Exploring the antimicrobial effects of a phenolic-rich extract from jabuticaba depulping waste against enterotoxigenic Escherichia coli.

This study evaluated the effects of a phenolic-rich extract from jabuticaba [Myrciaria jaboticaba (Vell.) Berg] depulping waste (PEJ) on the survival, antibiotic susceptibility, virulence, and cellular functions of various enterotoxigenic Escherichia coli (ETEC) strains. The minimum inhibitory concentration of PEJ against the five tested ETEC strains was 125 mg mL-1. PEJ at 125 and 250 mg mL-1 caused reductions in viable cell counts of ≥ 3 and ≥ 5 log CFU mL-1 in ETEC over 24 h, respectively. PEJ at subinhibitory concentrations (31.25 and 62.5 mg mL-1) reduced the viable cell counts of ETEC when exposed to in vitro gastrointestinal conditions, besides decreasing the biofilm formation, cell surface hydrophobicity, mucin adhesion, and swimming and swarming motility. PEJ (31.25 and 62.5 mg mL-1) increased the susceptibility of the tested ETEC strains to various clinically relevant antibiotics. The exposure to PEJ (62.5 and 125 mg mL-1) impaired the membrane permeability and enzymatic and efflux pump activities in ETEC cells. PEJ effectively reduces survival, increases antibiotic susceptibility, and attenuates virulence in ETEC. These effects could be linked to a PEJ multi-target action disturbing various cellular functions in ETEC cells. PEJ could be a candidate for developing innovative solutions to prevent and treat ETEC infections.

Enterotoxigenic Escherichia coli targeting intestinal epithelial tight junctions: An effective way to alter the barrier integrity.

Enterotoxigenic Escherichia coli are responsible for causing secretory diarrhea in animal(s), including human(s). This group of microorganisms is classified on the basis of production of toxins acting on the intestinal epithelium of the small intestine. Various enterotoxins, heat-labile and heat-resistant, are produced by distinct strains of ETEC. Although the mechanisms of action of ETEC enterotoxins were shown to involve diverse ion channels recent data suggest that these molecules could also be involved in disruption of the permeability barrier of the intestinal epithelium. More precisely, the tight junctions directly responsible for the selective permeability of the intestinal tissue could be affected. Studies indicating a change in TJ following exposure of cell monolayers or animal models either to pure enterotoxins or to ETEC strains producing one or more of these toxic molecules will be discussed.

Escherichia coli heat-stable toxin b impairs intestinal epithelial barrier function by altering tight junction proteins.

Escherichia coli heat-stable toxin b (STb) causes diarrhea in animals. STb binds to sulfatide, its receptor, and is then internalized. In the cytoplasm, through a cascade of events, STb triggers the opening of ion channels, allowing ion secretion and water loss and leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells, and some bacterial toxins are known to affect adversely TJs. The present study aimed at determining the effect of STb on TJs. T84 cells were treated for 24 h with purified STb and a nontoxic STb mutant (D30V). Transepithelial resistance (TER), paracellular flux marker, and confocal microscopy were used to analyze the effect of STb on TJs. Purified STb caused a significant reduction of TER parallel to an increase in paracellular permeability compared to the results seen in untreated cells or mutant D30V. The increased paracellular permeability was associated with a marked alteration of F-actin stress fibers. F-actin filament dissolution and condensation were accompanied by redistribution and/or fragmentation of ZO-1, claudin-1, and occludin. These changes were also observed following treatment of T84 cells with an 8-amino-acid peptide found in the STb sequence corresponding to a consensus sequence of Vibrio cholerae Zot toxin. These effects were not observed with a scrambled peptide or mutant D30V. Our findings indicate that STb induces epithelial barrier dysfunction through changes in TJ proteins that could contribute to diarrhea.

The whole Shebang: the gastrointestinal tract, Escherichia coli enterotoxins and secretion.

