systematic and hierarchical regulation of bacterial biological processes, this review summarizes the OI-encoded regulatory proteins in EHEC O157:H7 characterized to date, emphasizing OI-encoded regulatory proteins for bacterial virulence, motility, and acid resistance. This summary will be significant for further exploration and understanding of the virulence and pathogenesis of EHEC O157:H7.

Enterohemorrhagic Escherichia coli effector EspF triggers oxidative DNA lesions in intestinal epithelial cells.

Attaching/effacing (A/E) pathogens induce DNA damage and colorectal cancer by injecting effector proteins into host cells via the type III secretion system (T3SS). EspF is one of the T3SS-dependent effector proteins exclusive to A/E pathogens, which include enterohemorrhagic Escherichia coli. The role of EspF in the induction of double-strand breaks (DSBs) and the phosphorylation of the repair protein SMC1 has been demonstrated previously. However, the process of damage accumulation and DSB formation has remained enigmatic, and the damage response is not well understood. Here, we first showed a compensatory increase in the mismatch repair proteins MutS homolog 2 (MSH2) and MSH6, as well as poly(ADP-ribose) polymerase 1, followed by a dramatic decrease, threatening cell survival in the presence of EspF. Flow cytometry revealed that EspF arrested the cell cycle at the G2/M phase to facilitate DNA repair. Subsequently, 8-oxoguanine (8-oxoG) lesions, a marker of oxidative damage, were assayed by ELISA and immunofluorescence, which revealed the accumulation of 8-oxoG from the cytosol to the nucleus. Furthermore, the status of single-stranded DNA (ssDNA) and DSBs was confirmed. We observed that EspF accelerated the course of DNA lesions, including 8-oxoG and unrepaired ssDNA, which were converted into DSBs; this was accompanied by the phosphorylation of replication protein A 32 in repair-defective cells. Collectively, these findings reveal that EspF triggers various types of oxidative DNA lesions with impairment of the DNA damage response and may result in genomic instability and cell death, offering novel insight into the tumorigenic potential of EspF.IMPORTANCEOxidative DNA lesions play causative roles in colitis-associated colon cancer. Accumulating evidence shows strong links between attaching/effacing (A/E) pathogens and colorectal cancer (CRC). EspF is one of many effector proteins exclusive to A/E pathogens with defined roles in the induction of oxidative stress, double-strand breaks (DSBs), and repair dysregulation. Here, we found that EspF promotes reactive oxygen species generation and 8-oxoguanine (8-oxoG) lesions when the repair system is activated, contributing to sustained cell survival. However, infected cells exposed to EspF presented 8-oxoG, which results in DSBs and ssDNA accumulation when the cell cycle is arrested at the G2/M phase and the repair system is defective or saturated by DNA lesions. In addition, we found that EspF could intensify the accumulation of nuclear DNA lesions through oxidative and replication stress. Overall, our work highlights the involvement of EspF in DNA lesions and DNA damage response, providing a novel avenue by which A/E pathogens may contribute to CRC.

Z3495, a LysR-Type Transcriptional Regulator Encoded in O Island 97, Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. The genome of EHEC O157:H7 contains 177 unique O islands (OIs). Certain OIs significantly contribute to the heightened virulence and pathogenicity exhibited by EHEC O157:H7. However, the function of most OI genes remains unknown. We demonstrated here that EHEC O157:H7 adherence to and colonization of the mouse large intestine are both dependent on OI-97. Z3495, which is annotated as a LysR-type transcriptional regulator and encoded in OI-97, contributes to this phenotype. Z3495 activated the locus of enterocyte effacement (LEE) gene expression, promoting bacterial adherence. Deletion of z3495 significantly decreased the transcription of ler and other LEE genes, the ability to adhere to the host cells, and colonization in the mouse large intestine. Furthermore, the ChIP-seq results confirmed that Z3495 can directly bind to the promoter region of rcsF, which is a well-known activator of Ler, and increase LEE gene expression. Finally, phylogenetic analysis revealed that Z3495 is a widespread transcriptional regulator in enterohemorrhagic and enteropathogenic Escherichia coli. As a result of this study, we have gained a deeper understanding of how bacteria control their virulence and provide another example of a laterally acquired regulator that regulates LEE gene expression in bacteria.

