Selective Increase in Hydrogen Peroxide Resistance of a Coagulase-positive Staphylococcus

Staphylococcus culture 105B was serially treated with 0.05% hydrogen peroxide at 54.4 C or without hydrogen peroxide at this temperature to determine changes in resistance to these conditions and in catalase activity of the surviving populations. Resistance of the final surviving populations to H(2)O(2) treatment and to heat treatment without H(2)O(2) was 5.6 and 4.5 times greater, respectively, than the parent culture. Catalase activities of the cell-free extracts of survivors of the H(2)O(2) treatments and of the heat treatments were 33.56 and 2.69 times greater, respectively, than the control. The untreated control cultures grew in Peptonized Milk (Difco), but addition of sodium pyruvate to the medium was necessary to support growth of survivors.     

Factors Affecting the Resistance of Staphylococcus aureus to Hydrogen Peroxide Treatments in Milk

Staphylococcus aureus 196E was treated with 0.05% hydrogen peroxide in milk under varying conditions to determine the effects of treatment conditions and characteristics of the culture on bactericidal effectiveness of hydrogen peroxide. Time intervals required for 90 to 99.99% destruction of S. aureus decreased significantly as treatment temperatures increased from 37.8 to 57.2 C. Plots of survivor curves showed extended lags in destruction at 37.8 C, slight lags followed by logarithmic rates of destruction at 48.9 C, and logarithmic rates at 54.4 and 57.2 C except for trials in which there was very rapid initial destruction followed by logarithmic rates. S. aureus 196E was significantly more resistant to heat treatments at 54.4 C without added hydrogen peroxide than to treatment with 0.05% hydrogen peroxide at this temperature. Cultures grown at 37 C for 16 hr in milk were more resistant to hydrogen peroxide than were cultures grown at 35 C. Storage of cultures for 96 hr in milk at 4 C caused a decrease in the resistance of the culture. Numbers of staphylococci being treated had little effect on rates of destruction.     

Production of catalases byComamonas spp. and resistance to oxidative stress

Bacterial isolates Comamonas terrigena N3H (from soil contaminated with crude oil) and C. testosteroni (isolated from the sludge of a wastewater treatment plant), exhibit much higher total catalase activity than the same species from laboratory collection cultures. Electrophoretic resolution of catalases revealed only one corresponding band in cell-free extracts of both C. testosteroni cultures. Isolates of C. terrigena N3H exhibited catalase-1 and catalase-2 activity, whereas in the collection culture C. terrigena ATCC 8461 only catalase-1 was detected. The environmental isolates exhibited much higher resistance to exogenous H2O2 (20, 40 mmol/L) than collection cultures, mainly in the middle and late exponential growth phases. The stepwise H2O2-adapted culture of C. terrigena N3H, which was more resistant to oxidative stress than the original isolate, exhibited an increase of catalase and peroxidase activity represented by catalase-1. Pretreatment of cells with 0.5 mmol/L H2O2 followed by an application of the oxidative agent in toxic concentrations (up to 40 mmol/L) increased the rate of cell survival in the original isolate, but not in the H2O2-adapted variant. The protection of bacteria caused by such pretreatment corresponded with stimulation of catalase activity in pretreated culture.     

Microbial resistance in relation to catalase activity to oxidative stress induced by photolysis of hydrogen peroxide

The purpose of the present study was to evaluate the mechanism of microbial resistance to oxidative stress induced by photolysis of hydrogen peroxide (H(2)O(2)) in relation to microbial catalase activity. In microbicidal tests, Staphylococcus aureus and Candida albicans were killed and this was accompanied by production of hydroxyl radicals. C. albicans was more resistant to hydroxyl radicals generated by photolysis of H(2)O(2) than was S. aureus. A catalase activity assay demonstrated that C. albicans had stronger catalase activity; accordingly, catalase activity could be one of the reasons for the resistance of the fungus to photolysis of H(2)O(2). Indeed, it was demonstrated that C. albicans with strong catalase activity was more resistant to photolysis of H(2)O(2) than that with weak catalase activity. Kinetic analysis using a modified Lineweaver-Burk plot also demonstrated that the microorganisms reacted directly with hydroxyl radicals and that this was accompanied by decomposition of H(2)O(2). The results of the present study suggest that the microbicidal effects of hydroxyl radicals generated by photolysis of H(2)O(2) can be alleviated by decomposition of H(2)O(2) by catalase in microorganisms.     

