Search

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) can colonize the gastrointestinal tract and cause bloody diarrhea in children. Previous studies showed that its pathogenesis could be mediated by metabolites from both the host and microbiota. L-Glutamine (Gln) was found to be depleted in intestinal tissues as the main energy source according to previous studies. Hence, we aimed to determine the effects of Gln on EHEC infection and its underlying mode of action. In this study, a Gln-limited signal was found to activate the type 3 secretion system (T3SS), which is crucial for EHEC infection via perturbation of central metabolism. By shifting the phosphorylation of NtrC, a key regulator in bacterial nitrogen metabolism, Gln stimulates ler transcripts in a σS-PchA-dependent manner. Our in vivo experiments further demonstrated that Gln supplementation can reduce EHEC colonization in the gastrointestinal tract by repressing T3SS. Moreover, Gln could further attenuate bacterial infection by boosting host defense, which might be dependent on multiple pathways. Besides, our experiments demonstrated that Gln did not induce Shiga-like toxin (Stx) production or cause impairment of gut flora. In conclusion, our study presented evidence that Gln could act against EHEC infection by reducing bacterial virulence and strengthening host defense. Therefore, Gln serves as a promising therapeutic agent for EHEC infection. 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.

Microbiota, host and dietary metabolites/signals compose the rich gut chemical environment, which profoundly impacts virulence of enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) engages a syringe-like machinery named type-III secretion system (T3SS) to inject effectors within host cells that lead to intestinal colonization and disease. We previously conducted a high-throughput screen to identify metabolic pathways that affect T3SS expression. Here we show that in the presence of arginine, the arginine sensor ArgR, identified through this screen, directly activates expression of the genes encoding the T3SS. Exogenously added arginine induces EHEC virulence gene expression in vitro. Congruently, a mutant deficient in arginine transport (ΔartP) had decreased virulence gene expression. ArgR also augments murine disease caused by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. The source of arginine sensed by C. rodentium is not dietary. At the peak of C. rodentium infection, increased arginine concentration in the colon correlated with down-regulation of the host SLC7A2 transporter. This increase in the concentration of colonic arginine promotes virulence gene expression in C. rodentium Arginine is an important modulator of the host immune response to pathogens. Here we add that arginine also directly impacts bacterial virulence. These findings suggest that a delicate balance between host and pathogen responses to arginine occur during disease progression.

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.

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.

IntroductionBacteriophages 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.MethodsT. 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.ResultsHere, 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.ConclusionThe 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.

Colonocytes possess a specific carrier-mediated uptake process for the microbiota-generated thiamin (vitamin B1) pyrophosphate (TPP) that involves the TPP transporter (TPPT; product of the SLC44A4 gene). Little is known about the effect of exogenous factors (including enteric pathogens) on the colonic TPP uptake process. Our aim in this study was to investigate the effect of Enterohemorrhagic Escherichia coli (EHEC) infection on colonic uptake of TPP. We used human-derived colonic epithelial NCM460 cells and mice in our investigation. The results showed that infecting NCM460 cells with live EHEC (but not with heat-killed EHEC, EHEC culture supernatant, or with non-pathogenic E. Coli) to lead to a significant inhibition in carrier-mediated TPP uptake, as well as in level of expression of the TPPT protein and mRNA. Similarly, infecting mice with EHEC led to a significant inhibition in colonic TPP uptake and in level of expression of TPPT protein and mRNA. The inhibitory effect of EHEC on TPP uptake by NCM460 was found to be associated with reduction in the rate of transcription of the SLC44A4 gene as indicated by the significant reduction in the activity of the SLC44A4 promoter transfected into EHEC infected cells. The latter was also associated with a marked reduction in the level of expression of the transcription factors CREB-1 and ELF3, which are known to drive the activity of the SLC44A4 promoter. Finally, blocking the ERK1/2 and NF-kB signaling pathways in NCM460 cells significantly reversed the level of EHEC inhibition in TPP uptake and TPPT expression. Collectively, these findings show, for the first time, that EHEC infection significantly inhibit colonic uptake of TPP, and that this effect appears to be exerted at the level of SLC44A4 transcription and involves the ERK1/2 and NF-kB signaling pathways.

