A novel micellar formulation based on natural plant extracts enhances the efficacy of hydrogen peroxide against biofilms of Staphylococcus spp. and Pseudomonas aeruginosa

Abstract

The antibacterial efficacy of hydrogen peroxide encapsulated in micelles (mH₂O₂) against biofilms was compared with that of hydrogen peroxide alone and of three commercially available aqueous biocides. The activity of mH₂O₂ on 24-h biofilms of reference strains of Staphylococcus spp. and Pseudomonas aeruginosa was tested in a static microtiter plate model. The biofilms were incubated with mH₂O₂ (17% v/v H₂O₂, 2% lactic acid, 0.3% phytoextract, H₂O) and its individual ingredients and compared with three aqueous biocides at different concentrations and times of exposure. After 5-min exposure, 10% mH₂O₂ (corresponding to 1.7% v/v H₂O₂) achieved > 8 log₁₀ reductions against all the test strains, while 1.7% H₂O₂ achieved a maximum of 1.5 log₁₀ reduction. After 5-min exposure, none of the commercially available biocides tested showed themselves to be capable of completely eliminating the test strains embedded in biofilms. Hydrogen peroxide encapsulated in micelles demonstrated enhanced activity against planktonic cells and biofilms of Staphylococcus spp. and P. aeruginosa.     

Ligninolytic enzymes activities of Oyster mushrooms cultivated on OMW (olive mill waste) supplemented media, spawn and substrates

Ligninolytic enzymes activities (laccases, peroxidases (total, MnP and MiP) and aryl-alcohol oxidase (AAO)) were measured during the cultivation of six commercial Pleurotus sp. strains on MMP media, on cereal grains (spawn) and on straw substrates (the three commonly utilized cultivation steps to obtain fruiting bodies) supplemented with several concentrations of autoclaved (OMW) or gamma-irradiated (iOMW) olive mill waste. Results indicated that all the strains were able to grow on MMP media and spawn containing up to 30% OMW and iOMW and on straw substrates mixed with 50% OMW. None of the strains showed AAO activity and there was not a single strain which showed the highest laccases and peroxidases activities, independently of the utilized substrate. Pleurotus mycelia adjusted their enzymatic mechanisms depending on their variety, type of substrate, concentration of OMW or iOMW added. OMW was a better supplement to use than iOMW because OMW induced higher exo-enzymes activities.     

Decolorization of melanin by lignin peroxidase from<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Melanin was decolorized by lignin peroxidase fromPhanerochaete chrysosporium. This decolorization reaction showed a Michaelis-Mentens type relationship between the decolorization rate and concentration of two substrates: melanin and hydrogen peroxide. Kinetic constants of the decolorization reaction were 0.1 OD₄₇₅/min (V _max) and 99.7 mg/L (K _m) for melanin and 0.08 OD₄₇₅/min (V _max) and 504.9 μM (K _m) for hydrogen peroxide, respectively. Depletion of hydrogen peroxide interrupted the decolorization reaction, indicating the essential requirement of hydrogen peroxide. Pulsewise feeding of hydrogen peroxide continued the decolorizing reaction catalyzed by lignin peroxidase. These results indicate that enzymatic decolorization of melanin has applications in the development of new cosmetic whitening agents.     

Intrinsic and inducible resistance to hydrogen peroxide in <Emphasis Type="Italic">Bifidobacterium</Emphasis> species

Interest in, and use of, bifidobacteria as a probiotic delivered in functional foods has increased dramatically in recent years. As a result of their anaerobic nature, oxidative stress can pose a major challenge to maintaining viability of bifidobacteria during functional food storage. To better understand the oxidative stress response in two industrially important bifidobacteria species, we examined the response of three strains of B. longum and three strains of B. animalis subsp. lactis to hydrogen peroxide (H₂O₂). Each strain was exposed to a range of H₂O₂ concentrations (0–10 mM) to evaluate and compare intrinsic resistance to H₂O₂. Next, strains were tested for the presence of an inducible oxidative stress response by exposure to a sublethal H₂O₂ concentration for 20 or 60 min followed by challenge at a lethal H₂O₂ concentration. Results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H₂O₂ resistance of all strains tested and B. animalis subsp. lactis BL-04 had the highest resistance among B. lactis strains. Inducible H₂O₂ resistance was detected in four strains, B. longum NCC2705, B. longum D2957, B. lactis RH-1, and B. lactis BL-04. Other strains showed either no difference or increased sensitivity to H₂O₂ after induction treatments. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific in B. longum and B. lactis and suggest that for some strains, sublethal H₂O₂ treatments might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.     

