Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.

In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies.

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein-protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Sub-inhibitory stress with essential oil affects enterotoxins production and essential oil susceptibility in Staphylococcus aureus.

Fourteen wild strains of Staphylococcus aureus positive for gene sea were tested for enterotoxins production and the minimum inhibitory concentration of Leptospermum scoparium, Origanum majorana, Origanum vulgare, Satureja montana and Thymus vulgaris essential oils (EOs) were determined. After this trial, bacteria stressed with sub-inhibitory concentration of each EO were tested for enterotoxins production by an immunoenzymatic assay and resistance to the same EO. Oregano oil exhibited the highest antibacterial activity followed by manuka and thyme oils. After the exposure to a sub-inhibitory concentration of EOs, strains displayed an increased sensitivity in more than 95% of the cases. After treatment with oregano and marjoram EOs, few strains showed a modified enterotoxins production, while 43% of the strains were no longer able to produce enterotoxins after treatment with manuka EO. The results obtained in this study highlight that exposure to sub-inhibitory concentration of EO modifies strains enterotoxins production and EOs susceptibility profile.

Colon-Targeted Delivery of IgY Against Clostridium difficile Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads.

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl2 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).

A human gut ecosystem protects against C. difficile disease by targeting TcdA.

BACKGROUND: A defined Microbial Ecosystem Therapeutic (MET-1, or "RePOOPulate") derived from the feces of a healthy volunteer can cure recurrent C. difficile infection (rCDI) in humans. The mechanisms of action whereby healthy microbiota protect against rCDI remain unclear. Since C. difficile toxins are largely responsible for the disease pathology of CDI, we hypothesized that MET-1 exerts its protective effects by inhibiting the effects of these toxins on the host. METHODS: A combination of in vivo (antibiotic-associated mouse model of C. difficile colitis, mouse ileal loop model) and in vitro models (FITC-phalloidin staining, F actin Western blots and apoptosis assay in Caco2 cells, transepithelial electrical resistance measurements in T84 cells) were employed. RESULTS: MET-1 decreased both local and systemic inflammation in infection and decreased both the cytotoxicity and the amount of TcdA detected in stool, without an effect on C. difficile viability. MET-1 protected against TcdA-mediated damage in a murine ileal loop model. MET-1 protected the integrity of the cytoskeleton in cells treated with purified TcdA, as indicated by FITC-phalloidin staining, F:G actin assays and preservation of transepithelial electrical resistance. Finally, co-incubation of MET-1 with purified TcdA resulted in decreased detectable TcdA by Western blot analysis. CONCLUSIONS: MET-1 intestinal microbiota confers protection against C. difficile and decreases C. difficile-mediated inflammation through its protective effects against C. difficile toxins, including enhancement of host barrier function and degradation of TcdA. The effect of MET-1 on C. difficile viability seems to offer little, if any, contribution to its protective effects on the host.

Campomanesia adamantium Peel Extract in Antidiarrheal Activity: The Ability of Inhibition of Heat-Stable Enterotoxin by Polyphenols.

Campomanesia adamantium (Myrtaceae) is a medicinal plant distributed in Brazilian Cerrado. Different parts of this plant are used in popular medicine for treatment of several diseases like fever, diarrhea, hypercholesterolemia and rheumatism. The aim of this work was to evaluate the inhibition of heat-stable enterotoxin type A (STa) by gallic acid present in the peel of C. adamantium fruit and assays to assess the antidiarrheal activity, anti-inflammatory and cytotoxic properties of peel extract using the T84 cell line model. The possible inhibition exerted by the gallic acid of the peel extract on the STa peptide was inferred by molecular dynamics simulations. The antidiarrheal effects were investigated measuring cGMP accumulation in cells after stimulation by STa toxin and antibacterial activity was assessed. The anti-inflammatory activity was assessed by inhibition of COX-1 and COX-2. MTT and LDH assays were used to evaluate any possible cytotoxic action while the CyQUANT test was used to investigate the effect on cell proliferation. A representation showing how the possible interactions between STa and the gallic acid of the extract might reduce the action of the enterotoxin is presented. C. adamantium peel extract significantly decreased the levels of cGMP in T84 cells. However, no effect on the species of microorganisms was observed. The extract also inhibited COX-1 (IC50 255.70 ± 0.04 ng/mL) and COX-2 (IC50 569.50 ± 0.11 ng/mL) enzymes. Cytotoxicity assay have shown significant changes in cells treated with the extract, which inhibited the cell proliferation until 72 hours of treatment. Direct interactions of phenolic compounds present in the extract with the STa toxin may limit its activity. Curative effect in the diarrhea treatment and its anti-inflammatory action is based on the pharmacological properties, mechanism of action of the C. adamantium peel extract, and no toxic effects of the peel extract presented on this work.

