The American cockroach peptide periplanetasin-4 inhibits Clostridium difficile toxin A-induced cell toxicities and inflammatory responses in the mouse gut.

Many reports have shown that crude extracts of the American cockroach have therapeutic effects on inflammation. In a previous study, our research group showed that an antimicrobial peptide (Periplanetasin-2) derived from the American cockroach via de novo transcriptome analysis inhibited apoptosis of human colonocytes and inflammatory responses of the mouse gut caused by Clostridium difficile toxin A. Here, we examined whether Periplanetasin-4 (Peri-4), another antimicrobial peptide identified via de novo transcriptome analysis of the American cockroach, could also inhibit the various toxicities induced by C. difficile toxin A. We found that Peri-4 significantly reduced the cell viability loss and cell apoptosis caused by toxin A in vitro. Peri-4 also ameliorated the severe inflammatory responses seen in the toxin A-induced mouse enteritis model, rescuing the villus disruption and interleukin-6 production induced by luminal injection of toxin A into the mouse gut. Mechanistically, we found that Peri-4 could reduce toxin A-induced reactive oxygen species production to inhibit the activations of p38MAPK and p21Cip1/Waf1 , which are critical for the cell damages induced by toxin A. These results collectively suggest that the Peri-4 may be a potential therapeutic agent for treating toxin A-induced pseudomembranous colitis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

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.

A Tetraspecific VHH-Based Neutralizing Antibody Modifies Disease Outcome in Three Animal Models of Clostridium difficile Infection.

Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd.

Neutralization of staphylococcal enterotoxin B by an aptamer antagonist.

Staphylococcal enterotoxin B (SEB) is a major virulence factor for staphylococcal toxic shock syndrome (TSS). SEB activates a large subset of the T lymphocytic population, releasing proinflammatory cytokines. Blocking SEB-initiated toxicity may be an effective strategy for treating TSS. Using a process known as systematic evolution of ligands by exponential enrichment (SELEX), we identified an aptamer that can antagonize SEB with nanomolar binding affinity (Kd = 64 nM). The aptamer antagonist effectively inhibits SEB-mediated proliferation and cytokine secretion in human peripheral blood mononuclear cells. Moreover, a PEGylated aptamer antagonist significantly reduced mortality in a "double-hit" mouse model of SEB-induced TSS, established via sensitization with d-galactosamine followed by SEB challenge. Therefore, our novel aptamer antagonist may offer potential therapeutic efficacy against SEB-mediated TSS.

In vitro and in vivo antibacterial evaluation of cadazolid, a new antibiotic for treatment of Clostridium difficile infections.

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 µg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.

Grape extracts inhibit multiple events in the cell biology of cholera intoxication.

Vibrio cholerae produces cholera toxin (CT), an AB5 protein toxin that is primarily responsible for the profuse watery diarrhea of cholera. CT is secreted into the extracellular milieu, but the toxin attacks its Gsα target within the cytosol of a host cell. Thus, CT must cross a cellular membrane barrier in order to function. This event only occurs after the toxin travels by retrograde vesicular transport from the cell surface to the endoplasmic reticulum (ER). The catalytic A1 polypeptide then dissociates from the rest of the toxin and assumes an unfolded conformation that facilitates its transfer to the cytosol by a process involving the quality control system of ER-associated degradation. Productive intoxication is blocked by alterations to the vesicular transport of CT and/or the ER-to-cytosol translocation of CTA1. Various plant compounds have been reported to inhibit the cytopathic activity of CT, so in this work we evaluated the potential anti-CT properties of grape extract. Two grape extracts currently sold as nutritional supplements inhibited CT and Escherichia coli heat-labile toxin activity against cultured cells and intestinal loops. CT intoxication was blocked even when the extracts were added an hour after the initial toxin exposure. A specific subset of host-toxin interactions involving both the catalytic CTA1 subunit and the cell-binding CTB pentamer were affected. The extracts blocked toxin binding to the cell surface, prevented unfolding of the isolated CTA1 subunit, inhibited CTA1 translocation to the cytosol, and disrupted the catalytic activity of CTA1. Grape extract could thus potentially serve as a novel therapeutic to prevent or possibly treat cholera.

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.

Effect of Eleutherine americana Merr. extract on enzymatic activity and enterotoxin production of Staphylococcus aureus in broth and cooked pork.