This review focuses on diarrhea caused by toxins released by enterotoxigenic Escherichia coli. These bacteria are known to produce toxins that have adverse effects on the intestinal tissue in Man and animals. E. coli is contracted through the ingestion of water or food contaminated by this bacterium. Generally, E. coli colonizes the intestinal mucosa where it multiplies and causes damage to the target cells or interferes with the homeostasis that prevails in the gastrointestinal tract. Enteropathogens such as E. coli are only able to exhibit their effects after colonization of the intestinal mucosa from where they release their toxins. These bacteria mainly affect chloride ions secretion through second messenger pathways resulting in secretory diarrhea. In this review, the association of bacteria with the gastrointestinal tract as pathogens and the resulting effects on the various systems of the intestine, including the nervous system and mediators leading to secretion and diarrhea are examined.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines.

A previous study conducted in our laboratory demonstrated that cells having internalized Escherichia coli STb toxin display apoptotic-like morphology. We therefore investigated if STb could induce programmed cell death in both a human and an animal intestinal epithelial cell lines. HRT-18 (Human Colon Tumor) and IEC-18 (Rat Ileum Epithelial Cells) cell lines were used. As STb is frequently tested in a rat model, the IEC-18 cell line was most relevant to our work. The cell lines were treated with various amounts of purified STb (nanomole range) for a period of 24 h after which cells were harvested and examined for apoptotic characteristics. Caspase-9, the initiator of mitochondrion-mediated apoptosis, and caspase-3, an effector of caspase-9, were both activated following STb intoxication of HRT-18 and IEC-18 cells whereas caspase-8, the initiator caspase of the extrinsic pathway, was not activated. For both cell lines, agarose gel electrophoresis of the cell DNA content reveals laddering of DNA, resulting from DNA fragmentation, a characteristic of apoptosis. Hoechst 33342-stained DNA of STb-treated cell lines, observed using fluorescence microscopy, revealed condensation and fragmentation of the nuclei. Apoptotic indexes calculated from fragmented nuclei of Hoechst 33342-stained DNA for HRT-18 and IEC-18 cells showed an STb dose-dependent response. Overall, these data indicate that STb toxin induces a mitochondrion-mediated caspase-dependent apoptotic pathway.

Pig vaccination strategies based on enterotoxigenic Escherichia coli toxins.

Enterotoxigenic Escherichia coli (ETEC) are responsible for diarrhea in humans as well as in farm animals. ETEC infections in newborn, suckling, and especially in post-weaning piglets are associated with reduced growth rate, morbidity, and mortality. ETEC express virulence factors as adhesin and enterotoxins that play a central role in the pathogenic process. Adhesins associated with pigs are of diverse type being either fimbrial or non-fimbrial. Enterotoxins belong to two groups: heat-labile (LT) and heat-stable (ST). Heterogeneity of ETEC strains encompass expression of various fimbriae (F4, F5, F6, F18, and F41) and enterotoxins (LT, STa, STb, and EAST1). In the late years, attempts to immunize animals against neonatal and post-weaning diarrhea were focused on the development of anti-adhesin strategies as this is the initial step of ETEC pathogenesis. Although those vaccines demonstrated some protection against ETEC infections, as enterotoxins are pivotal to the virulence of ETEC, a new generation of vaccinal molecules, which include adhesin and one or more enterotoxins, were recently tested. Some of these newly developed chimeric fusion proteins are intended to control as well human diarrhea as enterotoxins are more or less common with the ones found in pigs. As these could not be tested in the natural host (human), either a mouse or pig model was substituted to evaluate the protection efficacy. For the advancement of pig vaccine, mice were sometimes used for preliminary testing. This review summarizes advances in the anti-enterotoxin immunization strategies considered in the last 10 years.

STb and AIDA-I: the missing link?