Effect of hydrogen peroxide on antioxidant enzyme activities in Saccharomyces cerevisiae is strain-specific

The effect of hydrogen peroxide on the survival and activity of antioxidant and associated enzymes in Saccharomyces cerevisiae has been studied. A difference found in the response of wild-type yeast strains treated with hydrogen peroxide was probably related to the different protective effects of antioxidant enzymes in these strains. Exposure of wild-type YPH250 cells to 0.25 mM H₂O₂ for 30 min increased activities of catalase and superoxide dismutase (SOD) by 3.4-and 2-fold, respectively. However, no activation of catalase in the EG103 strain, as well as of SOD in the YPH98 and EG103 wild strains was detected, which was in parallel to lower survival of these strains under oxidative stress. There is a strong positive correlation (R ² = 0.95) between activities of catalase and SOD in YPH250 cells treated with different concentrations of hydrogen peroxide. It is conceivable that catalase would protect SOD against inactivation caused by oxidative stress and vice versa. Finally, yeast cell treatment with hydrogen peroxide can lead to either a H₂O₂-induced increase in activities of antioxidant and associated enzymes or their decrease depending on the H₂O₂ concentration used or the yeast strain specificity. Published in Russion in Biokhimiya, 2006, Vol. 71, No. 9, pp. 1243–1252.     

Required characteristics of Paenibacillus polymyxa JB-0501 as potential probiotic

The ability of Paenibacillus polymyxa to inhibit the growth of Escherichia coli generic ATCC 25922 (Escherichia coli ATCC 25922) and to adhere to monolayers of the enterocyte-like human cell line Caco-2 was evaluated. P. polymyxa JB-0501 (P. polymyxa JB-0501), found in a livestock feed probiotic supplement, was compared to P. polymyxa reference strain ATCC 43685 and ATCC 7070 (P. polymyxa ATCC) in terms of carbohydrate utilization and resistance to lysozyme, acid, bile salts, and hydrogen peroxide. JB-0501 grew at pH 4.5 and at H₂O₂ concentrations less than 7.3 μg/ml and presented a higher affinity to hexadecane and decane. Bile salts at 0.2 % inhibited the growth of all three strains. P. polymyxa JB-0501 and P. polymyxa ATCC 43865 adhered to Caco-2 cell monolayers. The percentage of cells that adhered ranged from about 0.35 to 6.5 % and was partially proportional to the number applied. Contact time (from 15 min to 1 h) had little impact on adhesion. P. polymyxa JB-0501 inhibited the growth of E. coli ATCC 25922, as proven by the diffusion tests in agar. Taken together, these results suggested that P. polymyxa JB-0501 has the potential probiotic properties to justify its consideration as a livestock feed supplement.     

Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. Strains with different virulence

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H₂O₂) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H₂O₂ concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H₂O₂ production by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H₂O₂ concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.     

The study of the effects of carbon dioxide-induced elevation of hydrogen peroxide toxicity on microbes as a novel tool for water purification

The supercritical concentration of CO₂ (SCCO₂) and a high concentration (3.0%) of molecules of hydrogen peroxide (H₂O₂) are currently being used as antiseptic and antibacterial agents. The fact that low concentrations of CO₂ have an activation effect on functional activity of microbes allows us to predict that CO₂ could elevate the toxic effect of H₂O₂ on cells. To check this hypothesis the dependency of the toxic effect of H₂O₂ on wild type of Escherichia coli K-12 on soluble concentration of CO₂ in culture media was studied. The obtained data show that culture media enriched with CO₂ leads to the increase of toxic effect of H₂O₂ on microbes at both cases when pH is constant and when it changes. So CO₂ in non-supercritical concentration could elevate the toxic effect of H₂O₂ on microbes by the activation of the metabolic processes in microbes. During the experiments we used classical microbiological methods (indirect viable cell counts or counting colony forming units (CFUs)), as well as the method of measuring hydrogen peroxide content in aqueous solution by means of enhanced chemiluminescence method in a peroxidase-luminol-p-iodophenol system. This discovery is concerning to use CO₂/H₂O₂ combination system, which could have implication in the inhibition of growth of microbes in water and the microbiological monitoring of water could provide valuable information for managing the health of exhibition of aqua ecosystems.     

