Characterisation of physicochemical parameters and antibacterial properties of New Caledonian honeys.

Honey is an attractive natural product with various health benefits. A few honey-based commercial products have successfully been adopted in clinics to improve wound healing. However, screening of other potential sources of medical-grade honey, in particular, honeys from territories with high floral species diversity and high endemicity, is highly needed. The goal of this study was to characterise the physicochemical and antibacterial properties of New Caledonian honey samples (n = 33) and to elucidate the major mechanism of their antibacterial action. Inhibitory antibacterial activity of honeys against Staphylococcus aureus and Pseudomonas aeruginosa was determined with a minimum inhibitory concentration (MIC) assay. Enzymatic activity of glucose oxidase and the content of hydrogen peroxide (H2O2) in honey samples were analysed. Furthermore, total protein content of honeys together with their electrophoretic protein profiles were also determined in the study. The antibacterial efficacy of 24% of the tested honey samples was slightly superior to that of manuka honey with unique manuka factor 15+. The antibacterial activity of catalase-treated honey sample solutions was significantly reduced, suggesting that H2O2 is a key antibacterial compound of diluted honeys. However, the kinetic profiles of H2O2 production in most potent honeys at a MIC value of 6% was not uniform. Under the experimental conditions, we found that a H2O2 concentration of 150 µM in diluted honeys is a critical concentration for inhibiting the growth of S. aureus. In contrast, 150 µM H2O2 in artificial honey solution was not able to inhibit bacterial growth, suggesting a role of phytochemicals in the antibacterial activity of natural honey. In addition, the continuous generation of H2O2 in diluted honey demonstrated an ability to counteract additional bacteria in re-inoculation experiments. In conclusion, the tested New Caledonian honey samples showed strong antibacterial activity, primarily based on H2O2 action, and therefore represent a suitable source for medical-grade honey.

Physicochemical Characterization and Biological Properties of Pine Honey Produced across Greece.

Pine honey is a honeydew honey produced in the East Mediterranean region (Greece and Turkey) from the secretions of the plant sucking insect Marchalina hellenica (Gennadius) (Coccoidea: Marchalini-dae) feeding on living parts of Pinus species. Nowadays, honeydew honey has attracted great attention due to its biological activities. The aim of this study was to study unifloral pine honey samples produced in Greece regarding their physicochemical parameters and antioxidant and antibacterial activity against five nosocomial and foodborne pathogens. These honeys showed physicochemical and microscopic characteristics within the legal limits, except for diastase activity, a parameter known to be highly variable, depending on various factors. Substantially higher levels of H2O2 were estimated compared to other types of honeydew honey, whereas protein content was similar. The total phenolic content was 451.38 ± 120.38 mg GAE/kg and antiradical activity ranged from 42.43 to 79.33%, while FRAP values (1.87 to 9.43 mmol Fe+2/kg) were in general higher than those reported in the literature. Various correlations could be identified among these parameters. This is the first attempt to investigate in depth the antibacterial activity of pine honey from Greece and correlate it with honey quality parameters. All tested honeys exerted variable but significant antibacterial activity, expressed as MIC and MBC values, comparable or even superior to manuka honey for some tested samples. Although honey antibacterial activity is mainly attributed to hydrogen peroxide and proteins in some cases (demonstrated by elevated MICs after catalase and Proteinase K treatment, respectively), no strong correlation between the antibacterial activity and hydrogen peroxide concentration or total protein content was demonstrated in this study. However, there was a statistically significant correlation of moisture, antioxidant and antibacterial activity against Klebsiella pneuomoniae, as well as antioxidant and antibacterial activity against Salmonella ser. Typhimurium. Interestingly, a statistically significant negative correlation has been observed between diastase activity and Staphylococcus aureus antibacterial activity. Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey.

Effects of the Carbohydrate Sources Nectar, Sucrose and Invert Sugar on Antibacterial Activity of Honey and Bee-Processed Syrups.

Honey is a functional food with health-promoting properties. Some types of honey are used in wound care for the treatment of acute and chronic infected wounds. Increased interest in using honey as a functional food and as a base for wound care products causes limited availability of raw honey. Numerous studies suggest that the protein content of honey is mainly comprised of bee-derived proteins and peptides, with a pronounced antibacterial effect. Therefore, the aim of our study was to characterize for the first time the antibacterial activity of raw honeys and bee-processed syrups which were made by processing sucrose solution or invert sugar syrup in bee colonies under field conditions. Furthermore, we compared the contents of glucose oxidase (GOX) and the levels of hydrogen peroxide (H2O2) in honey samples and bee-processed syrups. These parameters were also compared between the processed sucrose solution and the processed invert sugar syrup. Our results clearly show that natural honey samples possess significantly higher antibacterial activity compared to bee-processed syrups. However, no differences in GOX contents and accumulated levels of H2O2 were found between honeys and bee-processed syrups. Comparison of the same parameters between bee-processed feeds based on the two artificial carbohydrate sources revealed no differences in all measured parameters, except for the content of GOX. The amount of GOX was significantly higher in bee-processed sucrose solutions, suggesting that processor bees can secrete a higher portion of carbohydrate metabolism enzymes. Determination of honey color intensity showed that in bee colonies, bee-processed syrups were partially mixed with natural honey. Further research is needed to identify the key botanical compounds in honey responsible for the increased antibacterial potential of honey.