Escherichia coli enterotoxigenic strains produce one or more toxins which action result in production of diarrhea in animals including Man. One of these toxins, STb, has been mainly associated with colibacillosis in swine. Although highly prevalent in pigs with diarrhea, a relation between STb and disease was arduous to establish. With the recent recognition of a new adhesin, originally found in human E. coli isolates, named AIDA (adhesin involved in diffuse adherence) and its association with new E. coli pathotypes to which STb is linked, new light was shed on STb toxic potency. In this review, the association of STb and AIDA is examined according to the recent knowledge gained with newly described E. coli pathotypes.

Fruit extracts to control pathogenic Escherichia coli: A sweet solution.

Escherichia coli is a major cause of diarrhea and is as well responsible for extraintestinal infections in humans and animals. Many pathotypes have been defined for this ubiquitous microorganism on the basis of the virulence attributes. For the last 70 years, antibiotics have been used to control infections caused by E. coli. However, with the resistance observed with many strains these drugs are less recommended. Plant extracts, in particular fruit, represent a source of bioactive compounds that could be beneficial in the control of infectious diseases caused by E. coli. These could have bacteriostatic or bactericidal potential or could be used as synergic agents to amplify the activity of antibiotics for which the germs present some level of resistance. Certain studies also revealed that fruit extracts could act directly on virulence characters to attenuate the pathogenic capacity of microorganisms. This review intent to expose the scant but rapidly growing information available that shows that fruit, used as crude extracts or purified molecules, should be considered to manage diverse types of infections caused by E. coli.

EAST1 toxin: An enigmatic molecule associated with sporadic episodes of diarrhea in humans and animals.

EAST1 is produced by a subset of enteroaggregative Escherichia coli strains. This toxin is a 38-amino acid peptide of 4100 Da. It shares 50% homology with the enterotoxic domain of STa and interacts with the same receptor. The mechanism of action of EAST1is proposed to be identical to that of STa eliciting a cGMP increase. EAST1 is associated with diarrheal disease in Man and various animal species including cattle and swine. Nevertheless, as EAST1-positive strains as well as culture supernatants did not provoke unequivocally diarrhea either in animal models or in human volunteers, the role of this toxin in disease is today still debated. This review intent is to examine the role of EAST1 toxin in diarrheal illnesses.

Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process.

Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans. Once established in this harsh ecological niche, it remains there virtually for the entire life of the host. To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium. Nevertheless, a critical feature of H. pylori is its ability to establish a long-lasting infection. In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection. A large body of evidence has shown that the interaction between H. pylori and the host is very complex. In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H. pylori as a chronically persisting pathogen. Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant. This review examines the binding of host proteins (serum and extracellular matrix proteins) to H. pylori and considers the significance of these interactions in the infectious process. A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H. pylori infections and could represent an alternative to antibiotic treatment.

Modulation of hexa-acyl pyrophosphate lipid A population under Escherichia coli phosphate (Pho) regulon activation.

Environmental phosphate is an important signal for microorganism gene regulation, and it has recently been shown to trigger some key bacterial virulence mechanisms. In many bacteria, the Pho regulon is the major circuit involved in adaptation to phosphate limitation. The Pho regulon is controlled jointly by the two-component regulatory system PhoR/PhoB and by the phosphate-specific transport (Pst) system, which both belong to the Pho regulon. We showed that a pst mutation results in virulence attenuation in extraintestinal pathogenic Escherichia coli (ExPEC) strains. Our results indicate that the bacterial cell surface of the pst mutants is altered. In this study, we show that pst mutants of ExPEC strains display an increased sensitivity to different cationic antimicrobial peptides and vancomycin. Remarkably, the hexa-acylated 1-pyrophosphate form of lipid A is significantly less abundant in pst mutants. Among differentially expressed genes in the pst mutant, lpxT coding for an enzyme that transfers a phosphoryl group to lipid A, forming the 1-diphosphate species, was found to be downregulated. Our results strongly suggest that the Pho regulon is involved in lipid A modifications, which could contribute to bacterial surface perturbations. Since the Pho regulon and the Pst system are conserved in many bacteria, such a lipid A modification mechanism could be widely distributed among gram-negative bacterial species.

