Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes foodborne outbreaks leading to hemolytic uremic syndrome (HUS) via Shiga toxin (Stx). During HUS pathogenesis, EHEC O157:H7 is phagocytized by intestinal macrophages, although only a small proportion of internalized bacteria survive within the phagosome. To investigate this persistence, we performed global RNA-seq analysis of intracellular EHEC O157:H7 within murine macrophage phagosomes, complemented by in vitro assays simulating key phagosomal stressors. Our results reveal that EHEC employs a sophisticated survival strategy characterized by metabolic reprogramming and stress resistance. Inside macrophages, EHEC activates the SOS pathway, synthesizes membrane lipids, and engages NO detoxification mechanisms while repressing energetically costly virulence systems —motility, Type 3 Secretion System (T3SS), and Type 6 Secretion System (T6SS)—to prioritize survival. Notably, Shiga toxin gene regulation proved highly context-dependent: phagocytosis selectively upregulated the stx2a A subunit via the SOS response, whereas stx1a remained uninduced, likely restrained by NO-mediated repression. In contrast, under in vitro multi-stress conditions (pH 4.5) engaged the AR2–AR5 acid resistance systems and triggered robust expression of both stx1a subunits (A and B). These findings highlight the dynamic, stress-specific regulatory networks EHEC relies on to persist within the host.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic foodborne pathogen responsible for different cases and outbreaks of severe enteric diseases in humans worldwide. Our group previously showed that Argentine O22:H8 isolates (155 and 182) interfere with the colonization of O157:H7 in an experimental infection bovine model. In this study, we applied a comparative genomic and genome-wide association study (GWAS) analysis to explore the differences between the genetic bases of O22:H8 and O157:H7 strains isolated from cattle. The whole-genome sequence of O22:H8 155 and 182 isolates was compared with 33 O22:H8 and 156 O157:H7 isolates from bovine. Our results showed that O22:H8 isolates harbor genes associated with specific metabolic pathways related to the metabolism of products of vegetal origin, which are absent in O157:H7 isolates. These O22:H8 isolates also carry classical genes of the locus of adhesion and autoaggregation related to the adhesion of non-O157 isolates. In addition, we showed that the T6SS-1 cluster related to bacterial competition predicted in the Argentine O22:H8 isolates is absent both in O157:H7 and other O22:H8 isolates. We also predicted for the first time the locus of type VI secretion system 1 (LT6SS-T1), a genomic island that harbors both T6SS-1 cluster genes and genes related to STEC pathogenesis. This study reveals a subset of genes present in the Argentine O22:H8 isolates that could confer an advantage over O157:H7 during the colonization process in the bovine intestinal tract.

Background/Objectives: Enterohemorrhagic Escherichia coli (EHEC) O157:H7, a zoonotic pathogen primarily found in cattle, causes Hemolytic Uremic Syndrome (HUS) in humans, often through contaminated food. Its Type Three Secretion System (T3SS) facilitates gut colonization. In contrast, neonatal calf diarrhea (NCD) is mainly caused by pathogens like enterotoxigenic Escherichia coli (ETEC), Salmonella spp., Bovine Coronavirus (BCoV), and Bovine Rotavirus type A (BRoVA). This study engineered a chimeric protein combining EspB and Int280γ, two T3SS components, expressed in the membranes of Salmonella Dublin and ETEC. Methods: Immune responses in vaccinated mice and guinea pigs were assessed through ELISA assays. Results: Successful membrane anchorage and stability of the chimera were confirmed. Immune evaluations showed no enhancement from combining recombinant bacteria, indicating either bacterium suffices in a single formulation. Chimeric expression yielded immunogenicity equivalent to 10 µg of recombinant protein, with similar antibody titers. IgG1/IgG2a levels and Th1, Th2, and Th17 markers indicated a mixed immune response, providing broad humoral and cellular protection. Responses to BCoV, BRoVA, ETEC, and Salmonella antigens remained strong and did not interfere with chimera-specific responses, potentially boosting NCD vaccine efficacy. Conclusions: The chimera demonstrated robust immunogenicity, supporting its potential as a viable vaccine candidate against EHEC O157:H7. This approach could enhance NCD vaccine valency by offering broader protection against calf diarrhea while reducing HUS transmission risks to humans.