Crude ethanolic extract from the bulb of Eleutherine americana was investigated for its inhibitory activities against lipase and protease enzymes and enterotoxin production by Staphylococcus aureus. Eleven isolates that demonstrated high enzyme activity with three reference strains were selected to study the effect of extract on enzyme production. Exposure of the isolates to subminimal inhibitory concentrations, (1/2) minimum inhibitory concentration (MIC) (125 microg/mL), and (1/4)MIC (62.5 microg/mL) of the crude extract resulted in both partial and total inhibition of lipase and protease enzymes. About 15% of the 106 isolates were positive for enterotoxin production with staphylococcal enterotoxin A (11.3%), enterotoxin B (3.7%), and enterotoxin C (10.3%), and no enterotoxin D was produced. The production of staphylococcal enterotoxins A-D in the presence or absence of the crude extract was carried out. In the broth system, the extract reduced enterotoxin production at subminimal inhibitory concentrations compared with the control. At MIC, total enterotoxin inhibition was observed for enterotoxin C production, whereas synthesis of enterotoxins A, B, and D was totally eliminated at 2MIC. The food system study revealed that the extract could delay production of enterotoxins A, B, and C compared with the control. The extract at 2 mg/mL delayed production of toxins A and C for 8 and 4 h, while toxin B was not detected in the pork at 48 h. The ability of E. americana extract to inhibit lipase and protease enzymes and to delay enterotoxin production in food could present it as a novel food additive to combat the growth of S. aureus in food.

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity.

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo. In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.

Identification of Escherichia coli enterotoxin inhibitors from traditional medicinal herbs by in silico, in vitro, and in vivo analyses.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza uralensis has been used for the treatment of gastrointestinal disorders, such as diarrhea, in several ancient cultures. Glycyrrhizin is the principal component of liquorice and lots of pharmacological effects have been demonstrated. AIM OF THE STUDY: Heat-labile enterotoxin (LT), the virulence factor of enterotoxigenic Escherichia coli, induces diarrhea by initially binding to the GM1 on the surfaces of intestinal epithelial cells and consequently leading to the massive loss of fluid and ions from cells. Therefore, we evaluated the inhibitory effects of traditional medicinal herbs (TMH) on the B subunit of LT (LTB) and GM1 interaction. MATERIALS AND METHODS: The inhibitory effects of TMH on LTB-GM1 interaction were evaluated by GM1-enzyme-linked immunosorbent assay (ELISA). The likely active phytochemicals of these TMH were then predicted by in silico model (docking) and analyzed by in vitro (GM1-ELISA) and in vivo (patent mouse gut assay) models. RESULTS: We found that various TMH, which have been ethnomedically used for the treatment of diarrhea, inhibited the LTB-GM1 interaction. Docking data showed that triterpenoids were the most active phytochemicals and the oleanane-type triterpenoids presented better LTB-binding abilities than other types of triterpenoids. Moreover, by in vitro and in vivo models, we demonstrated that glycyrrhizin was the most effective oleanane-type triterpenoid that significantly suppressed both the LTB-binding ability (IC50=3.26+/-0.17 mM) and the LT-induced fluid accumulation in mice. CONCLUSIONS: We found an LT inhibitor, glycyrrhizin, from TMH by in silico, in vitro, and in vivo analyses.

Ginger and its bioactive component inhibit enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea in mice.

Ginger is one of the most commonly used fresh herbs and spices. Enterotoxigenic Escherichia coli heat-labile enterotoxin (LT)-induced diarrhea is the leading cause of infant death in developing countries. In this study, we demonstrated that ginger significantly blocked the binding of LT to cell-surface receptor G M1, resulting in the inhibition of fluid accumulation in the closed ileal loops of mice. Biological-activity-guided searching for active components showed that zingerone (vanillylacetone) was the likely active constituent responsible for the antidiarrheal efficacy of ginger. Further analysis of chemically synthesized zingerone derivatives revealed that compound 31 (2-[(4-methoxybenzyl)oxy]benzoic acid) significantly suppressed LT-induced diarrhea in mice via an excellent surface complementarity with the B subunits of LT. In conclusion, our findings provide evidence that ginger and its derivatives may be effective herbal supplements for the clinical treatment of enterotoxigenic Escherichia coli diarrhea.

Inhibitory effect of antimicrobial agents and anisodamine on the staphylococcal superantigenic toxin-induced overproduction of proinflammatory cytokines by human peripheral blood mononuclear cells.

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), produces superantigenictoxins, such as toxic shock syndrome toxin-1 (TSST-1). TSST-1 abnormally activates T cells to overproduce inflammatory cytokines (such as tumor necrosis factor-alpha, interleukin-2, and interferon-gamma) leading to shock. In this study, we investigated the inhibitory effect of antimicrobial agents and anisodamine (a Chinese herbal extract) on TSST-1-induced cytokine production. Among the macrolides and related agents examined, azithromycin and rokitamycin showed the greatest inhibitory activity against the TSST-1-induced cytokine production. This inhibitory effect was similar to that of anisodamine, which, however, had no inhibitory activity against bacterial growth. Vancomycin, teicoplanin, arbekacin, and linezolid (anti-MRSA and related agents) had no significant inhibitory effect on cytokine production. The inhibitory effect of the drugs on cell proliferation was not significant. These data indicate that some antimicrobial agents, e.g., azithromycin and rokitamycin, manifest anti-superantigenic toxin activity through the inhibition of cytokine production, just like anisodamine.

