Antibacterial Activity of the Lactoperoxidase System in Milk Against Pseudomonads and Other Gram-Negative Bacteria

Products of thiocyanate oxidation by lactoperoxidase inhibit gram-positive bacteria that produce peroxide. We found these products to be bactericidal for such gram-negative bacteria as Pseudomonas species and Escherichia coli, provided peroxide is supplied exogenously by glucose oxidase and glucose. By the use of immobilized glucose oxidase the bactericidal agent was shown to be dialyzable, destroyed by heat and counteracted, or destroyed by reducing agents. Because the system is active against a number of gram-negative bacteria isolated from milk, it may possibly be exploited to increase the keeping quality of raw milk.     

Hydrogen peroxide production during experimental protein glycation

The accumulation of hydrogen peroxide (H2O2) during incubations of protein with glucose (experimental glycation) has previously been too low for direct measurement although it is suggested to be the precursor of protein-damaging hydroxylating agents. We have thus developed a simple H2O2-measuring technique which relies upon the rapid peroxide-mediated oxidation of Fe2+ to Fe3+ (catalysed by sorbitol) under acidic conditions followed by reaction of the latter cation with the dye, xylenol orange. We have used the method to demonstrate that incubation mixtures of protein and glucose generates nanomolar levels of hydrogen peroxide in the presence of protein under physiological conditions of pH and temperature.     

Melatonin inhibits the contractile effect of vanadate in the isolated pulmonary arterial rings of rats: possible role of hydrogen peroxide

The effect and possible mechanism of action of vanadate on the isolated pulmonary arterial rings of normal rats were studied. Pulmonary arterial rings contracted in response to vanadate (0.1-1 mM) in a concentration-dependent manner. Preincubation of the pulmonary arterial rings with 1 mM melatonin significantly reduced the contractile effect of vanadate by more than 60%. Furthermore, addition of hydrogen peroxide (50 microM) or enzymatic generation of hydrogen peroxide by the addition of glucose oxidase (10 U/mL) to the medium containing glucose produced remarkable increases in the pulmonary arterial tension, 46.2 +/- 7.3 and 78.7 +/- 9.7 g tension/g tissue, respectively. Similarly, incubation of the pulmonary arterial rings with 1 mM melatonin significantly reduced the contractile responses of the arterial rings to hydrogen peroxide and glucose/glucose oxidase to 25.7 +/- 2.9 and 24.7 +/- 4.4 g tension/g tissue, respectively. Vanadate, in vitro, significantly stimulated the oxidation of NADH by xanthine oxidase, and the rate of oxidation was increased by increasing either time or vanadate concentration. Similarly, addition of melatonin to a reaction mixture containing xanthine oxidase and vanadate significantly inhibited the rate of NADH oxidation in a concentration-dependent fashion. The results of the present study indicated that vanadate induced contraction in the isolated pulmonary arterial rings, which was significantly reduced by melatonin. Furthermore, the contractile effect of vanadate on the pulmonary arterial rings may be attributed to the intracellular generation of hydrogen peroxide.     

Decontamination of dental unit water systems with hydrogen peroxide

AIMS: Transmission of microbial pathogens to patients from water in dental units is a concern. To reduce this risk, the decontaminating efficiency of hydrogen peroxide was evaluated. METHODS AND RESULTS: Three percent hydrogen peroxide diluted 1 : 4 in distilled water (contact time 15 min) was used daily to disinfect the waterlines of a pilot unit previously contaminated with Pseudomonas aeruginosa or Staphylococcus aureus. The behaviour of the test bacteria was seen to differ over time. Staph. aureus numbers slowly decreased until only low numbers were recovered, after which the levels remained stable. Ps. aeruginosa abatement was more rapid and the density of the bacteria reached a peak when the circuit was empty. CONCLUSIONS: Staph. aureus and Ps. aeruginosa treated with hydrogen peroxide fell from 6 to 4 log. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of dental unit waterlines with hydrogen peroxide was seen to be able to keep the number of the bacteria under control, as long as the treatment was repeated daily.     

Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humour

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH₂. 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH₂.     

