Effects of zinc supplementation on Shiga toxin 2e-producing Escherichia coli in vitro.

Swine edema disease is caused by Shiga toxin (Stx) 2e-producing Escherichia coli (STEC). Addition of highly concentrated zinc formulations to feed has been used to treat and prevent the disease, but the mechanism of the beneficial effect is unknown. The purpose of the present study was to investigate the effects of highly concentrated zinc formulations on bacterial growth, hemolysin production, and an Stx2e release by STEC in vitro. STEC strain MVH269 isolated from a piglet with edema disease was cultured with zinc oxide (ZnO) or with zinc carbonate (ZnCO3), each at up to 3,000 ppm. There was no effect of zinc addition on bacterial growth. Nonetheless, the cytotoxic activity of Stx2e released into the supernatant was significantly attenuated in the zinc-supplemented media compared to that in the control, with the 50% cytotoxic dose values of 163.2 ± 12.7, 211.6 ± 33.1 and 659.9 ± 84.2 after 24 hr of growth in the presence of ZnO, ZnCO3, or no supplemental zinc, respectively. The hemolytic zones around colonies grown on sheep blood agar supplemented with zinc were significantly smaller than those of colonies grown on control agar. Similarly, hemoglobin absorbance after exposure to the supernatants of STEC cultures incubated in sheep blood broth supplemented with zinc was significantly lower than that resulting from exposure to the control supernatant. These in vitro findings indicated that zinc formulations directly impair the factors associated with the virulence of STEC, suggesting a mechanism by which zinc supplementation prevents swine edema disease.

Improved porcine model for Shiga toxin-producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets.

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)-producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 1011  colony-forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum-deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.

Angiotensin-(1-7) protects from brain damage induced by shiga toxin 2-producing enterohemorrhagic Escherichia coli.

Shiga toxin 2 (Stx2)-producing enterohemorrhagic induced brain damage. Since a cerebroprotective action was reported for angiotensin (Ang)-(1-7), our aim was to investigate whether Ang-(1-7) protects from brain damage induced by Stx2-producing enterohemorrhagic Escherichia coli The anterior hypothalamic area of adult male Wistar rats was injected with saline solution or Stx2 or Stx2 plus Ang-(1-7) or Stx2 plus Ang-(1-7) plus A779. Rats received a single injection of Stx2 at the beginning of the experiment, and Ang-(1-7), A779, or saline was administered daily in a single injection for 8 days. Cellular ultrastructural changes were analyzed by transmission electron microscopy. Stx2 induced neurodegeneration, axonal demyelination, alterations in synapse, and oligodendrocyte and astrocyte damage, accompanied by edema. Ang-(1-7) prevented neuronal damage triggered by the toxin in 55.6 ± 9.5% of the neurons and the Stx2-induced synapse dysfunction was reversed. In addition, Ang-(1-7) blocked Stx2-induced demyelination in 92 ± 4% of the axons. Oligodendrocyte damage caused by Stx2 was prevented by Ang-(1-7) but astrocytes were only partially protected by the peptide (38 ± 5% of astrocytes were preserved). Ang-(1-7) treatment resulted in 50% reduction in the number of activated microglial cells induced by Stx2, suggesting an anti-inflammatory action. All these beneficial effects elicited by Ang-(1-7) were blocked by the Mas receptor antagonist and thus it was concluded that Ang-(1-7) protects mainly neurons and oligodendrocytes, and partially astrocytes, in the central nervous system through Mas receptor stimulation.

Nitrosylation: an adverse factor in Uremic Hemolytic Syndrome. Antitoxin effect of Ziziphus mistol Griseb.

Toxins of Escherichia coli (STEC) causing Uremic Hemolytic Syndrome (UHS) generate oxidative stress in human blood with more production of nitric oxide (NO) than reactive oxygen species (ROS). Shiga toxin (Stx) together with the hemolysin (Hly) increased lipid oxidation, as evaluated by malondialdehyde MDA and oxidation of proteins. The addition of Ziziphus mistol Griseb extracts decreased NO, ROS, MDA and simultaneously caused an increase in the degradation of oxidized proteins to advanced oxidation protein products (AOPPs) in controls and samples with toxins. Furthermore, the nitrosylated proteins/AOPP ratio was reduced, due to the increase of AOPP. Z. mistol Griseb extracts exhibited a high proportion of polyphenols and flavonoids, with evident correlation with ferrous reduction antioxidant potential (FRAP). The plasma of eight children with UHS showed oxidative stress and NO stimulus, comparable to the effect of toxins during the assays in vitro. UHS children presented high levels of nitrosylated proteins respect to control children of similar age. Although the degradation of oxidized proteins to AOPP rose in UHS children, the nitrosylated proteins/AOPP rate increased as a consequence of the elevated nitrosative stress observed in these patients.

Minimizing human infection from Escherichia coli O157:H7 using GUMBOS.

OBJECTIVES: Reduction in faecal shedding of Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) in food-producing animals is a viable strategy to minimize human disease initiated by exposure to these microorganisms. To this end, an intervention strategy involving the electrostatic hybridization of two commonly used anti-infective agents for veterinary practice (i.e. chlorhexidine and ampicillin) was evaluated to curtail EHEC-transmitted disease from ruminant sources. Chlorhexidine di-ampicillin is a novel group of uniform material based on organic salts (GUMBOS) with inherent in vitro antibacterial activity that comes from its parent antimicrobial ions, chlorhexidine and ampicillin. METHODS: Antibacterial activities for chlorhexidine diacetate, sodium ampicillin, chlorhexidine di-ampicillin and stoichiometrically equivalent 1 : 2 chlorhexidine diacetate : sodium ampicillin were assessed using the serial 2-fold dilution method and time-kill studies against seven isolates of E. coli O157:H7 and one non-pathogenic E. coli 25922. Further studies to investigate synergistic interactions of reacted and stoichiometrically equivalent unreacted antimicrobial agents at MICs and possible mechanisms were also investigated. RESULTS: Synergism and in vitro antibacterial activities against EHEC were observed in this study, which suggests chlorhexidine di-ampicillin could be a useful reagent in reducing EHEC transmission and minimizing EHEC-associated infections. Likewise, chlorhexidine di-ampicillin reduced HeLa cell toxicity as compared with chlorhexidine diacetate or the stoichiometric combination of antimicrobial agents. Further results suggest that the mechanisms of action of chlorhexidine di-ampicillin and chlorhexidine diacetate against E. coli O157:H7 are similar. CONCLUSIONS: Reacting antimicrobial GUMBOS as indicated in this study may enhance the approach to current combination drug therapeutic strategies for EHEC disease control and prevention.

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment.

BACKGROUND: The bacteriophage life cycle has an important role in Shiga toxin (Stx) expression. The induction of Shiga toxin-encoding phages (Stx phages) increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. METHODOLOGY/PRINCIPAL FINDINGS: The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA) were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg(2+). In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. CONCLUSIONS/SIGNIFICANCE: Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) and in the emergence of new pathogenic strains.