Triptolide attenuates endotoxin- and staphylococcal exotoxin-induced T-cell proliferation and production of cytokines and chemokines.

Proinflammatory cytokines mediate the toxic effects of superantigenic staphylococcal exotoxins (SE) and bacterial lipopolysaccharide (LPS). Triptolide, an oxygenated diterpene derived from a traditional Chinese medicinal herb, Tripterygium wilfordii, inhibited SE-stimulated T-cell proliferation (by 98%) and expression of interleukin 1beta, interleukin 6, tumor necrosis factor, gamma interferon, monocyte chemotactic protein 1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta by human peripheral blood mononuclear cells (PBMC). It also blocked the production of these cytokines and chemokines by LPS-stimulated PBMC in a dose-dependent manner. These results suggest that triptolide has potent immunosuppressive effects even counteracting the effects of superantigens and LPS. It also may be therapeutically useful for mitigating the pathogenic effects of these microbial products by downregulating the signaling pathways activated by both bacterial exotoxins and endotoxins.

Specific inhibitory action of anisodamine against a staphylococcal superantigenic toxin, toxic shock syndrome toxin 1 (TSST-1), leading to down-regulation of cytokine production and blocking of TSST-1 toxicity in mice.

Toxic shock syndrome toxin 1 (TSST-1), produced by Staphylococcus aureus (including methicillin-resistant S. aureus), is a superantigenic toxin responsible for toxic shock syndrome as well as neonatal TSS-like exanthematous disease. TSST-1 exhibits its deleterious effects by leading to the abnormal proliferation of, e.g., Vbeta2+ T cells and overproduction of proinflammatory cytokines. In the present study we examined the inhibitory effect of a Chinese herbal extract, anisodamine, on TSST-1 using human peripheral blood mononuclear cells (PBMCs). Anisodamine inhibited the production of proinflammatory cytokines better than interleukin-10 (an anti-inflammatory cytokine). The inhibitory effect of anisodamine was greater than that of any tropane alkaloid examined. Anisodamine acted directly on both monocytes and T cells in human PBMCs, and the effect was confirmed at the transcriptional level. Inhibition of NF-kappaB activation was also demonstrated. In contrast, no significant inhibition of Vbeta2+ T-cell proliferation was observed. In mice injected with TSST-1, anisodamine treatment significantly decreased serum proinflammatory cytokine levels and prevented TSST-1-induced death. These results suggest that anisodamine specifically acts against the production of cytokines (inflammatory cytokines in particular) and not against Vbeta2+ T-cell proliferation and that anisodamine may have a beneficial effect on TSST-1-associated disease.

Zinc inhibits cholera toxin-induced, but not Escherichia coli heat-stable enterotoxin-induced, ion secretion in human enterocytes.

BACKGROUND: Because zinc deficiency in malnourished children is associated with severe diarrhea, use of zinc supplementation has been proposed as an adjunct to oral rehydration. However, the effects of zinc on enterocyte ion transport are largely unknown. The objective of the present study was to investigate the effects of zinc on transepithelial ion transport under basal conditions and under conditions of enterotoxin-induced ion secretion. METHODS: Ion transport was investigated by monitoring electrical parameters in human intestinal Caco-2 cells that were mounted in Ussing chambers and exposed to increasing concentrations of zinc, both in the absence and presence of either cholera toxin (CT) or Escherichia coli heat-stable enterotoxin (ST). Intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) concentrations were also determined. RESULTS: The addition of zinc to the luminal or basolateral side of enterocytes induced a chloride-dependent, dose-related decrease in short-circuit current, indicating ion absorption. It also resulted in a substantial reduction in CT-induced ion secretion and in cAMP concentration. E. coli ST-induced ion secretion and cGMP concentration were not affected. Ion absorption peaked at 35 mu mol/L zinc, whereas excess zinc load induced active ion secretion. CONCLUSIONS: By causing a decrease in cAMP concentration, zinc directly promotes ion absorption and substantially reduces CT-induced, but not E. coli ST-induced, ion secretion.

Pomegranate extract inhibits Staphylococcus aureus growth and subsequent enterotoxin production.