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a highly adaptive pathogen and has acquired diverse genetic elements, such as genomic islands and prophages, via horizontal gene transfer to promote fitness in vivo. Two-component signaling systems (TCSs) allow bacteria to sense, respond to, and adapt to various environments. This study identified a putative two-component signaling system composed of the histidine kinase EDL5436 (renamed LmvK) and the response regulator EDL5428 (renamed LmvR) in EHEC. lmvK and lmvR along with EDL5429 to EDL5434 (EDL5429–5434) between them constitute the OI167 genomic island and are highly associated with the EHEC pathotype. EDL5429–5434 encode transporters and metabolic enzymes that contribute to growth on mannose and are directly upregulated by LmvK/LmvR in the presence of mannose, as revealed by quantitative PCR (qPCR) and DNase I footprint assays. Moreover, LmvR directly activates the expression of the type III secretion system in response to mannose and promotes the formation of attaching and effacing lesions on HeLa cells. Using human colonoid and mouse infection models, we show that lmvK and lmvR contributed greatly to adherence and microcolony (MC) formation ex vivo and colonization in vivo. Finally, RNA sequencing and chromatin immunoprecipitation coupled with sequencing analyses identified additional direct targets of LmvR, most of which are involved in metabolism. Given that mannose is a mucus-derived sugar that induces virulence and is preferentially used by EHEC during infection, our data revealed a previously unknown mechanism by which EHEC recognizes the host metabolic landscape and regulates virulence expression accordingly. Our findings provide insights into how pathogenic bacteria evolve by acquiring genetic elements horizontally to adapt to host environments. IMPORTANCE The gastrointestinal tract represents a complex and challenging environment for enterohemorrhagic Escherichia coli (EHEC). However, EHEC is a highly adaptable pathogen, requiring only 10 to 100 CFUs to cause infection. This ability was achieved partially by acquiring mobile genetic elements, such as genomic islands, that promote overall fitness. Mannose is an intestinal mucus-derived sugar that stimulates virulence and is preferentially used by EHEC during infection. Here, we characterize the OI167 genomic island of EHEC, which encodes a novel two-component signaling system (TCS) and transporters and metabolic enzymes (EDL5429–5434) involved in mannose utilization. The TCS directly upregulates EDL5429–5434 and genes encoding the type III secretion system in the presence of mannose. Moreover, the TCS contributes greatly to EHEC virulence ex vivo and in vivo. Our data demonstrate an elegant example in which EHEC strains evolve by acquiring genetic elements horizontally to recognize the host metabolic landscape and regulate virulence expression accordingly, leading to successful infections.

We aimed to investigate whether a selective pre-PCR enrichment step improves test performance of RIDA®GENE EHEC/EPEC to detect diarrheagenic Escherichia coli from stool samples. Each of the 250 stool samples was analyzed for the presence of stx1/2 and eae both with and without pre-PCR enrichment in selective broth. In comparison to a reference method, sensitivities for stx1/2 and eae with and without pre-PCR enrichment were 84% (95%CI 70–93) and 89% (stx1/2, 95%CI 76–96), and 71% (95%CI 58–81) and 72% (eae, 95%CI 60–82), respectively. Specificity exceeded 97% for both methods and target genes. In summary, pre-PCR broth enrichment did not improve test performance.

There have been large foodborne outbreaks related to Enterohemorrhagic Escherichia coli (EHEC) around the world. Among its virulence proteins, the EspF encoded by locus of enterocyte effacement is one of the most known functional effector proteins. In this research, we infected the HT-29 cells with the EHEC wild type strain and EspF-deficient EHEC strain. Via the emerging technique isobaric tags for relative and absolute quantitation (iTRAQ), we explored the pathogenic characteristics of EspF within host cells. Our data showed that the differences regarding cellular responses mainly contained immune regulation, protein synthesis, signal transduction, cellular assembly and organization, endoplasmic reticulum (ER) stress, and apoptosis. Notably, compared with the EspF-deficient strain, the protein processing in the ER and ribosome were upregulated during wild type (WT) infection. Our findings proved that the EspF of Enterohemorrhagic Escherichia coli induced ER stress in intestinal epithelial cells; the ER stress-dependent apoptosis pathway was also activated within the host cells. This study provides insight into the virulence mechanism of protein EspF, which will deepen our general understanding of A/E pathogens and their interaction with host proteins.

