Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Magnesium Sensing Regulates Intestinal Colonization of Enterohemorrhagic Escherichia coli O157:H7.

The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections.IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

Effect of Lactobacillus acidophilus Fermented Broths Enriched with Eruca sativa Seed Extracts on Intestinal Barrier and Inflammation in a Co-Culture System of an Enterohemorrhagic Escherichia coli and Human Intestinal Cells.

Lactic acid bacteria (LAB) "fermentates" confer a beneficial effect on intestinal function. However, the ability of new fermentations to improve LAB broth activity in preventing pathogen-induced intestinal inflammation and barrier dysfunction has not yet been studied. The objective of this study was to determine if broths of LAB fermented with Eruca sativa or Barbarea verna seed extracts prevent gut barrier dysfunction and interleukin-8 (CXCL8) release in vitro in human intestinal Caco-2 cells infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7. LAB broths were assayed for their effects on EHEC growth and on Caco-2 viability; thereafter, their biological properties were analysed in a co-culture system consisting of EHEC and Caco-2 cells. Caco-2 cells infected with EHEC significantly increased CXCL8 release, and decreased Trans-Epithelial Electrical Resistance (TEER), a barrier-integrity marker. Notably, when Caco-2 cells were treated with LAB broth enriched with E. sativa seed extract and thereafter infected, both CXCL8 expression and epithelial dysfunction reduced compared to in untreated cells. These results underline the beneficial effect of broths from LAB fermented with E. sativa seed extracts in gut barrier and inflammation after EHEC infection and reveal that these LAB broths can be used as functional bioactive compounds to regulate intestinal function.

Antibiofilm activities of norharmane and its derivatives against Escherichia coli O157:H7 and other bacteria.

BACKGROUND: Bacterial biofilms exhibit reduced sensitivity to conventional antibiotics and host defence systems and contribute to the persistence of chronic bacterial infections. HYPOTHESIS: The antibiofilm approach using plant alkaloids provides an alternative to antibiotic strategies. STUDY DESIGN: In this study, the antibiofilm activities of various plant alkaloids were investigated against enterohemorrhagic Escherichia coli O157:H7 and Pseudomonas aeruginosa. In the subsequent investigation, the effects of five norharmane derivatives were investigated. RESULT: Harmaline significantly inhibited biofilm formation by E. coli O157:H7, P. aeruginosa PAO1, P. aeruginosa PA14, and Klebsiella oxytoca, and norharmane (ß-carboline) was found to have antibiofilm activity. It was also found that functional groups at the C-1 and C-7 positions of norharmane could play important roles in its antibiofilm activity. Confocal and electron microscopic observations confirmed biofilm inhibition by harmaline and norharmane, and both reduced fimbriae production and swarming and swimming motilities. Furthermore, harmaline and norharmane attenuated the virulence of E. coli O157:H7 in a Caenorhabditis elegans nematode model. CONCLUSION: These findings strongly suggest that harmaline and norharmane could have potential use in antibiofilm strategy against persistent bacterial infections.

Highly sensitive Escherichia coli shear horizontal surface acoustic wave biosensor with silicon dioxide nanostructures.

Surface acoustic wave mediated transductions have been widely used in the sensors and actuators applications. In this study, a shear horizontal surface acoustic wave (SHSAW) was used for the detection of food pathogenic Escherichia coli O157:H7 (E.coli O157:H7), a dangerous strain among 225 E. coli unique serotypes. A few cells of this bacterium are able to cause young children to be most vulnerable to serious complications. Presence of higher than 1cfu E.coli O157:H7 in 25g of food has been considered as a dangerous level. The SHSAW biosensor was fabricated on 64° YX LiNbO3 substrate. Its sensitivity was enhanced by depositing 130.5nm thin layer of SiO2 nanostructures with particle size lesser than 70nm. The nanostructures act both as a waveguide as well as a physical surface modification of the sensor prior to biomolecular immobilization. A specific DNA sequence from E. coli O157:H7 having 22 mers as an amine-terminated probe ssDNA was immobilized on the thin film sensing area through chemical functionalization [(CHO-(CH2)3-CHO) and APTES; NH2-(CH2)3-Si(OC2H5)3]. The high-performance of sensor was shown with the specific oligonucleotide target and attained the sensitivity of 0.6439nM/0.1kHz and detection limit was down to 1.8femto-molar (1.8×10-15M). Further evidence was provided by specificity analysis using single mismatched and complementary oligonucleotide sequences.