Escherichia coli STb toxin and colibacillosis: knowing is half the battle.

Expression of both adherence and enterotoxin expression are required for enterotoxigenic Escherichia coli (ETEC) strains to cause colibacillosis. ETEC strains are responsible for diarrhea in humans and animals by production of various enterotoxins. For many years, the role of the heat-stable E. coli enterotoxin STb as a diarrhea-causing toxin in animals, and in particular in swine, has been controversial. In fact, although the presence of STb-positive E. coli strains and diarrhea in animals is frequently observed, the difficulty of reproducing the pathology in an animal model was interpreted as a lack of toxicity. Recently, new light was shed on the activity of STb in intestinal ligated loops and in pigs orally inoculated with STb-positive E. coli strains. This minireview revisits the effects of STb on the intestinal epithelium and enlightens the significance of STb in swine colibacillosis. The interaction of STb toxin with other E. coli enterotoxins and dual ETEC/enteropathogenic E. coli or ETEC/attaching effacing E. coli infections are also discussed.

Escherichia coli STb toxin binding to sulfatide and its inhibition by carragenan.

Escherichia coli heat-STb is an important cause of diarrhea in piglets. STb was shown to interact specifically with sulfatide (3'-sulfogalactosyl-ceramide) present on the surface of epithelial cells of piglet jejunum. Basic data are lacking on STb binding to sulfatide in solution and more precisely on the possible inhibition of this interaction. Using surface plasmon resonance technology, we compare binding of STb to sulfatide and other glycoshingolipids previously shown, with a multiplate-binding assay, to also interact to various degrees with the enterotoxin. In addition, inhibition of STb-sulfatide binding was studied using free galactose, galactose-sulfate residues and a polymer of sulfated galactans known as carragenan. We determined a dissociation constant of 2.4+/-0.61 nM for the STb-sulfatide interaction. These data indicated that STb was binding to sulfatide with greater affinity than previously determined using radiolabeled toxin. Much lower affinities were observed for lactoceramide and glucoceramide. The binding of STb to sulfatide was clearly inhibited by lambda-carragenan but not by galactose, 4-SO(4)-galactose or 6-SO(4)-galactose. Inhibition of STb binding to its receptor was achieved using lambda-carragenan at picomolar concentrations. Then, using IPEC-J2 cells in culture and flow cytometry, we showed that lambda-carragenan was able to inhibit the permeabilization process associated with STb.

Heterogeneity of Escherichia coli STb enterotoxin isolated from diseased pigs.

To investigate the presence and frequency of estB variant(s), a collection of 100 STb-positive enterotoxigenic Escherichia coli (ETEC) strains isolated from 1980 to 2007 inclusively and randomly selected from diseased pigs in Québec, Canada, was analysed. A wide diversity of virulence gene profiles (virotypes) was detected in the strain collection. The estB gene was amplified by PCR using primers designed from the signal sequence and the C-terminal end, and the amplified fragment was sequenced using the forward primer. The translated DNA sequence revealed a His(12)-->Asn change in 23 of the 100 ETEC isolates tested. The STb-variant strains were observed throughout the sampling period covered in the study. No other STb-variant type was found in this study. All 23 variant strains were also positive for the STa enterotoxin and were resistant to tetracycline, as for strain 2173. The STb variant was associated with Stx2-positive strains (5/6) and STa : STb strains that did not harbour any of the tested porcine fimbrial adhesins (13/17). The remaining variant strains were associated with fimbriae F4 (1/40), F5 (1/6), F6 (1/1) and F18 (2/7; excluding F18 : Stx2 strains).

Animal Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors: adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17, and F18 fimbriae. Once established in the animal small intestine, ETEC produce enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes: heat-labile toxins that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This review describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics, and the identification of potential new targets by genomics are presented in the context of animal ETEC.