The Screening of Hydrogen Peroxide-Producing Lactic Acid Bacteria and Their Application to Inactivating Psychrotrophic Food-Borne Pathogens

Lactic acid bacteria were isolated from various food samples and evaluated for hydrogen peroxide (H₂O₂) production. Cells suspended in 0.5% (wt/vol) glucose plus 0.5% (wt/vol) lactate (pH 7.0) were incubated for 5 h at 37°C under aeration. Among 193 strains, 27 strains accumulated 201-300 ppm H₂O₂, and 4 strains accumulated more than 301 ppm H₂O₂ in the cell suspensions. Among the 9 high-level H₂O₂-producing strains, 8 strains were identified as Lactococcus lactis subsp. lactis. The cell-free filtrate from Lc. lactis subsp. lactis AI 62, which contained approximately 350 ppm H₂O₂, was evaluated for antimicrobial activity against Enterococcus faecalis, Ent. faecium, enterotoxigenic Escherichia coli, Listeria ivanovii, Staphylococcus aureus, Yersinia enterocolitica, and Aeromonas hydrophila. After 1 h incubation at 30°C in the cell-free filtrate, the initial viable cell counts of the target bacteria (5.53–6.00 log cfu/mL) were reduced by 0.12-5.00 log units, except in the case of enterococci. The sensitivity varied with the bacterial species and pH. The enterococci were resistant to the treatment. Our results show that H₂O₂ accumulated by lactic acid bacteria in a cell suspension is very effective in reducing the viable cell count of food-borne pathogens.Received: 7 October 2002 / Accepted: 4 November 2002     

Localization changes of endogenous hydrogen peroxide during cell division cycle of Xanthomonas

Production and localization of endogenous hydrogen peroxide (H₂O₂) were investigated in strains of Xanthomonas by histochemical analysis under electron microscopy. Even though the levels of endogenous H₂O₂ production were different among various strains, the produced H₂O₂ was localized in the cell wall of all Xanthomonas strains tested. The impairment of the level of endogenous H₂O₂ accumulation resulted in a significantly decreased growth rate of bacteria, regardless if the difference of the H₂O₂ level is originally present between wild type strains or caused by mutation of the ahpC gene of Xanthomonas. The endogenous accumulation of H₂O₂ positively correlates with the cell division. Interestingly, the accumulated H₂O₂ was also localized in the mesosome-like structure and nucleoids during the cell division cycle. Furthermore, results revealed quantitative and dimensional changes of H₂O₂ accumulation in the two additional locations. These findings indicated that the additional locations of the accumulated H₂O₂ were closely associated with the process of cell division. Together, these results suggest that the endogenous H₂O₂ production plays an important role in cell proliferation of Xanthomonas.     

Metabolism of hydrogen peroxide in univoltine and polyvoltine strains of silkworm (Bombyx mori)

Recent work has demonstrated that hydrogen peroxide functions as a signaling molecule controlling different essential processes in plants and mammals, which can be produced by superoxide dismutase (SOD) and xanthine oxidase (XO) and decomposed by catalase (CAT), respectively. Progeny diapause of the silkworm, Bombyx mori, is induced by diapause hormone (DH) and the expression of DH gene in the maternal generation has been determined. In order to investigate the relationship between the metabolism of H₂O₂ and the expression of DH gene, level of H₂O₂ and activities of SOD, XO and CAT between univoltine and polyvoltine strains, which can produce diapause and non-diapause eggs, respectively, at embryonic and pupal stages were measured. Our results showed that there were significant differences in the metabolism of hydrogen peroxide between two strains and between embryonic and pupal stages. Compared to polyvoltine strain, level of hydrogen peroxide in univoltine strain was significantly higher from stage 19 to stage 21 but lower from stage 24 to stage 29 and the whole pupal stage (Fig. 1). Variations of hydrogen peroxide indicated that hydrogen peroxide may be involved in the active release of DH and the progeny diapause decision by DH rather than the expression of DH gene.     

Yeasts isolated from olive mill wastewaters from southern Italy: technological characterization and potential use for phenol removal

Olive mill wastewater (OMW) samples from two traditional varieties (Peranzana and Ogliarola Garganica) of Apulian region (southern Italy) and produced through continuous and traditional methods were microbiologically and chemically examined; thus, 104 yeasts were isolated and selected for further analyses. The strains were identified as Candida boidinii, Pichia holstii, Pichia membranifaciens, and Saccharomyces cerevisiae and analyzed to assess their suitability to metabolize phenols. Based on phenol metabolism, 27 strains were selected and inoculated into OMW aliquots to determine their ability to reduce phenols in vivo; then, five strains (identified with the codes 682—C. boidinii and 625, 642, 647, and 941—P. holstii) were used as a cocktail in wastewaters for a final validation step. In this last experiment, the effects of the temperature (10–30°C) and (NH₄)₂SO₄ (0.0–6.0 g l⁻¹) were studied through a central composite design approach, and the results highlighted that the cocktail was able to reduce phenols by 40% at 10°C with 6.0 g l⁻¹ of (NH₄)₂SO₄ added.     