Background, Context or Rationale Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans may cause bloody diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome (HUS). Cattle are the main reservoir of EHEC and a source of infection for humans. It has been previously demonstrated that hyperimmune bovine colostrum (HC) against virulence factors of EHEC was efficiently transferred to newborn calves through lactation. Also, it is well described that human consumption of HC can provide therapeutic effects on several gastrointestinal infections. Aim(s) The objectives of the present work were to produce HC using two recombinant virulence factors of EHEC as immunogen: C-terminal fragment of Intimin (C280-Int) and Shiga toxin type 2 (Stx2), and then to evaluate specific IgG levels and correlate them with the ability of HC to neutralise the cytotoxic activity of Stx2 and EHEC O157:H7 adhesion. Methods For this, 12 pregnant Aberdeen Angus breed cows were immunised with two doses of C280-Int and Stx2 at 40 and 20 days before calving. Colostrum was obtained within 24 h after delivery and specific IgG against Stx2 and Intimin was evaluated by ELISA. Stx2 neutralisation capacity of HC was evaluated on Vero cells. EHEC O157:H7 adherence inhibition was evaluated on human intestinal epithelial cells (HCT-8). Major Findings Stx2 neutraliation capacity was observed in nine of the HC obtained, and 10 colostrum could inhibit adherence of EHEC O157:H7 to HCT-8 cells. A correlation was observed between Stx2 neutralisation and adherence inhibition of EHEC with specific IgG levels thus indicating that specific IgG levels may predict the neutralisation capacity of colostrum. Scientific or Industrial Implications HC against EHEC virulence factors is effective in neutralising the virulence activity of EHEC O157:H7. This opens a perspective on the utilisation of HC for controlling EHEC colonisation in calves and to prevent the development HUS in humans.

AbstractEHEC O157:H7 is responsible for Hemolytic Uremic Syndrome (HUS) outbreaks in humans and its virulence is associated with Shiga toxin (Stx). During the pathogenesis of HUS, EHEC O157:H7 is phagocytized by the intestinal host macrophage into the phagosome. According to our study, a low percentage of phagocytized bacteria survived in the murine macrophage phagosome. To understand the initials mechanisms involved in bacterial persistence, we performed a global profile of bacterial RNAseq analysis in the early phagosome of murine macrophage andin vitroassays with more extreme stress conditions. The defense strategy of an early murine phagosome consisted on producing DNA damage, membrane damage, acid pH and nitric oxide (NO) agents. These hostile conditions triggering the bacterial SOS response, lipid biosynthesis and NO detoxification enzymes in response to DNA damage, membrane damage and nitric oxide agents, respectively. In turn, to survive, the bacterium conserves energy by downregulating flagellar biosynthesis, T3SS and T6SS virulence mechanisms. In contrast,stx2aexpression is upregulated. At the same time, it increases the ribosomal levels and amino acid synthesis to react more effectively against adverse conditions. Under more extreme stress conditions, EHEC O157:H7 expressed genes related to an acidic environment (pH 4.5) upregulating the acid stress response pathways AR2-5, whereas, upon high concentrations of hydrogen peroxide, it transcribed higher levels of genes of the OxyR pathway and subsequently hydrogen peroxide oxidative stress-related genes.

Bovine tuberculosis (bTB), a global zoonotic disease, causes negative effects on human and animal health. PhoP protein is a key regulator of pathogenic phenotypes in members of the Mycobacterium tuberculosis complex, which includes the causative agent of bTB. Despite extensive research on this protein focused in deciphering its regulatory role, little was explored about it as a diagnostic antigen. In humans, a novel role of anti-PhoP antibodies as a possible marker for the diagnosis of TB was demonstrated. However, this issue was not addressed in bovines. In this study, antigenic properties of the PhoP protein were evaluated in naturally Mycobacterium bovis (M. bovis) infected bovines. A high homology of PhoP (>= 75 %) was observed in environmental mycobacterial species and other genera such as Salmonella and Pasteurella. Using the IFN-gamma release assay (IGRA), we detected cellmediated immune response against PhoP in cattle from infected herds (25 %; IC 95 % 3.2-65.1), although it was significantly lower than that evoked by the reference antigens, ESAT-6/CFP-10/Rv3615c (75 %; IC95 % 34.9-96.8), and the purified protein derivative (87.5 %; IC 95 % 47.4-99.7) (p < 0.05)). Animals from a bTB free area showed no response against PhoP when analyzed by IGRA. Although, the humoral response detected 62.5 % (CI95% 24.5-91.5) of naturally infected animals, there was 100 % cross-reactivity among TB-free cattle. These results suggest that the PhoP protein is not a promising candidate for bTB diagnosis, due to it had relatively low levels of test sensitivity in the IGRA test, and very low specificity in a humoral antibody western blot assay.

