Nickel chelation therapy as an approach to combat multi-drug resistant enteric pathogens.

The nickel (Ni)-specific chelator dimethylglyoxime (DMG) has been used for many years to detect, quantitate or decrease Ni levels in various environments. Addition of DMG at millimolar levels has a bacteriostatic effect on some enteric pathogens, including multidrug resistant (MDR) strains of Salmonella Typhimurium and Klebsiella pneumoniae. DMG inhibited activity of two Ni-containing enzymes, Salmonella hydrogenase and Klebsiella urease. Oral delivery of nontoxic levels of DMG to mice previously inoculated with S. Typhimurium led to a 50% survival rate, while 100% of infected mice in the no-DMG control group succumbed to salmonellosis. Pathogen colonization numbers from livers and spleens of mice were 10- fold reduced by DMG treatment of the Salmonella-infected mice. Using Nuclear Magnetic Resonance, we were able to detect DMG in the livers of DMG-(orally) treated mice. Inoculation of Galleria mellonella (wax moth) larvae with DMG prior to injection of either MDR K. pneumoniae or MDR S. Typhimurium led to 40% and 60% survival, respectively, compared to 100% mortality of larvae infected with either pathogen, but without prior DMG administration. Our results suggest that DMG-mediated Ni-chelation could provide a novel approach to combat enteric pathogens, including recalcitrant multi-drug resistant strains.

Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food-borne bacterial pathogens.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.

Composition of Herba Pogostemonis water extract and protection of infected mice against Salmonella Typhimurium-induced liver damage and mortality by stimulation of innate immune cells.

GC-MS analysis of a hot water extract of Herba Pogostemonis (HP) revealed the presence of 131 compounds. HP slightly inhibited Salmonella Typhimurium bacteria in culture and stimulated uptake of the bacteria into RAW 264.7 murine macrophage cells as indicated by both increased fluorescence from internalized FITC-dextran and increased colony-forming unit (CFU) counts of the lysed macrophages. Postinfection, the HP-treated cells showed lower bacterial counts than the control. HP elicited altered morphology, elevated inducible NO synthase (iNOS) mRNA, and reduced pro-inflammatory cytokine expression in macrophage cells. Salmonella induced increased expression of iNOS mRNA, cognate polypeptides, and NO. Histology of mice infected with a sublethal dose (1 × 10(4) CFU) of Salmonella showed that intraperitoneally administered HP protected against necrosis of the liver, a biomarker of in vivo salmonellosis. The lifespan of mice infected with a lethal dose (1 × 10(5) CFU) was significantly extended. These results suggest that the activity of HP against bacterial infection in mice occurs through the activation of innate immune macrophage cells. The relationship of composition of HP to bioactivity is discussed.

Efficacy of crude extract of Aloe secundiflora against Salmonella gallinarum in experimentally infected free-range chickens in Tanzania.

The ethnoveterinary use of Aloe species extract in free-range local chickens against fowl typhoid was investigated. Five-months-old local chickens, free of antibodies against fowl typhoid were used. The chickens were randomly assorted into five groups including pretreated and infected (G1, n=21), infected and untreated (G2, n=21), infected and treated (G3, n=21), untreated and uninfected (G4, n=10) and treated uninfected (G5, n=10). Groups 1, 2 and 3 were inoculated with 5.0 x 10(8) c.f.u/ml of Salmonella gallinarum, following which the chickens were monitored for 15 days. There was a delay on the occurrence of the clinical signs and reduced severity of the disease in the Aloe treated chickens (G1 and G3). The mortality rates were 23.8% (5/21) in G1 (pretreated and infected), 42.8% (9/21) in G2 (infected and untreated) and 14.2% (3/21) in G3 (infected and treated). Antibody levels were raised among the infected and untreated group (G2) while they remained relatively low in the Aloe treated groups (G1 and G3). However, there was a sharp increase in the levels of interleukin 6 (IL-6) in the Aloe treated groups (G1 and G3) as compared to the untreated group (G2) until day 9 post-infection. The results of this work indicate that the extract of Aloe secundiflora may be used in the control of fowl typhoid in chickens. Studies to determine the active ingredients of the plant extract are in progress.

High doses of dietary arginine during repletion impair weight gain and increase infectious mortality in protein-malnourished mice.

There is considerable evidence for the beneficial effects of dietary arginine, a conditionally-essential amino acid that enhances anabolism and T-cell function. However, the safety and efficacy of higher doses of arginine supplementation following infection have not been investigated completely. These issues were explored therefore, in a murine model of malnutrition and infection. Severe protein malnutrition was induced by feeding mice for 6 weeks on an isoenergetic diet containing only 10 g protein/kg. Mice were then allowed to consume diets with normal amounts of protein (200 g/kg) with 50 g/kg provided as amino acid mixtures of glycine and arginine in which the arginine content ranged from 0 to 50 g/kg. During the repletion period a significant weight gain was noted in the groups fed on diets with either 10 or 20 g arginine/kg, but not in the group fed on the diet with 50 g arginine/kg, compared with the diet with 0 g arginine/kg. Mortality rates after infection with Salmonella typhimurium were not decreased by the addition of 10 or 20 g arginine/kg to the diet, and were in fact worsened by supplementation with 50 g arginine/kg. The results of the present study showed that not only are the beneficial effects of arginine supplementation after infection lost when high doses are administered, but also that these high doses become toxic. Mice fed on higher doses showed significant impairment of weight gain and an increase in mortality rates.