Bacterial ligases reveal fundamental principles of polyubiquitin specificity.

Homologous to E6AP C terminus (HECT) E3 ubiquitin (Ub) ligases direct substrates toward distinct cellular fates dictated by the specific form of monomeric or polymeric Ub (polyUb) signal attached. How polyUb specificity is achieved has been a long-standing mystery, despite extensive study in various hosts, ranging from yeast to human. The bacterial pathogens enterohemorrhagic Escherichia coli and Salmonella Typhimurium encode outlying examples of "HECT-like" (bHECT) E3 ligases, but commonalities to eukaryotic HECT (eHECT) mechanism and specificity had not been explored. We expanded the bHECT family with examples in human and plant pathogens. Three bHECT structures in primed, Ub-loaded states resolved key details of the entire Ub ligation process. One structure provided a rare glimpse into the act of ligating polyUb, yielding a means to rewire polyUb specificity of both bHECT and eHECT ligases. Studying this evolutionarily distinct bHECT family has revealed insight into the function of key bacterial virulence factors as well as fundamental principles underlying HECT-type Ub ligation.

Possible solution of capturing viable Enterohemorrhagic Escherichia coli (EHEC) in clinical patient stool.

For clinical diagnosis of enterohemorrhagic Escherichia coli (EHEC）, it needs to capture viable EHEC cells from stool sample in the view of medical fee points. However, there is no comprehensive solution for the detection of viable EHEC cells since there are wide variety of serotype and susceptibility against potassium tellurite which is commonly used for selective agent in selective medium for EHEC. In these background, EHEC Clear-HT System (EHEC-CHT）, a novel effective chromogenic medium system for screening comprehensive viable EHEC, was developed. When EHEC-CHT was assessed using 128 microbes including 49 clinical isolated EHEC strains, EHEC-CHT detected all 49 EHEC strains as typical blue-colored colony regardless of both serotype and susceptibility to potassium tellurite. EHEC-CHT was compared with Japanese commercially available tellurite-based EHEC selective media using 107 clinical patient stool samples. EHEC-CHT showed higher detection ratio than conventional tellurite-based selective media compared, and 7% improvement at least in detection ratio in this study.

L-glutamine protects against enterohemorrhagic Escherichia coli infection by inhibiting bacterial virulence and enhancing host defense concurrently.

IMPORTANCE: The type 3 secretion system (T3SS) was obtained in many Gram-negative bacterial pathogens, and it is crucial for their pathogenesis. Environmental signals were found to be involved in the expression regulation of T3SS, which was vital for successful bacterial infection in the host. Here, we discovered that L-glutamine (Gln), the most abundant amino acid in the human body, could repress enterohemorrhagic Escherichia coli (EHEC) T3SS expression via nitrogen metabolism and therefore had potential as an antivirulence agent. Our in vitro and in vivo evidence demonstrated that Gln could decline EHEC infection by attenuating bacterial virulence and enhancing host defense simultaneously. We repurpose Gln as a potential treatment for EHEC infection accordingly.

Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers.

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.

Protective immunization against Enterohemorrhagic Escherichia coli and Shigella dysenteriae Type 1 by chitosan nanoparticle loaded with recombinant chimeric antigens comprising EIT and STX1B-IpaD.