Staphylococcal enterotoxin A gene-carrying Staphylococcus aureus isolated from foods and its control by crude alkaloid from papaya leaves.

Staphylococcus aureus is a known pathogen causing intoxication by producing enterotoxins in food. Staphylococcal enterotoxin A is one of the enterotoxins commonly implicated in staphylococcal food poisoning. The ability of crude alkaloid extract from papaya leaves to inhibit the growth of S. aureus and staphylococcal enterotoxin A synthesis was investigated. Staphylococcal enterotoxin A gene-carrying S. aureus was isolated from raw milk and ready-to-eat foods. Crude alkaloid was extracted from ground, dried papaya leaves using ultrasonic-assisted extraction, and a MIC of the alkaloid was determined by the broth macrodilution method. Furthermore, S. aureus isolate was exposed to the crude alkaloid extract at one- and twofold MIC, and the expression of sea was subsequently analyzed using a quantitative reverse transcription real-time PCR. Ten isolates of S. aureus were obtained, and nine of those isolates were sea carriers. The yield of crude alkaloid extract was 0.48 to 1.82% per dry weight of papaya leaves. A MIC of crude alkaloid to S. aureus was 0.25 mg/ml. After exposure to the alkaloid at 0.25 and 0.5 mg/ml for 2 h, a significant increase in cycle threshold values of sea was observed. The sea was expressed 29 and 41 times less when S. aureus was exposed to crude alkaloid at one- and twofold MIC, respectively. This study revealed that crude alkaloid of papaya leaves could control staphylococcal enterotoxin A gene-carrying S. aureus by suppressing the expression of sea, in addition to the ability to inhibit the growth of S. aureus. The expression of sea was successfully quantified.

A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions.

Screening of tea extract and theaflavins for inhibitory effects on the biological activity and production of staphylococcal enterotoxin A.

This study aimed to develop a novel method with tea extracts and its components, to reduce the risk of foodborne illnesses caused by the bacterial toxin staphylococcal enterotoxin A (SEA). The potential effect of tea extracts, theaflavins, and epitheaflagallin on staphylococcal growth was studied. A broth microdilution method was used to determine the minimum inhibitory concentration of these samples against an SEA-producing strain, Staphylococcus aureus C-29. The following assays were performed to evaluate various effects on concentrations of no effect on staphylococcal growth. The interactions of theaflavin-rich green tea extracts (TGE), theaflavins, and epitheaflagallin to cultured S. aureus C-29 were determined using Western blot analysis. As a result, all samples suppressed the binding affinity of the anti-SEA antibody to SEA. Since these samples could react directly with SEA, we examined whether they could bind to SEA. Our results demonstrated that binding of the anti-SEA antibody to 4 theaflavins-treated SEA was inhibited in a dose-dependent manner. On the other hand, the production of SEA was significantly decreased by treatment with TGE and epitheaflagallin. Based on the finding that TGE and epitheaflagallin inhibit the production of SEA, we further examined the relative expression levels of sea toxin-encoding genes after treatment with TGE and epitheaflagallin with real-time RT-PCR. TGE and epitheaflagallin significantly supressed the gene transcription of SEA in S. aureus C-29. We then tested whether the samples block the biological activity of SEA in murine spleen cells. TGE, theaflavins, and epitheaflagallin became inactivated the biological activity of SEA. These results suggest that edible and safe compounds in tea can be used to inactivate both pathogens and toxins.