Liposome-mediated augmentation of catalase in alveolar type II cells protects against H2O2 injury

Oxidant injury to the alveolar epithelium can be mediated by exposure to oxidant gases such as O2 at high concentrations and O3, inflammatory cell-derived reactive O2 species, and the intracellular metabolism of xenobiotics such as paraquat. An in vitro model of alveolar epithelial oxidant injury was developed based on exposure of cultured rat type II pneumocytes to superoxide and hydrogen peroxide (H2O2) enzymatically generated in the culture medium. Cytotoxicity was assessed by the release of lactate dehydrogenase (LDH) into the culture medium, which was a more reliable indicator of damage than release of 51Cr by prelabeled cells. Incubation of cells for 6-8 h with xanthine plus xanthine oxidase and glucose plus glucose oxidase induced the release of greater than 50% of total intracellular LDH. Oxidant exposure also resulted in significant detachment of cells from culture dishes. Modulation of oxidant damage was accomplished using liposomes as vectors for the delivery of catalase. Treatment of cells with catalase liposomes for 2 h resulted in augmentation of cellular catalase specific activities up to 631% of controls. Catalase was partitioned into intracellular and surface-associated compartments in catalase liposome-treated cells. Partial and complete protection against oxidant injury, induced by xanthine plus xanthine oxidase and glucose plus glucose oxidase, respectively, was achieved by pretreatment of cells with catalase liposomes. LDH release during oxidant exposure was inversely related to augmentation of cellular catalase activities. Catalase liposome-treated cells also exhibited an enhanced ability to scavenge enzymatically generated H2O2 from the culture medium. These observations suggest a useful approach to modulation of alveolar injury induced by reactive O2 species.     

Is catalase activity one of the factors associated with maize resistance to Aspergillus flavus?

Plant responses to biotic and abiotic stresses are usually accompanied by the release of reactive oxygen species including hydrogen peroxide. Hydrogen peroxide plays a direct role in defense and is involved in many signal transduction pathways that lead to the proliferation of other defenses. Because catalase helps to maintain reactive oxygen homeostasis during biotic and abiotic stress, its activity was measured in various cob tissues during maize ear development. Catalase activity was determined in immature and mature embryos, pericarp, and rachis tissues of maize lines that are resistant and susceptible to Aspergillus flavus infection. The effect of fungal inoculation on catalase activity was also measured. Over two years of field experimentation, a correlation was observed between resistance and the level of catalase-specific activity in immature embryos, which was significantly higher in resistant lines (P < 0.0001). Furthermore, catalase activity in the resistant lines was significantly higher in immature embryos from inoculated ears (P = 0.0199). No correlation was observed between resistance and catalase activity in other ear tissues. Levels of hydrogen peroxide, the catalase substrate, and salicylic acid in the embryo were also determined. The resistant lines showed lower levels of H2O2 (P < 0.0001) and higher levels of salicylic acid (P < 0.0001) as compared with the susceptible lines. Catalase 3 was sequenced from the aflatoxin-resistant (Mp313E) and susceptible (SC212m) inbreds. The predicted amino acid sequence indicated that there was a 20-aa deletion in the resistant inbred that might affect enzymatic activity. Unlike many plant-pathogen interactions, it appears that lowering H2O2 levels helps to prevent A. flavus infection and subsequent aflatoxin accumulation.     

Heat inactivation of catalase from Staphylococcus aureus MF-31.

