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 role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.     

The macromolecule with antimicrobial activity synthesized by <Emphasis Type="Italic">Pseudoalteromonas luteoviolacea</Emphasis> strains is an <Emphasis Type="SmallCaps">l</Emphasis>-amino acid oxidase

Two purple pigmented bacterial strains, CPMOR-1 and CPMOR-2, have been newly isolated from the Mediterranean Sea. 16S RNA sequencing and phenotypic characteristics indicate that they belong to the species Pseudoalteromonas luteoviolacea. The synthesis of macromolecules with antimicrobial activity is a capacity described in many strains of this species although the nature of those macromolecules has not been reported up to now. The search for antimicrobial compounds in the two new strains described in this work shows that they synthesize a macromolecule with antimicrobial activity that can be inhibited by catalase, as it had been described in the type strain P. luteoviolacea NCIMB 1893(T). This work elucidates the nature of such macromolecule as a novel L-amino acid oxidase (LAO) with broad substrate specificity. The enzyme is most active with Met, Gln, Leu, Phe, Glu, and Trp. In growth media containing those amino acids, the hydrogen peroxide generated by the reaction catalyzed by the LAO mediates its antimicrobial activity.     

The antibacterial activity of honey derived from Australian flora

Chronic wound infections and antibiotic resistance are driving interest in antimicrobial treatments that have generally been considered complementary, including antimicrobially active honey. Australia has unique native flora and produces honey with a wide range of different physicochemical properties. In this study we surveyed 477 honey samples, derived from native and exotic plants from various regions of Australia, for their antibacterial activity using an established screening protocol. A level of activity considered potentially therapeutically useful was found in 274 (57%) of the honey samples, with exceptional activity seen in samples derived from marri (Corymbia calophylla), jarrah (Eucalyptus marginata) and jellybush (Leptospermum polygalifolium). In most cases the antibacterial activity was attributable to hydrogen peroxide produced by the bee-derived enzyme glucose oxidase. Non-hydrogen peroxide activity was detected in 80 (16.8%) samples, and was most consistently seen in honey produced from Leptospermum spp. Testing over time found the hydrogen peroxide-dependent activity in honey decreased, in some cases by 100%, and this activity was more stable at 4 °C than at 25 °C. In contrast, the non-hydrogen peroxide activity of Leptospermum honey samples increased, and this was greatest in samples stored at 25 °C. The stability of non-peroxide activity from other honeys was more variable, suggesting this activity may have a different cause. We conclude that many Australian honeys have clinical potential, and that further studies into the composition and stability of their active constituents are warranted.     

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A2B2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s-1 and the Km for hydrogen peroxide was 16 mM. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.     

A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honey.

Both honey and sugar are used with good effect as dressings for wounds and ulcers. The good control of infection is attributed to the high osmolarity, but honey can have additional antibacterial activity because of its content of hydrogen peroxide and unidentified substances from certain floral sources. Manuka honey is known to have a high level of the latter. Seven major wound-infecting species of bacteria were studied to compare their sensitivity to the non-peroxide antibacterial activity of manuka honey and to a honey in which the antibacterial activity was primarily due to hydrogen peroxide. Honeys with activity in the middle of the normal range were used. A comparison of the median response of the various species of bacteria showed no significant difference between the two types of activity overall, but marked differences between the two types of activity in the rank order of sensitivity of the seven bacterial species. The non-peroxide antibacterial activity of manuka honey at a honey concentration of 1.8% (v/v) completely inhibited the growth of Staphylococcus aureus during incubation for 8 h. The growth of all seven species was completely inhibited by both types of honey at concentrations below 11% (v/v).     

A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honey

Both honey and sugar are used with good effect as dressings for wounds and ulcers. The good control of infection is attributed to the high osmolarity, but honey can have additional antibacterial activity because of its content of hydrogen peroxide and unidentified substances from certain floral sources. Manuka honey is known to have a high level of the latter.
Seven major wound-infecting species of bacteria were studied to compare their sensitivity to the non-peroxide antibacterial activity of manuka honey and to a honey in which the antibacterial activity was primarily due to hydrogen peroxide. Honeys with activity in the middle of the normal range were used. A comparison of the median response of the various species of bacteria showed no significant difference between the two types of activity overall, but marked differences between the two types of activity in the rank order of sensitivity of the seven bacterial species. The non-peroxide antibacterial activity of manuka honey at a honey concentration of 1.8% (v/v) completely inhibited the growth of Staphylococcus aureus during incubation for 8 h. The growth of all seven species was completely inhibited by both types of honey at concentrations below 11% (v/v).     