Exogenous phosphatidylcholine supplementation improves intestinal barrier defense against Clostridium difficile toxin.

BACKGROUND: The incidence and severity of Clostridium difficile colitis have increased dramatically in the last decade. Disease severity is related to C. difficile virulence factors, including toxins A and B, as well as the patient's immune status. The intestinal mucus is an important component of innate barrier function in the intestine. Phosphatidylcholine (PC) is a key constituent of the intestinal mucus barrier, and exogenous PC administration has had therapeutic efficacy in patients with ulcerative colitis. We studied the protective function of exogenous PC on C. difficile toxin effects on the intestinal barrier in vitro. METHODS: Mucus-producing (HT29-MTX strain) and non-mucus-producing (HT29 strain) intestinal epithelial monolayers were cocultured with PC and C. difficile toxin A added to the apical media. Basal chamber culture supernatants were subsequently obtained, and tumor necrosis factor and interleukin 6 were quantitated by enzyme-linked immunosorbent assay. In other experiments, HT29 toxin A uptake, intestinal monolayer permeability, necrosis, and actin microfilament disruption were determined. RESULTS: There was a threefold to fourfold decrease in tumor necrosis factor and interleukin 6 levels and similar decreases in toxin A uptake and permeability changes in intestinal epithelial cells with mucus or PC versus control. Intestinal epithelial cell necrosis was reduced by more than 50% with either mucus or PC versus control. The integrity of HT29 cell cytoskeleton was demonstrated by both the mucus layer of the HT29-MTX strain and by exogenous PC administration by phalloidin staining of actin microfilaments. CONCLUSION: PC supplementation was effective in improving intestinal barrier defense against C. difficile toxin A challenge. PC administration may be a useful therapeutic adjunct in severe cases of C. difficile colitis or in patients who do not improve with conventional treatment.

Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) production and Lactobacillus species growth in a defined medium simulating vaginal secretions.

Lactobacillus species are commensal with the healthy vaginal environment and inhibit the growth of many pathogenic bacteria in the vaginal tract by a variety of mechanisms, such as the production of hydrogen peroxide, organic acids, and antimicrobial substances. Simulation of the vaginal environment is crucial for proper investigation of the effects of Lactobacillus species on pathogenic bacteria. In this study, we modified a medium used to simulate vaginal secretions to improve the growth of toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus clinical strains and Lactobacillus species so that interactions between these bacteria may be examined. A medium consisting of basal salts, vitamins, albumin, glycogen, mucin, urea, sodium bicarbonate, polyoxyethylene sorbitan monolaurate, and amino acids supported the growth of S. aureus and the production of TSST-1 as determined by Western analysis. Improved growth of the Lactobacillus species was seen when this same medium was supplemented with manganese chloride, sodium acetate, and an increase in glucose concentration. However, growth of S. aureus in the supplemented medium resulted in reduced levels of TSST-1. Production of TSST-1 was not detected in a medium routinely used for the growth of Lactobacillus species although S. aureus growth was not inhibited. The development of an improved genital tract secretion medium provides a more authentic environment in which to study the interactions of Lactobacillus species and vaginal pathogens, such as S. aureus.

The suppressive activities of six sources of medicinal ferns known as gusuibu on heat-labile enterotoxin-induced diarrhea.

Diarrheal disease is one of the most important worldwide health problems. Enterotoxigenic Escherichia coli (ETEC) is the most frequently isolated enteropathogen in diarrheal diseases. In developing countries, a very large number of people, especially children, suffer from diarrhea. To combat this problem, World Health Organization has constituted the Diarrhea Diseases Control Program which guides studies on traditional medicinal practices and preventive measures. Gusuibu, a traditional folk medicine, has been claimed to heal certain types of diarrhea. However, so far no scientific study has been carried out on the anti-diarrheal mechanism of Gusiubu. The present study was performed to examine the suppressive activities of ethanol extracts of six sources of folk medicinal ferns used as Gusuibu on heat-labile enterotoxin (LT)-induced diarrhea. Inhibitory effects of six sources were evaluated on the ETEC LT subunit B (LTB) and monosialotetrahexosylganglioside (GMI) interaction by GM1-enzyme linked immunosorbent assay and patent mouse gut assay. Our results indicated that Drynaria fortunei had no anti-diarrheal effect, while, among the remaining five folk medicinal ferns, four belonging to family Davalliaceae had significant abilities on both the blocking of LTB and GM1 interaction and the inhibition of LT-induced diarrhea. In conclusion, these findings suggested the potential application of Gusuibu as an anti-diarrheal remedy.