The effects of heat on catalase from Staphylococcus aureus lysates were examined. Catalase activity increased with increasing concentrations of potassium phosphate buffer, when heated at temperatures between 50 and 65 degrees C for 10 min. Inactivation of catalase by NaCl during heating was demonstrated. Extended heating of S. aureus cells at 52 degrees C resulted in a slight decrease in catalase activity of the resultant lysates. This decrease was more pronounced in the presence of salt. Heating at 62 degrees C caused a decrease in catalase activity, but not complete inactivation. These results implicate the combined effects of heat, and NaCl in the inactivation of catalase from S. aureus. The findings are consistent with the hypothesis that H2O2 may accumulate as a result of decreased catalase activity and be responsible for the decreased colony-forming ability of stressed S. aureus.     

Heat inactivation of catalase from Staphylococcus aureus MF-31.

The effects of heat on catalase from Staphylococcus aureus lysates were examined. Catalase activity increased with increasing concentrations of potassium phosphate buffer, when heated at temperatures between 50 and 65 degrees C for 10 min. Inactivation of catalase by NaCl during heating was demonstrated. Extended heating of S. aureus cells at 52 degrees C resulted in a slight decrease in catalase activity of the resultant lysates. This decrease was more pronounced in the presence of salt. Heating at 62 degrees C caused a decrease in catalase activity, but not complete inactivation. These results implicate the combined effects of heat, and NaCl in the inactivation of catalase from S. aureus. The findings are consistent with the hypothesis that H2O2 may accumulate as a result of decreased catalase activity and be responsible for the decreased colony-forming ability of stressed S. aureus.     

Inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus gasseri isolated from the vaginal tract of cattle

Infectious diseases are the major reproductive complication during postpartum. The bacteria that colonize the postpartum uterus of cattle are specific as well as opportunistic pathogens that include Staphylococcus aureus. The antibiotics and antiseptic agents used in the treatment of postpartum acute infections induce residues in foods, spread of bacterial resistance, increase in financial costs and failures in defense mechanisms of the host. Preventive treatment with probiotic products could decrease the use of antibiotics in dairy farming systems. Lactobacilli are present in the vaginal microflora of healthy cows. They can prevent pathogen colonization by mechanisms such as the production of antagonistic substances as lactic acid, H2O2, or bacteriocins. The aim of the present study was the selection of H2O2 generating lactobacilli from a group of 72 strains isolated from the vagina of cattle. Both Lactobacillus gasseri CRL1421 and Lactobacillus gasseri CRL1412, which share some probiotic properties, produce H2O2, detected by the plate colorimetric method. They were chosen to study the kinetics of H2O2 production under different culture conditions. Both microorganisms produced greater amounts of H2O2 in aerated than in static cultures. As L. gasseri CRL1421 had a greater capacity to generate H2O2, associative cultures with this strain and S. aureus were conducted. A significant decrease in the growth of the pathogen was detected after culture for 6h, this effect being greater under aerated conditions. The addition of catalase to mixed cultures partially abolished the inhibition, an effect that could be attributed to the combined action of H2O2 and other antagonistic metabolites. The simultaneous addition of catalase and NaOH to these cultures restored S. aureus growth. This observation suggests that the inhibition was produced by the combination of H2O2 and lactic acid, both released by the lactobacilli. Electron microphotographs showed the damage caused by the lactobacilli supernatant on the pathogenic cells. The treatment of S. aureus with lactic acid and hydrogen peroxide evidenced that each metabolite produced a different type of morphological damage. The number of viable cells obtained agrees with the electron microscopy observations. The results support the idea that L. gasseri CRL1421 could be successfully included in a probiotic product to prevent S. aureus infection in cows.     

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short term and prolonged oxidative stress

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short-term and prolonged oxidative stress was studied. An increase in the activity of intracellular catalase in fungal cells after short-term exposure to hydrogen peroxide was shown. Activation of fungal cells induced by H2O2 depends on H2O2 concentration, time of exposure, and the growth phase of the fungus. Variants of P. piceum F-648 that produced two forms of extracellular catalase with different catalytic properties were obtained due to prolonged adaptation to H2O2. Catalase with low affinity for substrate was produced predominantly by the parent culture and variant 3; however, a high substrate affinity of catalase was observed in variant 5. Variant 5 of P. peniceum F-648 displayed a high catalytic activity and operational stability of catalase in the presence of phosphate ions and the concentration of substrate less than 30 mM at pH more than 7.     

Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in Escherichia coli

Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H(2)O(2) whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H(2)O(2). These mutants excreted substantial amounts of H(2)O(2), and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H(2)O(2) than is catalase and therefore is likely to be the primary scavenger of endogenous H(2)O(2). Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10(-5) M) concentration of H(2)O(2). In contrast, catalase has a high K(m), and it therefore becomes the predominant scavenger when H(2)O(2) concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H(2)O(2). In sum, E. coli does indeed generate substantial H(2)O(2), but damage is averted by the scavenging activity of Ahp.     

Protective effects of vitamins and selenium compounds in yeast

Antimutagens and anticarcinogens are known to play an important role in decreasing damages induced by oxidants. In this study, we investigated the genotoxic and antimutagenic potential of two selenium compounds (sodium selenite: Na(2)SeO(3); seleno-DL-methionine: C(5)H(11)NO(2)Se) and Vitamins A and E in yeast cells of Saccharomyces cerevisiae. An oxidative mutagen (hydrogen peroxide (H(2)O(2)), HP) was chosen as positive control. We determined the enzymatic activities involved in the protection against oxidative damages (catalase: CAT; superoxide dismutase: SOD; glutathione peroxidase: GPx) in the cytosolic extract of yeast cells. The results demonstrated that selenium compounds exerted both mutagenic and antimutagenic effect at different concentrations. Antimutagenesis was evident both in stationary and in logarithmic phase cells. Catalase, SOD, and GPx were significantly increased in the presence of all the compounds assayed. Vitamins A (retinol) and E (alpha-tocopherol) did not have toxic or mutagenic action.     

Hydroperoxide metabolism in cyanobacteria

The enzymes involved in antioxidative activity and the cellular content of the antioxidants glutathione and ascorbate in the cyanobacteria Nostoc muscorum 7119 and Synechococcus 6311 have been examined for their roles in hydroperoxide removal. High activities of ascorbate peroxidase and catalase were found in vegetative cells of both species and in the heterocysts of N. muscorum. The affinity of ascorbate peroxidase for H2O2 was 15- to 25-fold higher than that of catalase. Increased activity of ascorbate peroxidase was observed in N. muscorum when H2O2 production was enhanced by photorespiration. Catalase activity was decreased in dilute cultures whereas ascorbate peroxidase activity increased. Ascorbate peroxidase activity also increased when the CO2 concentration was reduced. Ascorbate peroxidase appears to be a key enzyme in a cascade of reactions regenerating antioxidants. Dehydroascorbate reductase was found to regenerate ascorbate, and glutathione reductase recycled glutathione. In vegetative cells glutathione was present in high amounts (2-4 mM) whereas the ascorbate content was almost 100-fold lower (20-100 microM). Glutathione peroxidase was not detected in either cyanobacterium. It is concluded from the high activity of ascorbate peroxidase activity and the levels of antioxidants found that this enzyme can effectively remove low concentrations of peroxides. Catalase may remove H2O2 produced under photooxidative conditions where the peroxide concentration is higher.     

Effect of hydrogen peroxide on antibacterial activities of Canadian honeys

Honey is recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds. The antimicrobial activity in some honeys depends on the endogenous hydrogen peroxide content. This study was aimed to determine whether honey's hydrogen peroxide level could serve as a honey-specific, activity-associated biomarker that would allow predicting and assessing the therapeutic effects of honey. Using a broth microdilution assay, I analyzed antibacterial activities of 42 Canadian honeys against two bacterial strains: Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633). The MIC90 and MIC50 were established from the dose-response relationship between antibacterial activities and honey concentrations. The impact of H2O2 on antibacterial activity was determined (i) by measuring the levels of H2O2 before and after its removal by catalase and (ii) by correlating the results with levels of antibacterial activities. Canadian honeys demonstrated moderate to high antibacterial activity against both bacterial species. Both MIC90 and MIC50 revealed that the honeys exhibited a selective growth inhibitory activity against E. coli, and this activity was strongly influenced by endogenous H2O2 concentrations. Bacillus subtilis activity was marginally significantly correlated with H2O2 content. The removal of H2O2 by catalase reduced the honeys' antibacterial activity, but the enzyme was unable to completely decompose endogenous H2O2. The 25%-30% H2O2 "leftover" was significantly correlated with the honeys' residual antibacterial activity against E. coli. These data indicate that all Canadian honeys exhibited antibacterial activity, with higher selectivity against E. coli than B. subtilis, and that these antibacterial activities were correlated with hydrogen peroxide production in honeys. Hydrogen peroxide levels in honey, therefore, is a strong predictor of the honey's antibacterial activity.     