Essential Oils and Eugenols Inhibit Biofilm Formation and the Virulence of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) has caused foodborne outbreaks worldwide and the bacterium forms antimicrobial-tolerant biofilms. We investigated the abilities of various plant essential oils and their components to inhibit biofilm formation by EHEC. Bay, clove, pimento berry oils and their major common constituent eugenol at 0.005% (v/v) were found to markedly inhibit EHEC biofilm formation without affecting planktonic cell growth. In addition, three other eugenol derivatives isoeugenol, 2-methoxy-4-propylphenol, and 4-ethylguaiacol had antibiofilm activity, indicating that the C-1 hydroxyl unit, the C-2 methoxy unit, and C-4 alkyl or alkane chain on the benzene ring of eugenol play important roles in antibiofilm activity. Interestingly, these essential oils and eugenol did not inhibit biofilm formation by three laboratory E. coli K-12 strains that reduced curli fimbriae production. Transcriptional analysis showed that eugenol down-regulated 17 of 28 genes analysed, including curli genes (csgABDFG), type I fimbriae genes (fimCDH) and ler-controlled toxin genes (espD, escJ, escR, and tir), which are required for biofilm formation and the attachment and effacement phenotype. In addition, biocompatible poly(lactic-co-glycolic acid) coatings containing clove oil or eugenol exhibited efficient biofilm inhibition on solid surfaces. In a Caenorhabditis elegans nematode model, clove oil and eugenol attenuated the virulence of EHEC.

Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7.

This study evaluated the inhibitory effect of cinnamon oil against Escherichia coli O157:H7 Shiga toxin (Stx) production and further explored the underlying mechanisms. The MIC and minimum bactericidal concentration (MBC) of cinnamon oil against E. coli O157:H7 were 0.025% and 0.05% (vol/vol), respectively. Cinnamon oil significantly reduced Stx2 production and the stx2 mRNA expression that is associated with diminished Vero cell cytotoxicity. Consistently, induction of the Stx-converting phage where the stx2 gene is located, along with the total number of phages, decreased proportionally to cinnamon oil concentration. In line with decreased Stx2 phage induction, cinnamon oil at 0.75× and 1.0× MIC eliminated RecA, a key mediator of SOS response, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I), which positively regulate Stx-converting phages, contributing to reduced Stx-converting phage induction and Stx production. Furthermore, cinnamon oil at 0.75× and 1.0× MIC strongly inhibited the qseBC and luxS expression associated with decreased AI-2 production, a universal quorum sensing signaling molecule. However, the expression of oxidative stress response genes oxyR, soxR, and rpoS was increased in response to cinnamon oil at 0.25× or 0.5× MIC, which may contribute to stunted bacterial growth and reduced Stx2 phage induction and Stx2 production due to the inhibitory effect of OxyR on prophage activation. Collectively, cinnamon oil inhibits Stx2 production and Stx2 phage induction in E. coli O157:H7 in multiple ways. IMPORTANCE: This study reports the inhibitory effect of cinnamon oil on Shiga toxin 2 phage induction and Shiga toxin 2 production. Subinhibitory concentrations (concentrations below the MIC) of cinnamon oil reduced Stx2 production, stx2 mRNA expression, and cytotoxicity on Vero cells. Subinhibitory concentrations of cinnamon oil also dramatically reduced both the Stx2 phage and total phage induction in E. coli O157:H7, which may be due to the suppression of RNA polyadenylation enzyme PNPase at 0.25× to 1.0× MIC and the downregulation of bacterial SOS response key regulator RecA and RNA polyadenylation enzyme PAP I at 0.75× or 1.0× MIC. Cinnamon oil at higher levels (0.75× and 1.0× MIC) eliminated quorum sensing and oxidative stress. Therefore, cinnamon oil has potential applications as a therapeutic to control E. coli O157:H7 infection through inhibition of bacterial growth and virulence factors.

Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression.

Inhibition of verotoxin (VT) 2 absorption into systemic blood from intestine by repeated administration of bovine immune colostral antibody against VT2 in mice.

BACKGROUND/PURPOSE: Whether absorption of verotoxin (VT) 2 from the intestine in mice is inhibited by administration bovine immune colostral antibody against VT2 was investigated. METHODS: Three-week-old mice were administered VT2 solution at 477.8 ng/mL or 955.6 ng/mL, and bovine immune colostral antibody against VT2 was then administered three times. Whey without antibody against VT2 was administered to control mice. Serum levels of VT2 were measured by fluorescence enzyme immunoassay. RESULTS: Serum levels of VT2 in mice administered VT2 solution at 477.8 ng/mL and bovine immune colostral antibody against VT2 scarcely changed. By contrast, serum levels of VT2 in control mice increased and peaked 12 hours after administration. Peak values were 15.4 ± 5.04 ng/mL. Furthermore, serum levels of VT2 at 12 hours and 16 hours in control mice were significantly higher than in mice administered bovine colostral antibody against VT2. Serum levels of VT2 in mice administered antibody at 955.6 ng/mL showed no significant differences between repeated administration of bovine immune colostral antibody and controls. CONCLUSION: These results suggest that absorption of VT2 from the intestine was inhibited by repeated administration of bovine immune colostral antibody against VT2 at early stages of Escherichia coli O157:H7 infection, whereas VT2 in the intestine remained at low levels.

Coumarins reduce biofilm formation and the virulence of Escherichia coli O157:H7.

E. coli O157:H7 is the most common cause of hemorrhagic colitis, and no effective therapy exists for E. coli O157:H7 infection. Biofilm formation is closely related to E. coli O157:H7 infection and constitutes a mechanism of antimicrobial resistance. Hence, the antibiofilm or antivirulence approach provides an alternative to antibiotic strategies. Coumarin and its derivatives have a broad range of biological effects, and in this study, the antibiofilm activities of nine coumarins were investigated against E. coli O157:H7. Coumarin or umbelliferone at 50µg/ml was found to inhibit biofilm E. coli O157:H7 formation by more than 80% without affecting bacterial growth. Transcriptional analysis showed that coumarins repressed curli genes and motility genes in E. coli O157:H7, and these findings were in-line with observed reductions in fimbriae production, swarming motility, and biofilm formation. In addition, esculetin repressed Shiga-like toxin gene stx2 in E. coli O157:H7 and attenuated its virulence in vivo in the nematode Caenorhabditis elegans. These findings show that coumarins have potential use in antivirulence strategies against persistent E. coli O157:H7 infection.

Proposal for effective treatment of Shiga toxin-producing Escherichia coli infection in mice.

Previously, we reported that minocycline, kanamycin and norfloxacin improved the survival rate in the E32511 model that we developed (FEMS Immunol Med Microbiol 26, 101-108, 1999), but fosfomycin did not. In this study, we investigated the effectiveness of azithromycin (AZM) against Stx2d-producing EHEC O91:H21 strain B2F1 or Stx2c-producing Escherichia coli strain E32511 treated with mitomycin C in vivo. Recently, we reported the effectiveness of AZM in our model and AZM strongly inhibited the release of Stx2c from E32511 in vitro (PLOS ONE e58959, 2013). However, it was very difficult to completely eliminate E32511 in the mouse feces by treatment with AZM alone. In this report, only AZM or Daio effectively promoted survival of mice infected with B2F1 compared to untreated mice. Furthermore, Daio inhibited the colonization of GFP-expressing B2F1 in the mouse intestine. Similarly, a combination of AZM and Daio in the E32511-infected mice reduced E32511 in the mouse feces and significantly improved survival.