LC-MS analysis of pig intestine sulfatides: interaction with Escherichia coli STb enterotoxin and characterization of molecular species present.

STb, a 48-amino acid thermostable enterotoxin is produced by enterotoxigenic Escherichia coli strains and is responsible for diarrheal diseases in many animals, including man. Our laboratory recently identified a family of molecules, from a lipid extract of porcine intestinal epithelial cells, that could bind to STb. These molecules were identified as sulfatides as they reacted with a monoclonal antibody raised against this family of molecules. However, as the epitope recognized by this monoclonal antibody was the galactose 3-sulfate, a doubt could remain as to the exact nature of the identified receptors. The goal of this study was thus to confirm the chemical nature of the STb-binding molecule as sulfatides or as distinctive molecules comprising a sulfated galactosyl residue. Using a thin-layer chromatography-overlay method we confirmed using antibodies to STb that STb recognizes the commercial sulfatides and a band migrating at the same level from the intestinal tissue lipid extract obtained from an 8-week-old piglet. The compounds recovered from the silica gel plates were analyzed by mass spectrometry in electrospray negative-ionization mode. The most abundant ions observed had m/z values of 779, 795, 879 and 907. For commercial bovine brain sulfatides the ions 795, 879 and 907 have been attributed to hydroxylated sulfatides with a saturated fatty acid chain containing 16, 22 and 24 carbons, while the 779 ion contained a saturated fatty acid chain of 16 carbons. The general profile of the ions observed was similar to the already described commercial bovine brain sulfatides.

Effect of heparin binding on Helicobacter pylori resistance to serum.

The objective of this study was to evaluate the effect of heparin binding to Helicobacter pylori cells on their survival in the presence of fresh rabbit serum with or without active complement components. Three H. pylori strains were compared and the amounts of heparin added reflected the physiological concentrations that can be found in animal tissues. No growth of H. pylori was noted in the presence of serum. Serum with or without active complement produced a reduction in c.f.u. for strains SPM 326, CCUG 17874(T) and SS1. However, addition of heparin resulted in increased survival of bacterial cells in serum with or without active complement. It appears that heparin binding to H. pylori can prevent bacterial cell death due to the alternative complement system. Heparin binding could also protect from heated serum (complement-inactivated), indicating protection from other serum components besides complement. In vivo, the process of heparin binding could possibly result in facilitated colonization due to a higher survival rate.

Trypan blue uptake by chinese hamster ovary cultured epithelial cells: a cellular model to study Escherichia coli STb enterotoxin.

The thermostable enterotoxin b (STb) produced by enterotoxigenic Escherichia coli strains is responsible for diarrheal diseases mainly in weaning piglets. For now, the only available assay for biological activity of STb toxin was in the animal host (i.e. piglet) or in an animal model (i.e. rat, mouse). In this study, we developed a cellular model for the study of the biological activity of STb enterotoxin. Using a trypan blue vital stain method, we showed that STb-treated cells of three out of the five cell lines tested absorbed more vital stain than their controls. Of all the cell lines tested, the chinese hamster's ovary derivated cells (CHO) were the most sensitive, absorbing 50% more trypan blue than their control. Maximal stain uptake was observed after 2h. We then evaluated the trypan blue uptake for 16 STb mutants, produced in a previous work, on the CHO cell lines in order to compare it with the in vivo rat loop assay data. Interestingly, we observed a good correlation between the two bioassays. In fact, the biological activity observed in the rat could be correlated with the trypan blue uptake by the CHO cells (R(2)=0.78) for STb toxin and the 16 mutants. Using the variance analysis statistical test, we determined that the correlation between the two bioassays is significant (F(c)> or =F(0.005)). These results suggest that the trypan blue uptake bioassay could represent a new method to evaluate the biological activity and facilitate the elucidation of the mechanism of action of E. coli STb enterotoxin.