[Gamma-interferon and the antibacterial activity of mouse macrophages in a Salmonella infection]. / Gamma-interferon i antibakterial'naia aktivnost' makrofagov myshei pri sal'monelleznoi infektsii.

The influence of natural and recombinant mouse gamma-interferon (gamma-IF) on Salmonella typhimurium was experimentally studied. The preparations of gamma-IF were introduced into animals in a dose of 250 units per mouse according to different schedules before and after Salmonella inoculation. In these experiments the capacity of the animals for survival, the elimination of the infective agent from the body and the functional activity of peritoneal exudate macrophages were studied. Natural and recombinant gamma-IF was found to stimulate the ingestion activity and oxidizing metabolism of macrophages of the infected animals. At the same time the preparations of gamma-IF were not capable of enhancing the intracellular killing of salmonellae and tumor cells. Recombinant gamma-IF did not prevent the lethal outcome of S. typhimurium infection in mice and did not increase the elimination of the infective agent from the body. Natural gamma-IF, when introduced into mice in a dose of 250 units per mouse on days 1, 3 and 6 after Salmonella inoculation, ensured the survival rate of more than 80% of the animals, but did not essentially influence the dynamics of the elimination of the infective agent from the host body.

Short-term dietary lipid manipulation does not affect survival in two models of murine sepsis.

Dietary lipid manipulation has been shown to have various effects on the immune system, depending on the amount of fat, degree of saturation, and type of fat used. In this study we investigated the role of different sources of fat at different levels on the survival of mice in two models of peritonitis, one with Pseudomonas aeruginosa and the other with Salmonella typhimurium. CF1 mice were pair-fed diets with 5% or 40% of total calories as fat. The source of fat used was coconut oil, oleic acid, safflower oil, or fish oil. Three other diets were tested as well, one with no fat, one with only 0.5% of total calories linoleic acid as the only source of fat, and a control diet that had 12% of total calories as corn oil. At the end of 2 weeks of feeding the experimental diets, mice were challenged with Ps aeruginosa intraperitoneally and mortality was recorded over 1 week. After 3 weeks of feeding the experimental diets, mice were challenged with S typhimurium and mortality was recorded over 2 weeks. No significant differences were seen on survival among groups fed different levels of fat, as well as different sources of fat. We conclude that, overall, 2- and 3-week manipulation of dietary fat does not affect outcome from infection in these models.

[The effect of "Solco" lactobacteria (Lactobacillus acidophilus) on the survival and microflora of mice with a Salmonella infection]. / Vliianie laktobakterii "Solko" (Lactobacillus acidophilus) na vyzhivaemost' i mikrofloru myshei pri sal'monelleznoi infektsii.

The Solco lactobacterial strain L. acidophilus Lat 11/83 has been used for the normalization of intestinal microflora in experimental post-infectious intestinal dysbacteriosis in mice. The results of experiments indicate that the intragastric administration of live Solco lactobacteria contributes to an increase in the survival rate of infected animals and the normalization of their gastrointestinal microflora. This strain may be used as a bacterial preparation for the regulation of intestinal microbiocenosis.

Effect of Salmonella gallinarum infection on zinc metabolism in chicks.

The effect of Salmonella gallinarum infection in chicks on serum, liver, and kidney zinc concentrations was studied. Within 48 h after intraperitoneal administration of the organism, serum zinc declined to approximately one-half the control value. In one experiment, the serum zinc concentration remained low for the 12 days of the experiment, whereas in a second experiment, the concentration gradually increased after 6 days postinoculation but never returned to the control value. Feeding as much as 500 ppm supplemental zinc did not prevent the infection-induced decline in the serum zinc concentration. The infection resulted in a sequestering of zinc in the liver; the kidney remained relatively unresponsive in this system. Fractionation of liver homogenates by gel filtration column chromatography revealed that the zinc in the livers of the infected animals eluted in a volume characteristic of metallothionein, whereas that of control animals was associated with high molecular weight proteins. Increasing the zinc content of the serum by repeated subcutaneous injections of zinc had no effect on mortality from this infection. Restricting feed consumption of uninfected chicks to that of infected animals did mimic the influence of infection of serum zinc and hepatic metallothionein concentrations.

[The protective properties of myelopeptides in the development of infectious processes caused by bacteria of the genus Salmonella]. / Protektivnye svoistva mielopeptidov pri razvitii infektsionnykh protsessov, vyzvannykh bakteriiami roda Salmonella.