An immobilized two-enzyme system for the activation of the lactoperoxidase antibacterial system in milk

Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide and an intermediary product is formed with antibacterial properties. The components of this system, with the exception of hydrogen peroxide, are present in milk. H₂O₂ may be introduced by means of enzymatic, generation and thus make the system complete. A two-enzyme system consisting of β–galactosidase and glucose oxidase has been developed for this purpose. The coupled enzyme reaction is shown to work with high efficiency at the neutral pH of milk although the enzymes as such, particularly lactases suitable for immobilization, have optimal activities at much lower pH values. The results indicate that the lactoperoxidase system may in this way be employed to inactivate bacteria present in milk.     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

Methylene blue directly oxidizes glutathione without the intermediate formation of hydrogen peroxide

Methylene blue stimulates the oxidation of glutathione in red blood cells in vitro and in vivo. This oxidation has been attributed to hydrogen peroxide that is generated from the autooxidation of leucomethylene blue arising from the reduction of methylene blue by NADPH. In this report we present evidence that methylene blue directly oxidizes glutathione and that oxidation of glutathione by hydrogen peroxide is a secondary reaction. Moreover, superoxide dismutase has no effect on the oxidation. Under aerobic conditions, methylene blue oxidizes glutathione 30 times faster than the spontaneous autooxidation of glutathione. Under anaerobic conditions the stoichiometry of the reaction of methylene blue with glutathione supports a direct chemical reaction. The reaction rates between glutathione and methylene blue suggest a second order reaction over the conditions tested. That neither oxygen radical formation nor significant amounts of hydrogen peroxide are produced by methylene blue, even in the presence of added glucose, is further confirmed by the failure to detect significant amounts of lipid peroxidation products, or hemolysis, in red blood cells incubated with the dye.     

Microplate assay for detection of bacterial contamination in tissue culture media

In the present study, a rapid and simple colorimetric technique has been described to determine the presence of bacteria in tissue culture medium used in animal cell culture. The microplate assay is based on utilization of MTT [3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] by bacteria resulting in formation of formazan crystals which can be measured colorimetrically. Contaminated medium, a standard gram-negative and gram-positive bacteria produce formazan from MTT which is related to the bacterial load. The assay has utility in screening tissue culture reagents to detect the presence of bacteria.     

Use of Hydrogen Peroxide Treatment and Crystal Violet Agar Plates for Selective Recovery of Bacteriophages from Natural Environments

Hydrogen peroxide inactivated bacteriophages and bacteria at different rates. A concentration of 0.1% hydrogen peroxide reduced the numbers of several bacteria by an average of 94% but caused an average of 25% inactivation in the numbers of bacteriophages tested. Treating natural samples with hydrogen peroxide selectively reduced the indigenous bacterial flora and permitted better visualization of plaques of lawns of Escherichia coli C-3000. In some cases indigenous gram-positive bacteria were relatively resistant to hydrogen peroxide, but their growth could be limited by incorporation of crystal violet into the bottom agar used for plaque assays. The use of hydrogen peroxide treatment and crystal violet-containing plates permitted recovery of more phages from natural samples than did other procedures, such as chloroform pretreatment or the use of selective plating agar such as EC medium.     

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.     

The occurrence of denitrifying colourless sulphur-oxidising bacteria in marine waters and sediments as shown by the agar shake technique

Abstract The agar shake technique has been tested for the enumeration and isolation of bacteria involved in the anaerobic oxidation of reduced sulphur compounds. High numbers of colony forming units were observed from regions rich in sulphide, and the numbers of these forms were sometimes significantly correlated with the number of sulphate-reducing bacteria. The isolates could oxidise not only thiosulphate but also sulphide in liquid medium at the expense of nitrate. Addition of 1 mM glucose to the medium enhanced the rate and amount of thiosulphate oxidised by many of the isolates. Hence the use of the agar shake technique is recommended for the study of these little known facultatively or even obligately chemolithotrophic bacteria involved in the anaerobic oxidation of reduced inorganic sulphur compounds in the marine and estuarine environment.     

Polyvinyl pyridine metal complex as permanent antimicrobial finishing for viscose fabric

Viscose fabrics were treated with polyvinyl pyridine (PVP) using padding technique, followed by oxidation with hydrogen peroxide or peracetic acid, which was prepared by the reaction of tetra acetyl ethylene diamine (TAED) with hydrogen peroxide. Peracetic acid gives higher oxidation of PVP than hydrogen peroxide. FTIR study proved the formation of N-oxide as a result of oxidation. Incorporation of copper and silver ion onto oxidized PVP was also proved by FTIR. The antimicrobial study emphasise that Cu/oxidized PVP and Ag/oxidized PVP have retarded the growth of bacteria significantly, and Ag/oxidized PVP has a far better biocidal activity. The antibacterial activity of both metal ions survived after washing 10 times.     

Killing and Lysis of Gram-negative Bacteria Through the Synergistic Effect of Hydrogen Peroxide, Ascorbic Acid, and Lysozyme

A mixture of hydrogen peroxide and ascorbic acid has been found to generate an antibacterial mechanism which is active against gram-negative bacteria. It results in bacterial death and renders the organism sensitive to lysis by lysozyme. Under the conditions used, horseradish peroxidase did not augment the antibacterial effect. It is suggested that the effector mechanism involves the generation of short-lived free radicals which disturb the integrity of the cell wall. This effect alone might kill bacteria by interfering with selective permeability, but in the presence of lysozyme a further bactericidal activity is accomplished by complete disruption of the cell. It is proposed that a transient antibacterial system such as that described could exist within phagocytic cells. Free radicals would be formed through the interaction of certain oxidizable substances and hydrogen peroxide, which is produced during the enhanced metabolic activity that accompanies ingestion of bacteria. Such a system would help to explain why macrophages, which are apparently devoid of preformed bactericidins, are nonetheless very efficient in killing most phagocytosed bacteria.     