Bifidobacterium spp. influences the production of autoinducer-2 and biofilm formation by Escherichia coli O157:H7.

The effect of Bifidobacterium spp. on the production of quorum-sensing (QS) signals and biofilm formation by enterohemorrhagic Escherichia coli (EHEC) O157:H7 was investigated. In an AI-2 bioassay, cell extracts of Bifidobacterium longum ATCC 15707 resulted in a 98-fold reduction in AI-2 activity in EHEC O157:H7 as well as in the Vibrio harveyi reporter strain, even though they did not inhibit the growth of EHEC O157:H7. In addition, they resulted in a 36% reduction in biofilm formation by the organism. Consistently, the virulence of EHEC O157:H7 was significantly attenuated by the presence of cell extracts of B. longum ATCC 15707 in the Caenorhabditis elegans nematode in vivo model. By a proteome analysis using two dimensional electrophoresis (2-DE), we determined that seven proteins including formation of iron-sulfur protein (NifU), thiol:disulfide interchange protein (DsbA), and flagellar P-ring protein (FlgI) were differentially regulated in the EHEC O157:H7 when supplemented with cell extracts of B. longum ATCC 15707. Taken together, these findings propose a novel function of a dairy adjunct in repressing the virulence of EHEC O157:H7.

Inhibitory effects of broccoli extract on Escherichia coli O157:H7 quorum sensing and in vivo virulence.

Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key process for the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.

Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages and essential oils can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of this study was to determine the effect of a bacteriophage cocktail, BEC8, alone and in combination with the essential oil trans-cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on whole baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were Nal(R) mutants of EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on leaves and dried. EHEC cells were placed at low, medium, and high inoculum levels (10(4), 10(5), and 10(6) CFU/mL, respectively). Appropriate controls, BEC8 (approx. 10(6) PFU/leaf), and TC (0.5% v/v) were applied on treated leaves. The leaves were incubated at 4, 8, 23, and 37 °C in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid (50 µg/mL) Sorbitol MacConkey agar. No survivors were detected when both leaves were treated with BEC8 or TC individually at low inoculum levels after 24 h at 23 and 37 °C. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain.

Increased advanced oxidation of protein products and enhanced total antioxidant capacity in plasma by action of toxins of Escherichia coli STEC.

Shiga toxin (Stx) and hemolysin (Hly) of Escherichia coli O157:H7 produced an increase of reactive oxygen species (ROS) in normal human blood. In vitro assays showed that stimuli of ROS with these toxins oxidized proteins to carbonyls in plasma and raised the degradation of oxidized macromolecules, with the AOPP/carbonyl relationship also increasing. The oxidative stress generated by toxins during the Hemolytic Uremic Syndrome (HUS) produced oxidation of blood proteins with a rise in advanced oxidation protein products (AOPP) in children with HUS. There was a response from the antioxidant system in these patients, evaluated through the determination of the total antioxidant capacity of plasma by the Ferric Reducing Antioxidant Power (FRAP), which reduced the stimuli of ROS during in vitro incubation with Stx or Hly. The application of natural antioxidants was sufficient to reduce in vitro the oxidative stress provoked by both toxins in blood.

Bovine colostral antibody against verotoxin 2 derived from Escherichia coli O157:H7: resistance to proteases and effects in beagle dogs.