Hydrogen-peroxide-producing system ofPleurotus eryngii involving the extracellular enzyme aryl-alcohol oxidase

The effect of benzyl alcohol, benzaldehyde and benzoic acid on the production of extracellular hydrogen peroxide (H₂O₂) by the ligninolytic fungusPleurotus eryngii was investigated. It was found that an equilibrium between oxidative and reductive reactions of these compounds is established, leading to the continuous production of H₂O₂. A multienzymatic cyclic system is proposed in which H₂O₂ is produced extracellularly by the action of aryl-alcohol oxidase on benzyl alcohol, the most abundant compound after redox reactions, and to a lower extent on benzaldehyde. The oxidation products of these reactions, benzaldehyde and benzoic acid, are reduced by intracellular dehydrogenases.     

Effect of Prototheca zopfii on neutrophil function from bovine milk

This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H₂O₂) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative “California Mastitis Test” (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H₂O₂ production was measured using the phenol red method. Catalase activity was measured following H₂O₂ reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey’s test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H₂O₂ production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.     

Highly Specific Culture-Independent Detection of YGNGV Motif-Containing Pediocin-Producing Strains

A novel method based on (1) initial microbiological screening and (2) a highly specific PCR is described for selection of strains expressing YGNGV motif-containing pediocin. Initial screening is carried out using spot on the lawn assay for selection of acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity producing strains. This is followed by highly specific PCR for amplification of 406-bp fragment using forward primer: 5′-tggccaatatcattggtggt-3′ targeting signal peptide sequence of pediocin structural gene and reverse primer: 5′-ctactaacgcttggctggca-3′ encoding N-terminus of immunity gene. The assay was validated with Pediococcus pentosaceus NCDC273 and Pediococcus acidilactici NCDC252 using (1) digestion of amplified 406-bp fragment with HindIII restriction enzyme-producing two restriction fragments of expected sizes (227 and 179 bp), (2) nucleotide sequencing of 406-bp fragment from both strains found these pediocins identical to pediocin PA-1/AcH and (3) identification of both pediocins as pediocin PA-1 at protein level using RP-HPLC. The assay was used for screening six strains (3 pediococci, 2 lactobacilli and an Enterococcus faecium) producing acid-free, hydrogen peroxide (H₂O₂)-free and secreted heat-stable inhibitory activity. This resulted in the detection of three new strains (P. pentosaceus NCDC35, E. faecium NCDC124 and Lactobacillus plantarum NCDC20) producing YGNGV motif-containing pediocins.     

Hydrogen peroxide in artificial photosynthesizing systems

From the point of view of the concepts of hydrogen peroxide as a source of photosynthetic oxygen (hydrogen) coordination and photochemical properties of chlorophyll and its aggregates towards hydrogen peroxide were considered. The binding energy of H₂O and H₂O₂ with chlorophyll and chlorophyllide depending on their form (monomers, dimers and trimers) was estimated by quantum chemical calculations. It is shown that at an increase of the degree of the pigment aggregation binding energy of H₂O₂ was more than the energy of H₂O. Analysis of experimental results of the photochemical decomposition of hydrogen peroxide using chlorophyll was carried out. Estimates of the thermodynamic parameters (ΔG° and ΔH°) of the formation of organic compounds from CO₂ with water and hydrogen peroxide were compared. The interaction of CO₂ with H₂O₂ requires much less energy consumption than with water for all considered cases. The formation of organic products (formaldehyde, alcohols, carboxylic and carbonylic compounds) and simultaneous production of O₂ under the influence of visible light in the systems of inorganic carbon-hydrogen peroxide - chlorophyll (phthalocyanine) is detected by GC/MS method, FTIR spectroscopy, and chemical analysis.     