Effect of lectins from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability of lectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.     

The formation of hydrogen peroxide during the oxidation of reduced nicotinamide adenine dinucleotide by cytochrome o from Vitreoscilla.

The formation of hydrogen peroxide during the oxidation of NADH by purified preparations of cytochrome o has been demonstrated by employing three independent methods: polarographic, colorimetric, and fluorometric. The first two methods were used to assay for the accumulation of hydrogen peroxide and showed that hydrogen peroxide did accumulate as a product, but only about 30% of the oxygen consumed or 15 to 20% of the NADH oxidized was recoverable as hydrogen peroxide. This lack of 1:1 stoichiometry was not due to residual catalase activity in these preparations which could be eliminated by freeze-thawing. Thus, hydrogen peroxide may not be the sole or primary product of the NADH-cytochrome o oxidase reaction. The fluorometric assay could be coupled directly to the NADH-cytochrome o oxidase reaction in one medium, and this method showed that hydrogen peroxide was generated continuously from the beginning of the reaction in a 1:1 stoichiometry, hydrogen peroxide generated to NADH oxidized. This result suggests that hydrogen peroxide is an intermediate that can be trapped efficiently under the conditions of the fluorometric assay, whereas under the conditions of the first two assays most of the hydrogen peroxide generated undergoes further reaction. Exogenously added FAD or FMN increased the percentage of hydrogen peroxide that accumulated in the NADHcytochrome o oxidase reaction. Flavin is believed to act on the reductase side of cytochrome o so the increased percentage of hydrogen peroxide is not likely to result from the direct reaction of reduced flavin with oxygen.     

Campylobacter jejuni Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor

Expression of the peroxide stress genes alkyl hydroperoxide reductase (ahpC) and catalase (katA) of the microaerophile Campylobacter jejuni is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in C. jejuni, as these genes are still iron repressed in a C. jejuni fur mutant. An open reading frame encoding a Fur homolog (designated PerR for "peroxide stress regulator") was identified in the genome sequence of C. jejuni. The perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains. Subsequent characterization of the C. jejuni perR mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the perR mutant, fur mutant, and the fur perR double mutant showed that the perR mutation made C. jejuni hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium Bacillus subtilis. C. jejuni is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.     

Antimicrobial activities of some Bacillus spp. strains isolated from the soil

In this study a total of 29 Bacillus species isolated from the soil was analyzed using the agar diffusion method in terms of their general inhibition effects to some test bacteria. It has been found that isolates are effective against gram-positive and gram-negative bacteria whereas their extensive inhibition effect is particularly against gram-positive bacteria. On the other hand, B. cereus M15 strain has an inhibitory effect against both gram-positive and gram-negative bacteria. Furthermore some isolates are more effective against test bacteria when compared to some antibiotics.     

Optimization of activation conditions of Rhodobacter sphaeroides in hydrogen generation process

AIMS: To examine the effects of the culture age, illuminance intensity and changes in these parameters during activation on hydrogen generation process carried out by purple nonsulfur Rhodobacter sphaeroides bacteria. METHODS AND RESULTS: The following parameters were determined in all experiments: the amount of hydrogen evolved (measured using gas chromatography), biomass increase as dry mass, pH values and consumption of organic substance as chemical oxygen demand (COD). The medium used in the process of activation and hydrogen generation contained malic acid (15 mmol) and sodium glutamate (2 mmol). The optimum age of bacteria was 12-24 h and the best intensity of illuminance was found to be 5 cd sr m-2 on activation and 9 cd sr m-2 on hydrogen generation. These conditions provided hydrogen evolution of 1.39 l l-1 of the medium with the highest specific hydrogen production of 0.146 l H2 l-1 medium h-1 g-1 inoculum. An increase in the illuminance intensity resulted in a slight inhibition of the process. CONCLUSIONS: The activation stage of bacteria has a significant effect on the parameters of hydrogen photogeneration. The optimization of the activation stages allowed a shortening of the time of hydrogen generation and of the period after which hydrogen evolution starts. SIGNIFICANCE AND IMPACT OF THE STUDY: An innovative method of bacteria activation before the initiation of the hydrogen generation process has been used to optimize this process. The shortening of the process duration as well as the twice higher hydrogen yield can help in the designing of other systems (including also those operating under solar irradiation) in which R. sphaeroides bacteria are to be applied.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.