Shiga toxing-producing Escherichia coli (STEC) O22:H8 strain is a serotype occasionally isolated in Argentinian cattle. Preliminary works showed that the cattle carrying STEC O22:H8 strains could not be experimentally colonized by EHEC O157:H7 strain. The type 6 secretion system (T6SS) is one of the most versatile virulence mechanisms involved in delivering effectors, particularly the T6SS1 translocate antibacterial toxins effectors during bacterial competition for niche-space. In this work, we could evidence the molecular bases of the success of STEC O22:H8 (154) strain during bacterial competition against EHEC O157:H7 strains. The genome sequence of STEC O22:H8 (154) allowed us to identify a complete T6SS1 cluster. In addition, we identify and characterized several putative T6SS1-antibacterial effectors encoded inside the T6SS1 clusters and in genomic pathogenic islands. Competition assays against EHEC O157:H7 strain confirmed the antibacterial activity of STEC O22:H8 (154) strain in vitro . Considering the absent of T6SS1 in STEC strains, we proposed the recent horizontal transfer acquisition and the most probably donor belong to the same Escherichia coli species. A safe STEC O22:H8 (154) Δstx would be used as a new strategy to fight STECs in bovine intestinal colonization, leading in a reduction in beef contamination and consequently HUS cases in humans.### Competing Interest StatementThe authors have declared no competing interest.

Background/Objectives: Vaccines may improve the control and eradication of bovine tuberculosis. However, the evaluation of experimental candidates requires the assessment of the protection, excretion, transmission and biosafety. A natural transmission trial among likely infected animals was conducted. Methods: Seventy-four male heifers were randomly distributed (five groups) and vaccinated subcutaneously with attenuated strains (M. bovis Δmce2 or M. bovis Δmce2-phoP), a recombinant M. bovis BCG Pasteur (BCGr) or M. bovis BCG Pasteur. Then, they cohoused with a naturally infected bTB cohort under field conditions exposed to the infection. Results: A 23% of transmission of wild-type strains was confirmed (non-vaccinated group). Strikingly, first vaccination did not induce immune response (caudal fold test and IFN-gamma release assay). However, after 74 days of exposure to bTB, animals were re-vaccinated. Although their sensitization increased throughout the trial, the vaccines did not confer significant protection, when compared to the non-vaccinated group, as demonstrated by pathology progression of lesions and confirmatory tools. Besides, the likelihood of acquiring the infection was similar in all groups compared to the non-vaccinated group (p > 0.076). Respiratory and digestive excretion of viable vaccine candidates was undetectable. To note, the group vaccinated with M. bovis Δmce2-phoP exhibited the highest proportion of animals without macroscopic lesions, compared to the one vaccinated with BCG, although this was not statistically supported. Conclusions: This highlights that further evaluation of these vaccines would not guarantee better protection. The limitations detected during the trial are discussed regarding the transmission rate of M. bovis wild-type, the imperfect test for studying sensitization, the need for a DIVA diagnosis and management conditions of the trials performed under routine husbandry conditions. Re-vaccination of likely infected bovines did not highlight a conclusive result, even suggesting a detrimental effect on those vaccinated with M. bovis BCG.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, primarily found in cattle, is a zoonotic pathogen associated with Hemolytic Uremic Syndrome (HUS) in humans that is typically transmitted through contaminated food. Among its many virulence factors, the Type Three Secretion System (T3SS) is responsible for gut colonization. Neonatal calf diarrhea (NCD), in contrast, is primarily caused by pathogens such as enterotoxigenic Escherichia coli (ETEC), Salmonella spp, Bovine Coronavirus (BCoV) and Bovine Rotavirus type A (BRoVA). In this study, we engineered and expressed a chimeric protein combining EspB and Int280γ—two key components of the T3SS—in the membrane of Salmonella dublin and ETEC. We confirmed successful membrane anchorage, stability and preservation of the chimera and assessed its immunogenicity in murine and guinea pig models. Immune response evaluations showed that combining recombinant bacteria did not enhance immunogenicity, indicating either bacterium could be effective in a single formulation. Chimeric expression achieved equivalent immunogenicity to 10 µg of recombinant chimera protein, with similar antibody titers across doses, indicating that a single vaccination may suffice. IgG1 and IgG2a levels, along with Th1, Th2, and Th17 markers, suggest a mixed immune response, providing broad humoral and cellular protection. Additionally, the immune response to BCoV, BRoVA, ETEC and Salmonella antigens remained high and showed no interference with the chimera-specific responses, which could enhance the overall efficacy of an NCD vaccine. The results underscore the robust immunogenicity of the chimera, supporting its potential as a commercially viable and effective vaccine candidate against EHEC O157:H7. This strategy could enhance the valency of NCD vaccines by offering broader protection against calf diarrhea and contribute to public health by reducing the risk of HUS transmission from cattle to humans.