Deposition pattern of hydrogen peroxide in the leaf sheaths of rice under salt stress

Deposition pattern of hydrogen peroxide (H₂O₂) under salt stress (100 mM NaCl) was examined cytochemically in rice (Oryza sativa L. cv. Pokkali) through the reaction of H₂O₂ with cerium chloride (CeCl₃) to produce electron dense precipitates of cerium perhydroxide. The distribution pattern of cerium perhydroxide precipitates in leaf sheath was considerably different from other parts of rice under salinity stress. Cerium perhydroxide precipitates were mainly accumulated on the tonoplast of leaf sheath under salinity, although they were localized on the cell wall and plasma membrane in all other tissues such as leaf blade and root.     

Hydrogen production from aromatic acids byRhodopseudomonas palustris

The capability of five strains of the phototrophic bacteriumRhodopseudomonas palustris to produce molecular hydrogen (H₂) from the aromatic acids benzoate,p-hydroxybenzoate, cinnamate and D- and L-mandelate was investigated. Optimal H₂ production was achieved when the strains were grown anaerobically in the light at 10,000 lx under nitrogen (N) limitation using 1 mM L-glutamate as an N source. In the presence of 2 mM benzoate or L-mandelate as carbon and electron sources, strain DSM 131 produced 45% H₂ of the maximal theoretical value and strain F2 32%, respectively. Increased H₂ production correlated with increased nitrogenase activities, but H₂ formation was not further stimulated by inhibition of the H₂ uptake (hup) hydrogenase with ethylenediaminetetraacetic acid (EDTA).     

Screening of white-rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil

Soil samples from an agricultural field contaminated with 10 ppm¹⁴C-benz(a)anthracene in glass tubes were brought into contact with cultures of wood-rotting fungi, precultivated on wheat straw substrate. Forty-five strains of white-rot fungi and four brown-rot fungi were tested for their ability to colonize the soil and to mineralize¹⁴C-benz(a)anthracene to¹⁴CO₂ within a 20-week incubation time. Twenty-two white-rot fungi and all brown-rot fungi were unable to colonize the soil. Twenty-three strains of white-rot fungi, all belonging to the genusPleurotus, colonized the soil. During the experiment the noncolonizing fungi and their substrate disintegrated more and more to a nonstructured pulp from which water diffused into the soil. The same phenomenon was observed in the control which contained only straw without fungus and contaminated soil. In samples with colonizing fungi the substrate as well as the mycelia in the soil remained visibly unchanged during the entire experiment. Surprisingly, most samples with fungi not colonizing the soil and the control without fungus liberated between 40 and 58 % of the applied radioactivity as¹⁴CO₂ whereas the samples with the colonizing fungi respired only 15–25 % as¹⁴CO₂. This was 3–5 times more¹⁴CO₂ than that liberated from the control (4.9 %) which contained only contaminated soil without straw and fungus. A similar result was obtained with selected colonizing and noncolonizing fungi and soil contaminated with 10 ppm¹⁴C-pyrene. However, in pure culture studies in which¹⁴C-pyrene was added to the straw substrate,Pleurotus sp. (P2), as a representative of the colonizing fungi, mineralized 40.3 % of the added radioactivity to¹⁴CO₂. The noncolonizing fungiDichomitus squalens andFlammulina velutipes liberated only 17.2 or 1.7 %, respectively, as¹⁴CO₂. These results lead to the hypothesis that the native soil microflora stimulated by the formed products of straw lysis is responsible for high degradation rates found with noncolonizing fungi.     

Antioxidant Lactobacilli Could Protect Gingival Fibroblasts Against Hydrogen Peroxide: A Preliminary In Vitro Study

Oxidative stress and tissue destruction are at the heart of periodontal diseases. The dental research area is geared toward the prevention of free radicals by nutrient antioxidants. Lactic acid bacteria (LAB) have recently attracted attention in alternative dental therapies. We aimed at highlighting the antioxidative property of Lactobacilli and Bifidobacterium strains and at determining their protective effect on gingival fibroblasts (GFs). Two Lactobacilli and 2 Bifidobacterium strains were screened for their exopolysaccharide (EPSs) production. Antioxidative assays were conducted by spectrophotometer analysis. Resistance to different concentrations of hydrogen peroxide (H₂O₂) was determined by the serial dilution technique. The protective effect of strains on GFs on hydrogen peroxide exposure was also examined by a new trypan blue exclusion assay method. Bifidobacterium breve A28 showed the highest EPS production (122 mg/l) and remarkable antioxidant activity, which were demonstrated by its ability to scavenge 72 % α,α-diphenyl-1-picrylhydrazyl free radical and chelate 88 % of iron ion, respectively. Inhibition of lipid peroxidation was determined as 71 % for the A28 strain. We suggest that LAB with antioxidative activity could be a good natural therapy agent for periodontal disorders.