Role of catalase and superoxide dismutase in the yeast Saccharomyces cerevisiae response to hydrogen peroxide in exponential phase of growth

The role of catalase and superoxide dismutase (SOD) in response of the yeast Saccharomyces cerevisiae to oxidative stress induced by hydrogen peroxide in the middle-exponential phase has been investigated. It was shown that cell survival is significantly decreased after yeast exposure to hydrogen peroxide in the strains defective in cytosolic or peroxisomal catalases. Treatment of the wild-type cells with 0.5 mM H2O2 for 30 min causes an increase in the activity of catalase and superoxide dismutase, but the effect was not observed in all strains investigated. It was also shown that hydrogen peroxide leads to an increase in the activities of both catalases and Cu,Zn-containing SOD. The effect was cancelled by cycloheximide, an inhibitor of protein synthesis.     

The recA+ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity.

Various deoxyribonucleic acid repair-deficient strains of Escherichia coli K-12 were exposed to hydrogen peroxide under anaerobic conling of the strains was determined. The level of catalase, peroxidase, and superoxide dismutase in cell-free extracts of the strains as well as the capacity of intact cells to decompose hydrogen peroxide were assayed, recA strains were more rapidly killed than other strains with deoxyribonucleic acid repair deficiencies. There was no correlation between the killing rate of the strains and the capacity of intact cells to decompose hydrogen peroxide or the level of catalase and superoxide dismutase in cell-free extracts. The level of peroxidase in cell-free extract was too low to be determined.     

Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.     

Polarographic Assay of Hydrogen Peroxide Accumulation in Microbial Cultures

A method is described for determining low concentrations of hydrogen peroxide by using a polarographic oxygen electrode to measure the oxygen released into solution on addition of catalase. A sample can be assayed directly without prior manipulation in 3 min. The method is capable of assaying hydrogen peroxide concentrations as low as 7 μM. The method has proved extremely useful for the assay of hydrogen peroxide secreted into milk by lactic acid bacteria.     

A note on the use of the Catalasemetre in assessing the quality of milk

The Catalasemetre, for assessing the quality of raw and pasteurized milk, has been studied. No correlation was found between catalase activity and bacterial counts for farm bulk tank milks within the range 5.2 ± 10²-5.4 ± 10⁵ cfu/ml. Similarly, no relation was observed between catalase activity and somatic cell counts of milk (range of counts from 0.08 to 3.5). However, the catalase activity and bacterial count of pasteurized milks which had been pre-incubated at 21.C for 25 h in the presence of crystal violet-penicillin-nisin to inhibit Gram-positive bacterial growth were significantly related. Thus, the use of this pre-incubation procedure coupled with the Catalasemetre to estimate bacterial growth, has potential in assessing the keeping quality of pasteurized milk samples within 25.5 h of production. Results on the thermostability of native milk catalase are also presented.     

Regulation of catalase synthesis in Salmonella typhimurium.

The specific activity of catalase in Salmonella typhimurium and other enteric bacteria decreased during the logarithmic phase of growth and increased at the onset and during the stationary phase. The increase in catalase synthesis at the end of the exponential phase in S. typhimurium cells coincided with the lowest pH value reached by the culture. Maintenance of the pH at a constant neutral value did not alter the typical pattern of synthesis in contradiction of the results previously reported (McCarthy and Hinshelwood. 1959). A sudden decrease in the pH value of an S. typhimurium culture during exponential growth by addition of HC1 did not cause an alteration in the catalase synthesis pattern. Addition of hydrogen peroxide to S. typhimurium cultures within the range 1 muM TO 2MM during the exponential growth phase stimulated catalase synthesis. The extent of catalase synthesis depended on the concentration of hydrogen peroxide; the maximum stimulation was observed at 80 muM. Increased catalase synthesis was not detected for 10 to 15 min after hydrogen peroxide addition. Hydrogen peroxide was produced by S. typhimurium cultures during the exponential and stationary growth phases. However, no direct relationship between hydrogen peroxide accumulation and synthesis of catalase was observed.     

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk

Raw milk samples were stored for 1-4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.     

Role of Acinetobacter calcoaceticus 3,4-dihydrocoumarin hydrolase in oxidative stress defence against peroxoacids.

The physiological role of a bifunctional enzyme, 3,4-dihydrocoumarin hydrolase (DCH), which is capable of both hydrolysis of ester bonds and organic acid-assisted bromination of organic compounds, was investigated. Purified DCH from Acinetobacter calcoaceticus F46 catalysed dose- and time-dependent degradation of peracetic acid. The gene (dch) was cloned from the chromosomal DNA of the bacterium. The dch ORF was 831 bp long, corresponding to a protein of 272 amino acid residues, and the deduced amino acid sequence showed high similarity to those of bacterial serine esterases and perhydrolases. The dch gene was disrupted by homologous recombination on the A. calcoaceticus genome. The dch disruptant strain was more sensitive to growth inhibition by peracetic acid than the parent strain. On the other hand, the recombinant Escherichia coli cells expressing dch were more resistant to peracetic acid. A putative catalase gene was found immediately downstream of dch, and Northern blot hybridization analysis revealed that they are transcribed as part of a polycistronic mRNA. These results suggested that in vivo DCH detoxifies peroxoacids in conjunction with the catalase, i.e. peroxoacids are first hydrolysed to the corresponding acids and hydrogen peroxide by DCH, and then the resulting hydrogen peroxide is degraded by the catalase.     