Increasing evidence demonstrated that Enterohemorrhagic Escherichia coli (EHEC) and Shigella dysenteriae type 1 (S. dysenteriae1) are considered pathogens, that are connected with diarrhea and are still the greatest cause of death in children under the age of five years, worldwide. EHEC and S. dysenteriae 1 infections can be prevented and managed using a vaccination strategy against pathogen attachment stages. In this study, the chitosan nanostructures were loaded with recombinant EIT and STX1B-IpaD polypeptides. The immunogenic properties of this nano-vaccine candidate were investigated. The EIT and STX1B-IpaD recombinant proteins were heterologous expressed, purified, and confirmed by western blotting. The chitosan nanoparticles, were used to encapsulate the purified proteins. The immunogenicity of recombinant nano vaccine candidate, was examined in three groups of BalB/c mice by injection, oral delivery, and combination of oral-injection. ELISA and antibody titer, evaluated the humoral immune response. Finally, all three mice groups were challenged by two pathogens to test the ability of the nano-vaccine candidate to protect against bacterial infection. The Sereny test in guinea pigs was used to confirm the neutralizing effect of immune sera in controlling S. dysenteriae 1, infections. SDS-PAGE and western blotting, confirmed the presence and specificity of 63 and 27 kDa recombinant EIT and STX1B-IpaD, respectively. The results show that the nanoparticles containing recombinant proteins could stimulate the systemic and mucosal immune systems by producing IgG and IgA, respectively. The challenge test showed that, the candidate nano-vaccine could protect the animal model from bacterial infection. The combination of multiple recombinant proteins, carrying several epitopes and natural nanoparticles could evocate remarkable humoral and mucosal responses and improve the protection properties of synthetic antigens. Furthermore, compared with other available antigen delivery methods, using oral delivery as immune priming and injection as a booster method, could act as combinatorial methods to achieve a higher level of immunity. This approach could present an appropriate vaccine candidate against both EHEC and S. dysenteriae 1.

Evaluation of the Microbiological Status of Cattle Carcasses in Mongolia: Considering the Hygienic Practices of Slaughter Establishments.

The meat industry has received great attention in Mongolia, having over 70 million livestock, and is important to the nation's economy. Systematic microbiological testing of carcasses has not been mandatorily regulated in all abattoir premises, and the efficacy of the introduction of the Good Hygiene Practice and Hazard Analysis Critical Control Points (HACCP) to some plants has not yet been tested microbiologically in Mongolia. Therefore, samples were collected from two establishments: plant A with an HACCP certificate from a third party and plant B without an HACCP certificate. The rates and levels of the total bacterial count (TBC) as overall hygiene indicators, the Enterobacteriaceae count (EBC) as fecal contamination indicators, and the Staphylococcus spp. count (SC) as personal hygiene indicators were determined on different parts of beef carcasses. The contamination rates in most parts were lower in plant A than in plant B (e.g., TBC in the rump and flank: 103-105 and 105-107, in plant A vs. 104-106 and 105-108 in plant B, respectively). Plant A also had a lower EBC and SC (p < 0.001). Furthermore, 2 out of 100 beef carcasses (2%) were positive for enterohemorrhagic Escherichia coli as a foodborne pathogen indicator in plant A.

Influence of temperature and pH on induction of Shiga toxin Stx1a in Escherichia coli.

Shiga toxin-producing strains represent pathogenic group that is of concern in food production. The present study evaluated forty-eight E. coli isolates (11 with intact stx gene, while remaining isolates presented only stx-fragments) for Shiga toxin production. The four most expressive stx-producers (O26, O103, O145, and O157) were selected to evaluate effects of pH (3.5, 4.5, and 7) and temperature (35, 40, and 50°C). After determining acid stress effects in media on Stx-induction, we mimicked "in natura" conditions using milk, apple, and orange juices. Only isolates that showed the presence of intact stx gene (11/48) produced Shiga toxin. In addition, acid pH had a role in down-regulating the production of Shiga toxin, in both lactic acid and juices. In contrast, non-lethal heating (40°C), when in neutral pH and milk was a favorable environment to induce Shiga toxin. Lastly, two isolates (O26 and O103) showed a higher capacity to produce Shiga toxin and were included in a genomic cluster with other E. coli involved in worldwide foodborne outbreaks. The induction of this toxin when subjected to 40°C may represent a potential risk to the consumer, since the pathogenic effect of oral ingestion of Shiga toxin has already been proved in an animal model.