The effects of catalase gene overexpression on life span and resistance to oxidative stress in transgenic Drosophila melanogaster.

Oxygen free radicals and hydroperoxides have been postulated to play a causal role in the aging process, implying that antioxidant enzymes may act as longevity determinants. Catalase (H2O2:H2O2 oxidoreductase; EC1.11.1.6) is the sole enzyme involved in the elimination of H2O2 in Drosophila melanogaster; glutathione peroxidase being absent. A genomic fragment containing the Drosophila catalase gene was used to construct transgenic Drosophila lines by means of P element-mediated transformation. Enhanced levels of catalase (up to 80%) did not prolong the life span of flies, nor did they provide improved protection against oxidative stress induced by hyperoxia or paraquat treatment. However, enhanced resistance to hydrogen peroxide was observed in the overexpressors.     

Effect of heme on Bacteroides distasonis catalase and aerotolerance

Parallel increases in intracellular catalase activity and resistance to extracellular H2O2 and to hyperbaric O2 toxicity were observed when Bacteroides distasonis VPI 4243 (ATCC 8503, type strain) was grown in either complex or defined medium containing graded amounts of hemin. Virtually all of the cells with high catalase activity (greater than 200 U/mg) remained viable upon exposure at 37 degrees C to 100-lb/in2 O2 on agar surfaces for 1 h, whereas low-catalase cells (less than 10 U/mg) lost 1.2 log units of viable cells during that treatment. Upon exposure to 500 microM H2O2, high-catalase cells lost 0.4 log units of the initial viable colonies during the same period in which low-catalase cells lost 3 log units of viable cells. The superoxide dismutase activity was the same in each test culture. These data support the role of intracellular catalase in protecting B. distasonis from oxidative damage resulting from hyperbaric oxygenation or H2O2 exposure. Catalase activity elicited by adding hemin to cells grown previously in medium lacking hemin was inhibited only 40% by prior incubation of the cells with chloramphenicol (30 micrograms/ml) and only 22% with rifampin (5 micrograms/ml). A model which is consistent with these data involves the production of an apocatalase in cells grown in low-hemin medium. Addition of hemin to the cells would result in a rapid chloramphenicolor rifampin-insensitive stimulation of catalase activity followed by further de novo biosynthesis of catalase.     

Comparative kinetic characteristics of catalase of Penicillium species molds

Extracellular catalases produced by fungi of the genus Penicillium: P. piceum, P. varians and P. kapuscinskii were purified by consecutive filtration of culture liquids. The maximum reaction rate of H2O2 decomposition, the Michaelis constants and specific catalytic activities of isolated catalases were determined. The operational stability was characterized by effective rate of catalase inactivation during enzymatic reaction (kin at 30 degrees C). The thermal stability was determined by the rate of enzyme thermal inactivation at 45 degrees C (k*[symbol: see text]H, s-1). Catalase from P. piceum displayed the maximum activity, which was higher than the activity of catalase from bovine liver. The operational stability of catalase from P. piceum was twofold to threefold higher than the stability of catalase from bovine liver. The physicochemical characteristics of catalases of fungi are better than the characteristics of catalase from bovine liver and intracellular catalase of yeast C. boidinii.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation

Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA beta-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the beta-oxidation system and individual enzymes of beta-oxidation (fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the beta-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and beta-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal beta-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA beta-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.     

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.