The protective properties of myelopeptides in the development of bacterial infection in mice and young pigs, caused by S. typhimurium 415, S. cholerae-suis 1422 and 370, have been studied. Myelopeptides have been found to possess protective properties when injected into animals infected with S. typhimurium and S. cholerae-suis in lethal doses. The best protective effect (survival rate of 100%) has been achieved by the injection of myelopeptides 24 hours before challenge. Myelopeptides have also been found to promote the weight gain of young pigs infected with S. cholerae-suis.

In vivo inhibition of plasma protein leakage and Salmonella enteritidis-induced mortality in the rat by a specific paf-acether antagonist: BN 52021.

The effects of BN 52021, a new specific paf-acether receptor antagonist and the total Ginkgo Biloba extract (GBE 761) from which this product was isolated, were studied in the rat on paf-acether-induced permeability and cell number changes and on endotoxin-induced lethality. Their activities were compared to those of cyclooxygenase, 5-lipoxygenase and phospholipase A2 inhibitors. BN 52021 given s.c. or orally exerted a dose-related inhibition of paf-acether deleterious effects as well as of endotoxin lethality whereas the other drugs tested were poorly effective. These results strongly suggest paf-acether involvement in endotoxic and septic shock.

Influence of time of exposure to high levels of minerals on the susceptibility of chicks to Salmonella gallinarum.

Mercury, 500 ppm; cadmium, 100 ppm; vanadium, 25 ppm; copper, 1,000 ppm; or selenium, 20 ppm were fed to chicks for various times before and after inoculation with S. gallinarum. These levels have been found to be toxic to chicks as indicated by reduced growth. When the feeding of the elements was discontinued at the time of inoculation, early mortality was increased but the effects tended to diminish as the experiment progressed. The elements had no effect on mortality if the feeding was discontinued 1 week before inoculation. When the elements were fed starting at the time of inoculation, both early (1 week post inoculation) and final mortality were increased. Delaying the administration of the elements for 2 days after inoculation resulted in no effect on mortality 1 week after inoculation but there were increases in final mortality when mercury, cadmium or selenium were fed.

The effect of dietary protein levels on mineral toxicity in chicks.

Studies have been conducted to determine the influence of dietary protein levels of 10, 20, and 30% fed as soybean meal on the toxicity of selenium, cadmium, vanadium, cobalt, and nickel to chicks. The toxicity was evaluated in terms of growth retardation and a decrease in resistance to S. gallinarum infection as measured by mortality. The toxicity of selenium or cadmium was unaffected by the protein level of the diet while the toxicity of vanadium, cobalt and nickel was decreased by increased dietary protein as measured by the interactions on growth. The interactions apparent on growth were not as apparent on resistance to infection indicating that the latter parameter is much less sensitive to these dietary manipulations than is growth.

Enhancement of survival in acute experimental fowl typhoid in chicks by the administration of iron dextran.

Chicks aged 15 days were infected orally with 10(6-4) living Salmonella gallinarum. Iron from iron dextran or ferric ammonium citrate (DFe and CFe respectively), in doses of 2-0 or 0-4 mg/kg given intramuscularly at the time of infection, had no effect on subsequent mortality compared with infected controls. Increasing the amount of CFe to the maximum (20 mg/kg) that was non-toxic intramuscularly was also ineffective but when the dose of DFe was increased to 20 or 50 mg/kg the survival rate rose sharply. If given at other times in relation to the time of infection, or more frequently, 50 mg/kg or more of DFe was less active. The increased survival rate among infected chicks given 50 mg/kg of DFe at the time of infection was accompanied by decreases in the severity of the morbid changes in the disease, and by reductions of between c 10- to 100-fold in the numbers of viable S gallinarum in the liver, spleen or blood at the height of the acute infection. DFe (50 mg/kg) given at the time of infection also eliminated the difference in the survival rate noted previously between infected chicks fed respectively with diets containing fish or meat meal as the sole source of supplementary protein.

Experimental fowl typhoid in chicks impaired immunologically by treatment with cyclophosphamide.

Male chicks were injected intramuscularly with 6 mg of cyclophosphamide daily for three consecutive days, the initial dose being given on the day of hatching. This treatment impaired bursal development and lymphopoiesis and no circulating antibodies appeared after such chicks were injected with Salmonella gallinarum antigens at 19 and again at 26 days of age. In impaired chicks which were infected orally when 15 days old with S gallinarum the level of mortality was essentially the same as that in unimpaired animals. Impairment did not affect the lower mortality that accompanies feeding with a nutritionally adequate diet containing meat meal as the sole protein supplement in comparison with fish meal as the supplement. An anaemia was present in chicks examined seven days after infection. This anaemia was microcytic to a degree that either exceeded or approached statistical significance. As antibody-mediated hypersensitivity reactions involving the erythrocytes were absent in the impaired chicks, the anaemia must have differed in pathogenesis from the macrocytic anaemia of immunopathological orgin reported by others in adult females with acute fowl typhoid. A disturbance of iron metabolism could have accounted for the anaemia in the diseased chicks.