Selection of Escherichia coli heat-labile toxin (LT) inhibitors using both the GM1-ELISA and the cAMP Vero cell assay.

Weaned piglets are very susceptible to diarrhea caused by enterotoxigenic Escherichia coli. In the past, various natural components were proposed to have beneficial effects by reducing the effects of diarrheal infectious diseases in humans and animals, and thus may represent an alternative for the use of (prophylactic) antibiotics. Alternatives may inactivate enterotoxigenic Escherichia coli heat-labile toxin (LT) by interfering with toxin binding to the cellular receptor GM1. In this study, various plants and other natural substances were tested for inhibitory properties, in the GM1 binding assay, and in the LT-induced cAMP production in Vero cells. The toxic dose of each compound was determined in a cell viability assay, and the highest nontoxic concentrations were used in the GM1 and cAMP assays. Results demonstrated that only d-(+)-galactose, lactose, N-acetyl-d-galactosamine, and two tea extracts were able to inhibit the binding of LT to its GM1 receptor. In the cAMP assay, only the two tea extracts showed inhibitory activity. This shows that d-(+)-galactose, lactose, and N-acetyl-d-galactosamine can indeed inhibit LT binding to GM1 based on structural homology with GM1 in the absence of living cells. However, in the cAMP assay, d-(+)-galactose, and lactose, N-acetyl-d-galactosamine are apparently metabolized to below their effective inhibitory concentration, likely predicting limited practical applicability in vivo. Both tea extracts maintained their activity in the presence of cells. The active compounds in both are probably polyphenols, which are not easily metabolized, and most likely work by aggregating the toxin. In conclusion, the combination of methods used here is a convenient and fast method for preselecting natural substances containing potentially toxin-binding compounds. Furthermore, if antidiarrhea activity is attributed to compounds found inactive here, their activity is unlikely based on interference with toxin binding.

Modulation of Interleukin-8 and staphylococcal flora by Avène hydrotherapy in patients suffering from chronic inflammatory dermatoses.

BACKGROUND: A number of studies argue in favour of an important role of microbial colonization, in particular of Staphylococcus aureus, in triggering atopic dermatitis (AD) flare-up and psoriasis, in particular through the superantigenic properties of toxins generated by S. aureus. OBJECTIVES: The aim of this study was to assess the efficacy of a 3-week Avène hydrotherapy on the skin surface of patients suffering from psoriasis or atopic dermatitis. METHODS: Skin samples were taken from healthy subjects or atopic (n = 18) or psoriatic patients (n = 39) undergoing hydrotherapy at Avène at the beginning (D0) and the end of treatment (D18). The severity of the dermatosis was evaluated according to SCORing Atopic Dermatitis (SCORAD) or Psoriasis Area Severity Index (PASI) scores at D0 and D18. Marker of inflammation interleukin 8 (IL-8), S. aureus colonization (protein A) and enterotoxins were assessed in skin samples using RT-PCR. RESULTS: At D0, significant differences were observed between healthy subjects and atopic or psoriatic patients in all the parameters evaluated (IL-8, protein A). At the end of the hydrotherapy, a significant decrease in SCORAD was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin D in patients with atopic dermatitis. Similarly, a significant decrease in PASI was associated with a significant reduction of IL-8, S. aureus colonization and enterotoxin N in patients with psoriasis. CONCLUSIONS: This study demonstrates the positive effects of Avène hydrotherapy on the skin of patients suffering from chronic dermatosis, with decreased inflammation and reduced colonization by S. aureus.

Bacteriophage treatment significantly reduces viable Clostridium difficile and prevents toxin production in an in vitro model system.