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common foodborne pathogens. However, no drug that prevents the severe complications caused by this bacterium has been approved yet. This study showed that a macroporous magnesium oxide (MgO)-templated carbon material (MgOC150) adsorbs Shiga toxins, and Type III secretory EspA/EspB proteins responsible for EHEC pathogenesis, and decreases the extracellular levels of these proteins. On the other hand, this material did not affect the growth of EHEC. Citrobacter rodentium traditionally used to estimate Type III secretion system-associated virulence in mice is highly virulent. The survival period of infected mice was prolonged when MgOC150 was administered. This adsorbent disturbed neither mammalian cells nor normal intestinal bacteria, such as Enterococcus hirae, Lactobacillus acidophilus, and Lactobacillus casei. In contrast, MgOC150 adsorbed antimicrobial agents, including β-lactams, quinolones, tetracyclines, and trimethoprim/sulfamethoxazole. However, fosfomycin and amikacin were not adsorbed. Thus, MgOC150 can be used with fosfomycin and amikacin to treat infections. MgOC150 is used for industrial purposes, such as an electrode catalyst, a bioelectrode, and enzyme immobilization. The study proposed another potential application of MgOC150, assisting anti-EHEC chemotherapy.

Abstract Background This study describes the investigation of an outbreak of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) at a daycare center in southeastern Brazil, involving fourteen children, six staff members, six family members, and one nurse. All bacterial and viral pathogens detected were genetically characterized. Results Two isolates of a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O111:H8 were recovered, one implicated in a case of HUS and the other in a case of uncomplicated diarrhea. These isolates had a clonal relationship of 94% and carried the stx2a and eae virulence genes and the OI-122 pathogenicity island. The EHEC strain was determined to be a single-locus variant of sequence type (ST) 327. EHEC isolates were resistant to ofloxacin, doxycycline, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole and intermediately resistant to levofloxacin and ciprofloxacin. Rotavirus was not detected in any samples, and norovirus was detected in 46.7% (14/30) of the stool samples, three of which were from asymptomatic staff members. The noroviruses were classified as the recombinant GII.4 Sydney [P16] by gene sequencing. Conclusion In this outbreak, it was possible to identify an uncommon stx2a + EHEC O111:H8 strain, and the most recent pandemic norovirus strain GII.4 Sydney [P16]. Our findings reinforce the need for surveillance and diagnosis of multiple enteric pathogens by public health authorities, especially during outbreaks.

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 remains a pathogen of significance and high consequence around the world. This outcome is due in part to the high economic impact associated with massive, contaminated product recalls, prevalence of the pathogen in carrier reservoirs, disease sequelae, and mortality associated with several outbreaks worldwide. Furthermore, the contraindication of antibiotic use for the treatment of EHEC-related infections makes this pathogen a primary candidate for the development of effective prophylactic vaccines. However, no vaccines are approved for human use, and many have failed to provide a high degree of efficacy or broad protection, thereby opening an avenue for the use of new technologies to produce a safe, effective, and protective vaccine. Building on our previous studies using reverse vaccinology-predicted antigens, we refine a formulation, evaluate new immunogenic antigens, and further expand our understanding about the mechanism of EHEC vaccine-mediated protection. In the current study, we exploit the use of the nanotechnology platform based on gold nanoparticles (AuNP), which can act as a scaffold for the delivery of various antigens. Our results demonstrate that a refined vaccine formulation incorporating EHEC antigen LomW, EscC, LpfA1, or LpfA2 and delivered using AuNPs can elicit robust antigen-specific cellular and humoral responses associated with reduced EHEC colonization in vivo. Furthermore, our in vitro mechanistic studies further support that antibody-mediated protection is primarily driven by inhibition of bacterial adherence onto intestinal epithelial cells and by promotion of macrophage uptake and killing. IMPORTANCE Enterohemorrhagic E. coli O157:H7 remains an important human pathogen that does not have an effective and safe vaccine available. We have made outstanding progress in the identification of novel protective antigens that have been incorporated into the gold nanoparticle platform and used as vaccines. In this study, we have refined our vaccine formulations to incorporate multiple antigens and further define the mechanism of antibody-mediated protection, including one vaccine that promotes macrophage uptake. We further define the cell-mediated responses elicited at the mucosal surface by our nanovaccine formulations, another key immune mechanism linked to protection.

Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine β-hydroxylase knockout (Dbh(-/-)) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen. The epinephrine and norepinephrine neurotransmitters play important roles in gut physiology and motility. Of note, epinephrine and norepinephrine play a central role in stress responses in mammals, and stress has profound effects on GI function. Bacterial enteric pathogens exploit these neurotransmitters as signals to coordinate the regulation of their virulence genes. The bacterial QseC and QseE adrenergic sensors are at the center of this regulatory cascade. C. rodentium is a noninvasive murine pathogen with a colonization mechanism similar to that of EHEC, enabling the investigation of host signals in mice. The presence of these neurotransmitters in the gut is necessary for C. rodentium to fully activate its virulence program, in a QseC/QseE-dependent manner, to successfully colonize its murine host. Our study data provide the first example of epinephrine and norepinephrine signaling within the gut to stimulate infection by a bacterial pathogen in a natural animal infection.

ABSTRACT Many enteropathogenic bacteria express a needle-like type III secretion system (T3SS) that translocates effectors into host cells promoting infection. O antigen (OAg) constitutes the outer layer of Gram-negative bacteria protecting bacteria from host immune responses. Shigella constitutively shortens the OAg molecule in its three-dimensional conformation by glucosylation, leading to enhanced T3SS function. However, whether and how other enteropathogenic bacteria shorten the OAg molecule that probably facilitates infection remain unknown. For the first time, we report a smart mechanism by which enterohemorrhagic Escherichia coli specifically reduces the size of the OAg molecule at the infection site upon sensing mechanical signals of intestinal epithelial cell attachment via the membrane protein YgjI. YgjI represses expression of the OAg chain length regulator gene fepE via the global regulator H-NS, leading to shortened OAg chains and injection of more T3SS effectors into host cells. However, bacteria express long-chain OAg in the intestinal lumen benefiting their survival. Animal experiments show that blocking this regulatory pathway significantly attenuates bacterial virulence. This finding enhances our understanding of interactions between the surfaces of bacterial and host cells and the way this interaction enhances bacterial pathogenesis. IMPORTANCE Little is known about the regulation of cell wall structure of enteropathogenic bacteria within the host. Here, we report that enterohemorrhagic Escherichia coli regulates its cell wall structure during the infection process, which balances its survival in the intestinal lumen and infection of intestinal epithelial cells. In the intestinal lumen, bacteria express long-chain OAg, which is located in the outer part of the cell wall, leading to enhanced resistance to antimicrobial peptides. However, upon epithelial cell attachment, bacteria sense this mechanical signal via a membrane protein and reduce the OAg chain length, resulting in enhanced injection into epithelial cells of T3SS effectors that mediate host cell infection. Similar regulation mechanisms of cell wall structure in response to host cell attachment may be widespread in pathogenic bacteria and closely related with bacterial pathogenesis.

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli ("microvillus-minus," or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions.

A novel type II toxin of toxin–antitoxin systems (TAs), Gcn5-related N-acetyltransferase (GNAT) family, was reported recently. GNAT toxins are mainly present in pathogenic species, but studies of their involvement in pathogenicity are rare. This study discovered that the GANT toxin AtaT in enterohemorrhagic Escherichia coli (EHEC) can significantly enhance strain pathogenicity. First, we detected the virulence of ΔataT and ΔataR in cell and animal models. In the absence of ataT, strains showed a lower adhesion number, and host cells presented weaker attaching and effacing lesions, inflammatory response, and pathological injury. Next, we screened the acetylation substrate of AtaT to understand the underlying mechanism. Results showed that E. coli pore-forming protein EspB, which acts as a translocon in type III secretion system (T3SS) in strains, can be acetylated specifically by AtaT. The acetylation of K206 in EspB increases protein stability and maintains the efficiency of effectors translocating into host cells to cause close adhesion and tissue damage.