The Microaerophile SPirillum volutans: Cultivation on Complex Liquid and Solid Media

Spirillum volutans grows only under microaerobic conditions in a peptone-succinate-salts broth, but can grow aerobically when the peptone is replaced by vitamin-free acid-hydrolyzed casein broth. The addition of potassium metabisulfite, norepinephrine, catalase or superoxide dismutase (SOD) permitted aerobic growth in peptone-succinate-salts broth. A combination of catalase and SOD had a synergistic effect. S. volutans lacked catalase and had only a low level of peroxidase activity, but did possess SOD activity (12 to 14 U/mg of protein). The organism was found to be extraordinarily sensitive to exogenous hydrogen peroxide. Illumination of peptone-succinate-salts broth generated hydrogen peroxide and rendered the medium inhibitory to growth. A combination of catalase and SOD prevented this inhibition. Growth of S. volutans on solid media, not previously possible, was accomplished by the use of vitamin-free acid-hydrolyzed casein and peptone-succinate-salts agar media; maximum growth responses were dependent on the following combination of factors: addition of bisulfite, catalase, or SOD, protection of the media from illumination, incubation in a highly humid atmosphere, and incubation under atmospheres of 12% oxygen or less. The results indicate that the microaerophilic nature of S. volutans is attributable largely to the high sensitivity of the organism to exogenous hydrogen peroxide and, to a lesser extent, superoxide radicals occurring in the culture medium.     

Utilization of methanol by a catalase-negative mutant of Hansenula polymorpha

Summary In methanol-utilizing yeasts, catalase is an essential enzyme for the destruction of hydrogen peroxide generated by methanol oxidase (E.C. 1.1.3.13). It was found however that a catalase-negative mutant of Hansenula polymorpha is able to consume methanol in the presence of glucose in continuous cultures. At a dilution rate of 0.1 h^-1, stable continuous cultures could be obtained during growth on methanol/glucose mixtures with a molar ratio of methanol/glucose between 0 to 2.4. In these cultures methanol oxidase was induced up to a level of 40% of that obtained in the wild-type strain. The hydrogen peroxide-decomposition activity of the mutant was studied in more detail by pulsing methanol to samples of steady-state cultures. Only after the addition of excess methanol the hydrogen peroxide-decomposing system became saturated, and the cells excreted hydrogen peroxide. This was accompanied by excretion of formaldehyde and a rapid loss of viability. The presence of extracellular catalase during a methanol pulse prevented the loss of viability. The nature of the alternative hydrogen peroxide-decomposing enzyme system remains to be elucidated. Its capacity strongly depended on the cultivation conditions and pretreatment of the cells. Cells grown on formaldehyde/glucose mixtures showed a lower methanol tolerance than those grown on the methanol/glucose mixtures. Freeze-drying of cells drastically enhanced the excretion of hydrogen peroxide, probably as a result of an inactivation of the decomposing system.     

Relation of Catalase to Substrate Utilization by Mycoplasma pneumoniae

No catalase activity was detected in four strains of glucose-grown Mycoplasma pneumoniae at any time during the replication of the organism. Exogenous catalase dramatically increased the O(2) uptake with glycerol, presumably by releasing inhibition caused by hydrogen peroxide. The effect of added catalase on the O(2) uptake of washed organisms with glucose as substrate was moderate and variable in degree. The production of hydrogen peroxide was demonstrated by the quantitative enzymatic assay for inorganic peroxide and by the fact that added pyruvate, which is non-enzymatically oxidized by H(2)O(2) to acetic acid and CO(2) could mimic the action of catalase.     

Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity

A number of catalase-deficient mutants of Escherichia coli which exhibit no assayable catalase activity were isolated. The only physiological difference between the catalase mutants and their parents was a 50- to 60-fold greater sensitivity to killing by hydrogen peroxide. For comparison, mutations in the xthA and recA genes of the same strains increased the sensitivity of the mutants to hydrogen peroxide by seven- and fivefold, respectively, showing that catalase was the primary defense against hydrogen peroxide. One class of mutants named katE was localized between pfkB and xthA at 37.8 min on the E. coli genome. A second class of catalase mutants was found which did not map in this region.     

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk

Raw milk samples were stored for 1–4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.