A bovine colostral antibody against verotoxin (VT) 2 of Escherichia coli O157:H7 was administered orally to beagle dogs. The antibody remained in the dogs' small intestine for at least 2 h, whereas little serum antibody remained 1.5 h after administration. Furthermore, the antibody activity of secretory IgA did not change until 2 h after administration; however, the activity of IgG and IgM antibodies decreased by approximately 60% and 40% at 2 h after administration, respectively. Seven beagle dogs inoculated with Escherichia coli O157:H7 producing VT2 were administered bovine colostral antibody or bovine colostral whey without antibody. With administration of bovine colostral whey without antibody, the amount of VT2 in feces decreased gradually after administration and increased again at 5 d after inoculation, whereas bovine colostral antibody significantly reduced the amount of VT2 in feces on the day after administration. In addition, 9 beagle dogs were given bovine colostral antibody, bovine plasma antibody, or saline. The amount of VT2 in feces again decreased significantly more rapidly after administration of bovine colostral antibody than after administration of bovine plasma antibody or saline.

Antipathogenic properties of green tea polyphenol epigallocatechin gallate at concentrations below the MIC against enterohemorrhagic Escherichia coli O157:H7.

The inhibitory effects of green tea polyphenol epigallocatechin gallate (EGCG) on virulence phenotypes and gene expression regulated by quorum sensing (QS) in Escherichia coli O157:H7 were demonstrated at concentrations of 1 to 100 microg/ml, which are lower than the MIC (539 +/- 22 microg/ml). At 25 microg/ml, the growth rate was not affected, but autoinducer 2 concentration, biofilm formation, and swarm motility decreased to 13.2, 11.8, and 50%, respectively. Survival at 5 days of nematodes (Caenorhabditis elegans) that were fed the pathogen without and with EGCG were 47.1 and 76%, respectively. Real-time PCR data indicated decreased transcriptional level in many quorum sensing-regulated virulence genes at 25 microg/ml. Our results suggest that EGCG at concentrations below itsMIC has significant antipathogenic effects against E. coli O157:H7.

Novel cell-based method to detect Shiga toxin 2 from Escherichia coli O157:H7 and inhibitors of toxin activity.

Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.

Characterisation of Escherichia coli O157 strains from humans, cattle and pigs in the North-West Province, South Africa.

Escherichia coli O157 strains cause diseases in humans that result from the consumption of food and water contaminated with faeces of infected animals and/or individuals. The objectives of this study were to isolate and characterise E. coli O157 strains from humans, cattle and pigs and to determine their antibiotic resistant profiles as well as detection of virulence genes by PCR. Eight hundred faecal samples were analysed for typical E. coli O157 and 76 isolates were positively identified as E. coli O157 strains. 16S rRNA sequence data were used to confirm the identity of the isolates. Susceptibility profiles to 9 antibiotics were determined and the multiple antibiotic resistant (MAR) patterns were compiled. A large proportion (52.6%-92.1%) of the isolates from pigs, cattle and humans were resistant to tetracycline, sulphamethoxazole and erythromycin. Thus the phenotype Smx-T-E (sulphamethozaxole-tetracycline-erythromycin) was present in most of the predominant MAR phenotypes obtained. Cluster analysis of antibiotic resistances revealed a closer relationship between isolates from pig and human faeces than cattle and humans. PCR were performed to amplify STEC virulence and tetracycline resistance gene fragments. A tetB gene fragment was amplified among the isolates. Eighteen (60%) of the isolates possessed the hlyA gene and 7(23.3%) the eae gene while only 5(16.7%) possessed both genes. Although shiga toxin genes were detected in the E. coli O157:H7 positive control strain none of the isolates that were screened possessed these genes. In a related study we reported that the prevalence of E. coli O157 was higher in pigs than cattle and humans. A high market demand for pork and beef in South Africa amplifies the risk that diseased animals pose to human health. This highlighted the need for proper hygiene management to reduce the prevalence of E. coli O157 in farm animals and prevent the spread from animals to humans.