Bacterial resistance to known antibiotics comprises a serious threat to public health. Propagation of multidrug-resistant pathogenic strains is a reason for undertaking a search for new therapeutic strategies, based on newly developed chemical compounds and the agents present in nature. Moreover, antibiotic treatment of infections caused by enterotoxin toxin-bearing strain—enterohemorrhagic Escherichia coli (EHEC) is considered hazardous and controversial due to the possibility of induction of bacteriophage-encoded toxin production by the antibiotic-mediated stress. The important source of potentially beneficial compounds are secondary plant metabolites, isothiocyanates (ITC), and phytoncides from the Brassicaceae family. We reported previously that sulforaphane and phenethyl isothiocyanate, already known for their chemopreventive and anticancer features, exhibit significant antibacterial effects against various pathogenic bacteria. The mechanism of their action is based on the induction of the stringent response and accumulation of its alarmones, the guanosine penta- and tetraphosphate. In this process, the amino acid starvation path is employed via the RelA protein, however, the precise mechanism of amino acid limitation in the presence of ITCs is yet unknown. In this work, we asked whether ITCs could act synergistically with each other to increase the antibacterial effect. A set of aliphatic ITCs, such as iberin, iberverin, alyssin, erucin, sulforaphen, erysolin, and cheirolin was tested in combination with sulforaphane against E. coli. Our experiments show that all tested ITCs exhibit strong antimicrobial effect individually, and this effect involves the stringent response caused by induction of the amino acid starvation. Interestingly, excess of specific amino acids reversed the antimicrobial effects of ITCs, where the common amino acid for all tested compounds was glycine. The synergistic action observed for iberin, iberverin, and alyssin also led to accumulation of (p)ppGpp, and the minimal inhibitory concentration necessary for the antibacterial effect was four- to eightfold lower than for individual ITCs. Moreover, the unique mode of ITC action is responsible for inhibition of prophage induction and toxin production, in addition to growth inhibition of EHEC strains. Thus, the antimicrobial effect of plant secondary metabolites by the stringent response induction could be employed in potential therapeutic strategies.

The enterohemorrhagic Escherichia coli (EHEC) group is responsible for outbreaks and sporadic cases around the world annually. EHEC produces a potent protein known as Shiga toxin in the human intestine causing mild to bloody diarrhea. Some cases of EHEC infections may develop life-threatening symptoms, which may lead to human death. It also has other virulent factors that enable the EHEC cells to adhere to a target tissue and invade to some extent to crave more nutrition and escape the external extreme conditions, such as disinfection treatment. For those reasons, beef is not permitted for raw consumption unless guaranteed free of harmful bacteria, including EHEC, or the invading bacterial cells are completely removed or reduced to a safe level. A heat treatment that guarantees a sufficiently high temperature to reach inside the tissue of meat through the surface was established in Japan. This treatment may allow the core part of the meat to be consumed raw. However, it seemed to have some limitations. We aimed at developing a disinfection method with, hypothetically, nutrition-preserving property that is equivalent to the heat treatment or even superior. A combination of calcium hydroxide–ethanol–lactate-based food disinfectant and two proposed physical sterilization methods, assisted with microbial detection methods, exerted sufficient bactericidal activities against EHEC cells adhering to and/or invading the beef. These physical methods showed great usefulness in disinfecting fresh full-size boneless Round-beef of around 12 kg including fat on the outside. The first method applied a commercially available wide-drum washing machine (WM method) while the second method applied a specially designed plastic bag and a commercially available vibration machine (VV method). After trimming out the fat and the denatured surface of the beef (1 cm from the surface), the remaining meat mass showed no signs of denaturation and a significant reduction of viable EHEC cells by a factor of >104 CFU/ml. However, in the WM method, the disinfection process required a large amount of the disinfectant (150 L). The improved method, VV method, implemented a system that consumes a smaller amount of the disinfectant (50 L) while ensuring the targeted disinfection power degree.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT–transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.

Background and Aims: Probiotics are useful microorganisms for health of communities. Saccharomyces cerevisiae is one of the effective microorganisms for treating of functional and gastrointestinal diseases in order to control pathogens. Enterohemorrahgic Escherichia coli (EHEC) and Enterotoxogenic (ETEC) are common pathogenic strains in all the world. The aim of this study was to evaluate the inhibitory effect of S. cerevisiae probiotic yeast on the growth of ETEC and EHEC. Materials and Methods: For preparation of the supernatant extract, the yeast suspension was centrifuged, and then, the supernatant was filtered. Extraction with ethyl acetate was performed in three hours. For preparation of lysate, the precipitate was washed and centrifuged. The supernatant was removed and sterilize distilled water was added. Cell lysis was performed by sonication and the liquid was centrifuged and filtered. Then, the MIC and MBC were determined by micro dilution method. The concentration range was 16-8192 μg/ml. Results: The MIC and MBC of the supernatant against both ETEC and EHEC were 4096 μg/ml and 8192 μg/ml, respectively. Lysate in any of the concentrations showed no inhibitory effects on strains. Conclusions: The supernatant of S. cerevisiae has an inhibitory effect on growth of ETEC and EHEC. The lysate, probably due to the richness of the nutrients required for bacterial growth and not containing antibacterial compound, did not lead to such a repressive effect.