Clostridium difficile is primarily a nosocomial pathogen, causing thousands of cases of antibiotic-associated diarrhoea in the UK each year. In this study, we used a batch fermentation model of a C. difficile colonised system to evaluate the potential of a prophylactic and a remedial bacteriophage treatment regime to control the pathogen. It is shown that the prophylaxis regime was effective at preventing the growth of C. difficile (p = <0.001) and precluded the production of detectable levels of toxins A and B. The remedial treatment regime caused a less profound and somewhat transient decrease in the number of viable C. difficile cells (p = <0.0001), but still resulted in a lower level of toxin production relative to the control. The numbers of commensal bacteria including total aerobes and anaerobes, Bifidobacterium sp., Bacteroides sp., Lactobacillus sp., total Clostridium sp., and Enterobacteriaceae were not significantly decreased by this therapy, whereas significant detrimental effects were observed with metronidazole treatment. Our study indicates that phage therapy has potential to be used for the control of C. difficile; it highlights the main benefits of this approach, and some future challenges.

Screening of phenolic antioxidants for their inhibitory activity against foodborne Staphylococcus aureus strains.

Seventeen phenolic compounds that are allowed to be used in the European food industry as aromatizants or antioxidants or that are naturally present in plants were tested for their ability to inhibit 19 strains of Staphylococcus aureus by using a standardized paper disc assay. Most of the strains assayed were foodborne (dairy and meat products). Human isolates and/or strains recommended for testing antimicrobial agents were also included in the study, and some of the test strains were enterotoxin producers. When the content was 200 microg/disc, various phenolic compounds had shown antimicrobial activity against all (hydroquinone, thymol, carvacrol, butylated hydroxyanisole, octyl gallate, and tannic acid) or most (gallic acid, propyl gallate, and ellagic acid) of the S. aureus strains tested. Significant differences in the inhibition zones (p < 0.05) among strains of the same, or similar, origin and among the different origins were observed for most of the phenolic compounds that showed antimicrobial activity for all or most of the strains tested.

Clostridium difficile associated infection, diarrhea and colitis.

A new, hypervirulent strain of Clostridium difficile, called NAP1/BI/027, has been implicated in C. difficile outbreaks associated with increased morbidity and mortality since the early 2000s. The epidemic strain is resistant to fluoroquinolones in vitro, which was infrequent prior to 2001. The name of this strain reflects its characteristics, demonstrated by different typing methods: pulsed-field gel electrophoresis (NAP1), restriction endonuclease analysis (BI) and polymerase chain reaction (027). In 2004 and 2005, the US Centers for Disease Control and Prevention (CDC) emphasized that the risk of C. difficile-associated diarrhea (CDAD) is increased, not only by the usual factors, including antibiotic exposure, but also gastrointestinal surgery/manipulation, prolonged length of stay in a healthcare setting, serious underlying illness, immune-compromising conditions, and aging. Patients on proton pump inhibitors (PPIs) have an elevated risk, as do peripartum women and heart transplant recipients. Before 2002, toxic megacolon in C. difficile-associated colitis (CDAC), was rare, but its incidence has increased dramatically. Up to two-thirds of hospitalized patients may be infected with C. difficile. Asymptomatic carriers admitted to healthcare facilities can transmit the organism to other susceptible patients, thereby becoming vectors. Fulminant colitis is reported more frequently during outbreaks of C. difficile infection in patients with inflammatory bowel disease (IBD). C. difficile infection with IBD carries a higher mortality than without underlying IBD. This article reviews the latest information on C. difficile infection, including presentation, vulnerable hosts and choice of antibiotics, alternative therapies, and probiotics and immunotherapy. We review contact precautions for patients with known or suspected C. difficile-associated disease. Healthcare institutions require accurate and rapid diagnosis for early detection of possible outbreaks, to initiate specific therapy and implement effective control measures. A comprehensive C. difficile infection control management rapid response team (RRT) is recommended for each health care facility. A communication network between RRTs is recommended, in coordination with each country's department of health. Our aim is to convey a comprehensive source of information and to guide healthcare professionals in the difficult decisions that they face when caring for these oftentimes very ill patients.