Transgene with recombination sites to address biosafety concerns engineered into lettuce to produce EspB and γ-intimin C280 for oral vaccination against EHEC O157:H7. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen where ruminant farm animals, mainly bovine, serve as reservoirs. Bovine vaccination has been used to prevent disease outbreaks, and the current method relies on vaccines subcutaneously injected three times per year. Since EHEC O157:H7 colonizes mucosal surfaces, an oral vaccine that produces an IgA response could be more convenient. Here, we report on oral vaccination against EHEC O157:H7 in mice orally gavaged with transgenic lettuce that produces EHEC O157:H7 antigens EspB and γ-intimin C280. Younger leaves accumulated a higher concentration of antigens; and in unexpanded leaves of 30-day-old T2 plants, EspB and γ-intimin C280 were up to 32 and 51 μg/g fresh weight, respectively. Mice orally gavaged with lettuce powders containing < 3 µg antigens for 6 days showed a mucosal immune response with reduced colonization of EHEC O157:H7. This suggests that the transgenic lettuce has potential to be used for bovine vaccination. To promote the biosafety of crop plants producing medically relevant proteins, recombination sites were built into our transgenic lines that would permit optional marker removal by Cre-lox recombination, as well as transgene deletion in pollen by CinH-RS2 recombination. The ability to upgrade the transgenic lettuce by stacking additional antigen genes or replacing older genes with newer versions would also be possible through the combined use of Bxb-att and Cre-lox recombination systems.

Cattle vaccination is an attractive approach in compliance with control and eradication programs against Bovine Tuberculosis (bTB). Today, there is no anti bTB vaccine licensed. Two vaccine candidates, MbΔmce2 and MbΔmce2-phoP previously designed were evaluated in BALB/c mice, including the parental M. bovis NCTC10772 and a M. bovis hypervirulent Mb04-303 strains as controls. Sentinel mice (non-inoculated) cohoused with subcutaneous inoculated mice. Persistence, visible tuberculosis lesions (VTL) in lungs and spleens and bacillary load were investigated subcutaneously delivered at 60 and 90 days after inoculation (dpi) as well as their potential transmission to naïve mice. While a 100% survival was observed at 90 dpi without VTL in all groups, transmission was not evidenced in the sentinels mice. Vaccine candidates and control strains were isolated from the spleen of all inoculated mice, while Mb04-303 was isolated from the lungs of one inoculated mouse. Vaccine candidate's attenuation considering survival, lung bacillary load and VTL was confirmed, administrated by the subcutaneous route. Future experiments are necessary to demonstrate whether the persistence of both mutants in the spleen, with low CFU, remains over time to increase the potential increasing risk of dissemination to organs and subsequent transmission to other animals by airborne or other routes.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen capable of causing illness in humans. In a previous study, our group showed that a STEC isolate belonging to O22:H8 serotype (strain 154) can interfere with STEC O157:H7 colonization both in vitro and in vivo . Using whole-genome sequencing and genomic comparative, we predicted a subset of genes acquired by O22:H8 strain 154 through horizontal gene transfer that might be responsible for the phenotype previously described by our group. Among them were identified genes related to the pathogenesis of non-LEE (locus of enterocyte effacement) STEC, specific metabolic processes, antibiotic resistance and genes encoding for the T6SS-1 that is related to inter-bacterial competition. In addition, we showed that this strain carries stx1c and stx2d act , a mucus-inducible variant. The results obtained in this study provide insights into STEC genomic plasticity and the importance of genomic islands in the adaptation and pathogenesis of this pathogen.