In Brazil, pomegranate (Punica granatum L. (Punicaceae)) is widely used as a phytotherapeutic agent. This study evaluates the effect of pomegranate extract on Staphylococcus aureus FRI 722 growth and subsequent enterotoxin production. Bacterial susceptibility was determined by tube dilution method and production of enterotoxin was assessed using membrane-over-agar (MOA) plates. At a low extract concentration (0.01% v/v) bacterial growth was delayed, while a higher concentration (1% v/v) eliminated bacterial growth. Most interestingly, a 0.05% (v/v) concentration of extract was found to inhibit Staphylococcal enterotoxin (SE) A production. These data further implicate pomegranate extracts as potential antibacterial therapeutics with the added ability to inhibit enterotoxin production.

[Effect of xiaoyin jiedu yin on Staphylococcus aureus enterotoxin B induced serum interleukin 8 in mice].

OBJECTIVE: To explore the therapeutic mechanism of Xiaoyin Jiedu Yin (XYJDY) in treating psoriasis. METHODS: Abnormal elevation of interleukin 8 (IL-8) level in mice was induced by Staphylococcus aureus enterotoxin B (SEB), then the antagonizing effects of XYJDY and Composite Qingdai Capsule were observed. RESULTS: Serum IL-8 level arose and reached the peak 3-5 hrs after SEB induction. XYJDY could antagonize the induction significantly, and the effect was more significant when large dosage was given. CONCLUSION: XYJDY, in either large or small dose, could effectively antagonize the SEB induction on IL-8. Applying with superantigen theory, this fact could be used to elucidate the mechanism of XYJDY in removing Heat, cooling blood and detoxifying at cell biologic level.

[Bactericidal and anti-toxin activities of catechin on enterohemorrhagic Escherichia coli].

We examined the bactericidal activity of catechin, an astringent ingredient of tea, on enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the anti-toxin activity of catechin on vero toxin (VT), the main pathogenic factor of EHEC O157:H7. To examine bactericidal activity, we added 1 X 10(4) CFU/ml bacteria to 1.25 to 20 W/V% of green tea extract or the PBS solution containing 25 to 400 micrograms/ml of (-) epigallocatechin gallate (EGCg), which is the main catechin ingredient of green tea leaf, and counted the number of live bacteria at various intervals. After 3 to 5 hours, no live bacteria were seen in 1.25 to 2.5 (regular drinking concentration) % green tea extract. In the high concentrations of 100 to 400 micrograms/ml EGCg the number of live bacteria decreased with time and after 24 hours no survivors were seen. In the low concentrations of 25 to 50 micrograms/ml EGCg, however, no change was observed in the number of live bacteria during 5 hours. After 24 hours the bacteria in 50 micrograms/ml were killed and the number of bacteria in 25 micrograms/ml decreased to one tenth of that at the start. To examine the anti-toxin activity, we mixed equal volumes of 2 ng/0.1 ml VT2 and 0.5 to 2 mg/0.1 ml catechin in vitro and incubated them at 37 degrees C for various times. Then we inoculated 0.2 ml of the mixture intraperitonealy to BALB/c mice. One mg of catechin inhibited by 100% the lethal toxicity of 2 ng of VT2 (LD 100) to mice. The inhibition of lethal toxicity of VT2 by catechin depended on the incubation time. The rate of inhibition was 0, 40 and 100% for 9, 12 and 18-24 hours incubation, respectively. These results suggest that catechin has not only bactericidal activity on EHEC O157:H7 but also anti-toxin activity on vero toxin.

Effect of Merocel vaginal sponge on growth of Staphylococcus aureus and production of toxic shock syndrome-associated toxins.

BACKGROUND: Staphylococcus aureus toxic shock syndrome toxin-1 and enterotoxin B are the major causes of toxic shock syndrome. These toxins are produced in sufficient concentrations to produce illness in the presence of certain tampons. This necessitates evaluating tampons, as well as wound dressings for their effects on S. aureus growth and toxin production. STUDY DESIGN: In this study, the Merocel vaginal sponge was evaluated both in vitro and in vivo in a rabbit model for effect on S. aureus. The Merocel sponge was tested in Erlenmeyer shake flasks containing growth media and in dialysis tubing immersed in agar growth media for both effect on S. aureus plate counts compared to media alone and effect on production of toxic shock syndrome toxins. The in vivo test included placement of Merocel sponges subcutaneously along the flanks of rabbits with subsequent inoculation with toxic shock syndrome bacteria and evaluation for development of illness and death. RESULTS: In the two standard in vitro tests, the shake flask and tampon sac, the Merocel sponge inhibited both growth of toxic shock syndrome S. aureus and production of toxic shock syndrome toxin-1 and enterotoxin B. The Merocel sponge also prevented development of toxic shock syndrome in a rabbit model. CONCLUSIONS: The data suggest the Merocel sponge may reduce the risk of development of toxic shock syndrome in association with its use. These studies may serve as models for evaluation of other products that are intended to be used on mucosal and skin surfaces, for their effect on toxic shock syndrome toxins.