Acid tolerance of enterohemorrhagic Escherichia coli O157:H7 strain ATCC 43894 and its relationship with a large virulence plasmid pO157.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes a severe intestinal infection including hemolytic uremic syndrome in humans. Various factors contribute to its pathogenesis, including a large virulence plasmid pO157. This F-like 92-kb plasmid is isolated in virtually all clinical EHEC isolates, and is considered a hallmark of EHEC virulence. A previous report stated that removal of pO157 from EHEC ATCC 43894 induced overexpression of GadAB that are essential in glutamate-dependent acid resistance (GDAR) system, yet the mechanism remains elusive. Based on this observation, we surmised that pO157 is involved in the regulation of GDAR system. We comparatively analyzed 43894 and its pO157-cured (ΔpO157) mutant 277 for i) their acid resistance, ii) changes in the transcriptional profiles and iii) expression of GDAR associated genes/proteins. Survivability of 43894 upon exposure to acidic conditions was significantly lower than the ΔpO157 mutant. In addition, RNA-sequencing revealed that genes involved in GDAR were significantly down-regulated in 43894 when compared to the ΔpO157 mutant. Exogenous expression of GadE in 43894 led to expression of GadAB, suggesting possible intervention of pO157 in GDAR regulation. Despite these findings, reintroduction of pO157 into 277 did not reverted Gad overexpression. Likewise, removing pO157 from 43894 using the plasmid incompatibility method did not induce Gad overexpression as shown in 277. Taken together, the results suggest that variation in acid resistance among EHEC isolates exists, and the large virulence plasmid pO157 has no effect on weak acid resistance phenotype displayed in 43894.

Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier.

Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.

Application of BI-EHEC and BI-EPEC bacteriophages to control enterohemorrhagic and enteropathogenic escherichia coli on various food surfaces.

OBJECTIVES: The purposes of this study were to determine the Efficiency of Plating (EOP) value of Bacteriophage BI-EHEC and BI-EPEC and to evaluate the application of these bacteriophages in reducing population of EHEC and EPEC on various food samples. RESULTS: In this study, we used bacteriophage BI-EHEC and BI-EPEC, which were isolated from previous study. Both phages were tested with other multiple pathotypes of intestinal pathogenic E. coli to determine the efficiency of plating. BI-EHEC had high efficiency toward ETEC with an EOP value of 2.95 but low efficiency toward EHEC with an EOP value of 0.10, while BI-EPEC had high efficiency toward EHEC and ETEC with EOP values of 1.10 and 1.21, respectively. As biocontrol agents, both bacteriophages able to reduce CFU of EHEC and EPEC in several food samples using 1 and 6-days incubation times at 4 [Formula: see text]. BI-EHEC reduced the number of EHEC with an overall percentage of bacterial reduction value above 0.13 log10, while BI-EPEC reduced number of EPEC with reduction value above 0.33 log10.

Complete genome analysis of Tequatrovirus ufvareg1, a Tequatrovirus species inhibiting Escherichia coli O157:H7.

Introduction: Bacteriophages infecting human pathogens have been considered potential biocontrol agents, and studying their genetic content is essential to their safe use in the food industry. Tequatrovirus ufvareg1 is a bacteriophage named UFV-AREG1, isolated from cowshed wastewater and previously tested for its ability to inhibit Escherichia coli O157:H7. Methods: T. ufvareg1 was previously isolated using E. coli O157:H7 (ATCC 43895) as a bacterial host. The same strain was used for bacteriophage propagation and the one-step growth curve. The genome of the T. ufvareg1 was sequenced using 305 Illumina HiSeq, and the genome comparison was calculated by VIRIDIC and VIPTree. Results: Here, we characterize its genome and compare it to other Tequatrovirus. T. ufvareg1 virions have an icosahedral head (114 x 86 nm) and a contracted tail (117 x 23 nm), with a latent period of 25 min, and an average burst size was 18 phage particles per infected E. coli cell. The genome of the bacteriophage T. ufvareg1 contains 268 coding DNA sequences (CDS) and ten tRNA genes distributed in both negative and positive strains. T. ufvareg1 genome also contains 40 promoters on its regulatory regions and two rho-independent terminators. T. ufvareg1 shares an average intergenomic similarity (VIRIDC) of 88.77% and an average genomic similarity score (VipTree) of 88.91% with eight four reference genomes for Tequatrovirus available in the NCBI RefSeq database. The pan-genomic analysis confirmed the high conservation of Tequatrovirus genomes. Among all CDS annotated in the T. ufvareg1 genome, there are 123 core genes, 38 softcore genes, 94 shell genes, and 13 cloud genes. None of 268 CDS was classified as being exclusive of T. ufvareg1. Conclusion: The results in this paper, combined with other previously published findings, indicate that T. ufvareg1 bacteriophage is a potential candidate for food protection against E. coli O157:H7 in foods.