It is known that nitrate inhibits ruminal methanogenesis, mainly through competition with hydrogenotrophic methanogens for available hydrogen (H2) and also through toxic effects on the methanogens. However, there is limited knowledge about its effects on the others members of ruminal microbiota and their metabolites. In this study, we investigated the effects of dietary nitrate inclusion on enteric methane (CH4) emission, temporal changes in ruminal microbiota, and fermentation in Holstein calves. Eighteen animals were maintained in individual pens for 45 d. Animals were randomly allocated to either a control (CTR) or nitrate (NIT, containing 15 g of calcium nitrate/kg dry matter) diets. Methane emissions were estimated using the sulfur hexafluoride (SF6) tracer method. Ruminal microbiota changes and ruminal fermentation were evaluated at 0, 4, and 8 h post-feeding. In this study, feed dry matter intake (DMI) did not differ between dietary treatments (P > 0.05). Diets containing NIT reduced CH4 emissions by 27% (g/d) and yield by 21% (g/kg DMI) compared to the CTR (P < 0.05). The pH values and total volatile fatty acids (VFA) concentration did not differ between dietary treatments (P > 0.05) but differed with time, and post-feeding (P < 0.05). Increases in the concentrations of ruminal ammonia nitrogen (NH3–N) and acetate were observed, whereas propionate decreased at 4 h post-feeding with the NIT diet (P < 0.05). Feeding the NIT diet reduced the populations of total bacteria, total methanogens, Ruminococcus albus and Ruminococcus flavefaciens, and the abundance of Succiniclasticum, Coprococcus, Treponema, Shuttlewortia, Succinivibrio, Sharpea, Pseudobutyrivibrio, and Selenomona (P < 0.05); whereas, the population of total fungi, protozoa, Fibrobacter succinogenes, Atopobium and Erysipelotrichaceae L7A_E11 increased (P < 0.05). In conclusion, feeding nitrate reduces enteric CH4 emissions and the methanogens population, whereas it decreases the propionate concentration and the abundance of bacteria involved in the succinate and acrylate pathways. Despite the altered fermentation profile and ruminal microbiota, DMI was not influenced by dietary nitrate. These findings suggest that nitrate has a predominantly direct effect on the reduction of methanogenesis and propionate synthesis.

Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12–14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.

Aims & Objective: We studied the transmissibility in mice of two candidate vaccine M. bovis strains, M. bovis Δmce2 and M. bovis Δmce2ΔphoP, attenuated by gene knock out. The parental strain M. bovis UK (NCTC10772) and a M. bovis wild boar isolate (Mb04-303) were inoculated as controls. Methods: Groups of animals including 3 non-inoculated + 3 subcutaneously inoculated with every strain (1.105 CFU) were placed in the same cage (in duplicate). Necropsy was performed at 60 and 90 days post inoculation (dpi) in search of visible tuberculosis lesions (VTL) in lungs and spleens. Organs were analyzed to determine the bacterial load. Results: None of the animals presented VTL and no M. bovis was detectable in the non-inoculated group at 60 and 90 dpi. M. bovis was isolated from spleen of all the inoculated mice and from lungs of mice inoculated with control strains. The candidate vaccines were not isolated from lungs. Conclusions: This study shows the absence of transmissibility up to 90 days. Recovery of viable control strains from lungs and spleen suggests that the administration route would not alter the capacity of virulent strains to colonize organs. Contrasting, the candidate vaccines were only isolated from spleen. Longer periods should be assayed to evaluate transmissibility of these strains in mice.

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis, primarily affecting the lungs. The M. tuberculosis strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.

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The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine β defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.

Human pathogenic gram‐negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled‐coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha‐helical structures that play an important role in the protein‐protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled‐coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS‐dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking‐molecular dynamic simulations, and quantum‐mechanic calculations of the various peptide‐protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled‐coil domain of the C‐terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide‐protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad‐spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.

Bovines are the primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the main source of its transmission to humans. Here, we present a one-year longitudinal study of fecal shedding of E. coli O157. E. coli O157 obtained from recto-anal mucosal samples were characterized by multiplex PCR. The E. coli O157 prevalence ranged from 0.84% in July to 15.25% in November. The confinement within pens resulted in prevalence of 11%. Most animals (61.86%; 75/118) shed E. coli O157 at least in one sampling occasion. Of the positive animals, 82.19%, 16.44%, and 1.37% were stx positive on one, two and three sampling occasions, respectively. All the E. coli O157 isolated strains carried the genes eae and rfbO157, whereas 11%, 33% and 56% contained stx1, stx2 and stx1/stx2, respectively. The stx1/stx2 and stx2 types were significantly higher during the grazing and finishing periods, respectively, in comparison with the rearing and grazing periods. The presence of stx2a subtype was evident in four isolates, whereas stx2c was present in at least seven. However, both subtypes were present simultaneously in two isolates. The stx1/stx2c, stx1/stx2d and stx1/stx2NT genotypes occurred in 24, 2 and 15 isolates, respectively. The simultaneous occurrence of stx1 and stx2c significantly increased during grazing. Some cases of within-pen and between-pen transmission occurred throughout the study. Contagion levels during in-field grazing were higher than during permanent confinement in the pens. Thus, the individual patterns of shedding varied depending on the proportion of animals shedding the bacteria within pens and the time of shedding.