Salmonella and enterohemorrhagic Escherichia coli (EHEC) are of serious concern in wheat flour and its related products but little is known on their survival and thermal death kinetics. This study was undertaken to determine their long-term viability and thermal inactivation kinetics in flour. Inoculation was performed using mixtures of EHEC serogroups O45, O121, O145 and Salmonella followed by storage at room temperature (23°C) or 35°C (for Salmonella). Plate counting on tryptic soy agar (TSA) and enrichment were used to assess long-term survival. For thermal studies, wheat flour samples were heated at 55, 60, 65, and 70°C and cell counts of EHEC and Salmonella were determined by plating. The δ-values were calculated using the Weibull model. At room temperature, EHEC serovars and Salmonella were quantifiable for 84 and 112 days, and were detectable for the duration of the experiment after 168 and 365 days, respectively. The δ-values were 2.0, 5.54, and 9.3 days, for EHEC O121, O45, and O145, respectively, and 9.7 days for Salmonella. However, the only significant difference among all values was the δ-value for Salmonella and serogroup O121 (p ≤ 0.05). At 35°C, Salmonella counts declined to unquantifiable levels after a week and were not detected upon enrichment after 98 days. Heat treatment of inoculated wheat flour at 55, 60, 65, and 70°C resulted in δ-value ranges of 20.0–42.9, 4.9–10.0, 2.4–3.2, and 0.2–1.6 min, respectively, for EHEC. The δ-values for Salmonella at those temperatures were 152.2, 40.8, 17.9, and 17.4 min, respectively. The δ-values obtained for Salmonella at each temperature were significantly longer than for EHEC (p ≤ 0.05). Weibull model was a good fit to describe the thermal death kinetics of Salmonella and EHEC O45, O121 and O145 in wheat flour.HIGHLIGHTS-EHEC and Salmonella can survive for extended periods of time in wheat flour.-Long-term storage inactivation curves of EHEC and Salmonella were similar.-EHEC was more sensitive to heat than Salmonella.-Weibull model was a good fit to describe thermal death kinetics of EHEC and Salmonella.-Flour storage at 35°C may be a feasible method for microbial reduction.

Enterohemorrhagic Escherichia coli O157:H7 is a major human enteric pathogen capable of causing large outbreaks of severe infections that induce bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Its genome contains 177 unique O islands (OIs) including those carrying the main virulence elements, Shiga toxin-converting phages (OI-45 and OI-93) and locus for enterocyte effacement (OI-148). However, many of these islands harbor only genes of unknown function. Here, we demonstrate that OI-29 encodes a newly discovered transcriptional activator, Z0639 (named GmrA), that is required for motility and flagellar synthesis in O157:H7. GmrA directly binds to the promoter of fliA, an RNA polymerase sigma factor, and thereby regulates flagellar genes controlled by FliA. Expression of gmrA is maximal under host conditions (37°C, neutral pH, and physiological osmolarity), and in the presence of host epithelial cells, indicative of a role of this gene in infection by promoting motility. Finally, GmrA was found to be a widespread regulator of bacterial motility and flagellar synthesis in different pathotypes of E. coli. Our work largely enriches our understanding of bacterial motility control, and provides another example of regulators acquired laterally that mediate flagellar synthesis.

Innate immunity is an essential component in the protection of a host against pathogens. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are known to modulate the innate immune responses of infected cells. The interference is dependent on their type III secretion system (T3SS) and T3SS-dependent effector proteins. Furthermore, these cytosolically injected effectors have been demonstrated to engage multiple immune signaling pathways, including the IFN/STAT, MAPK, NF-κB, and inflammasome pathways. In this review, recent work describing the interaction between EPEC/EHEC and the inflammasome pathway will be discussed.