Demanding New Honey Qualitative Standard Based on Antibacterial Activity.

Honey is a functional food with health-beneficial properties and it is already used as a medical device in wound care management. Whether ingested orally or applied topically, honey must fulfill the requirements of international standards based on physicochemical characteristics. However, there is an urgent need for some additional standards reflecting biological properties. The aim of the study was to evaluate the antibacterial activity of 36 commercial honey samples purchased from supermarkets and local food shops and compare their efficacy to that of three honey samples from local beekeepers and three types of medical-grade honey. Furthermore, the hydrogen peroxide (H2O2) content and protein profile were assessed in all honey samples. Analysis of the antibacterial activity of commercial honeys revealed that 44% of tested samples exhibited low antibacterial activity, identical to the activity of artificial honey (sugars only). There was a significant correlation between the overall antibacterial activity and H2O2 content of honey samples. However, in some cases, honey samples exhibited high antibacterial activity while generating low levels of H2O2 and vice versa. Honey samples from local beekeepers showed superior antibacterial activity compared to medical-grade honeys. The antibacterial activity of honey can be easily altered by adulteration, thermal treatment or prolonged storage, and therefore it fulfils strict criteria to be suitable new additional quality standard.

Antibacterial potential of Swiss honeys and characterisation of their bee-derived bioactive compounds.

BACKGROUND: Antibacterial activity of honey is not only crucial characteristic in selection of honey for medical usage but also an important honey quality marker. The aim of the study was to characterise the antibacterial potential of 29 honey samples representing the main types of multi-floral blossom and honeydew honeys produced in Switzerland. Antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa was expressed as a minimum inhibitory and bactericidal concentrations (MIC and MBC). Furthermore, the content of bee-derived glucose oxidase (GOX) and its enzymatic product, H2 O2 , were also evaluated. RESULTS: All honey samples successfully met basic defined criteria (moisture and hydroxymethylfurfural (HMF)) tested in this study. Honeydew honeys were the most effective honey samples and generated the highest levels of H2 O2 . A strong significant correlation was found between the overall antibacterial activity and the level of H2 O2 among all honey samples. Interestingly, the content of GOX in honey samples did not correlate with their antibacterial activity as well as H2 O2 production capacity. A weak antibacterial activity was determined in five floral honeys, most likely due to increased enzymatic activity of pollen-derived catalase. CONCLUSION: This study showed that antibacterial effect of Swiss honey samples is associated mainly with H2 O2 . © 2019 Society of Chemical Industry.

Antibacterial Activity of Different Blossom Honeys: New Findings.

Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the content of GOX and level of H2O2. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H2O2 levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

The role of Bni5 in the regulation of septin higher-order structure formation.

Septins are a family of conserved cytoskeletal proteins playing an essential role in cytokinesis and in many other cellular processes in fungi and animals. In budding yeast Saccharomyces cerevisiae, septins form filaments and higher-order structures at the mother-bud neck depending on the particular stage of the cell cycle. Septin structures at the division plane serve as a scaffold to recruit the proteins required for particular cellular processes. The formation and localization of septin structures at particular stages of the cell cycle also determine functionality of these proteins. Many different proteins participate in regulating septin assembly. Despite recent developments, we are only beginning to understand how specific protein-protein interactions lead to changes in the polymerization of septin filaments or assembly of higher-order structures. Here, using fluorescence and electron microscopy, we found that Bni5 crosslinks septin filaments into networks by bridging pairs or multiple filaments, forming structures that resemble railways. Furthermore, Bni5 appears to be a substrate of the Elm1 protein kinase in vitro. Moreover, Elm1 induces in the presence of Bni5 disassembly of long septin filaments, suggesting that these proteins may participate in the hourglass to double ring transition. This work gives new insight into the regulatory role of Bni5 in the structural changes of septins.

Intracellular targeting of ascomycetous catalase-peroxidases (KatG1s).

Bifunctional catalase-peroxidases (KatGs) are heme oxidoreductases widely spread among bacteria, archaea and among lower eukaryotes. In fungi, two KatG groups with different localization have evolved, intracellular (KatG1) and extracellular (KatG2) proteins. Here, the cloning, expression analysis and subcellular localization of two novel katG1 genes from the soil fungi Chaetomium globosum and Chaetomium cochliodes are reported. Whereas, the metalloenzyme from Ch. globosum is expressed constitutively, Ch. cochliodes KatG1 reveals a slight increase in expression after induction of oxidative stress by cadmium ions and hydrogen peroxide. The intronless open reading frames of both Sordariomycetes katG1 genes as well as of almost all fungal katG1s possess two peroxisomal targeting signals (PTS1 and PTS2). Peroxisomal localization of intracellular eukaryotic catalase-peroxidases was verified by organelle separation and immunofluorescence microscopy. Co-localization with the peroxisomal enzyme 3-ketoacyl-CoA-thiolase was demonstrated for KatGs from Magnaporthe grisea, Chaetomium globosum and Chaetomium cochliodes. The physiological role of fungal catalase-peroxidases is discussed.

The effect of some factors of polluted environment on catalase responses and resistance of microbial isolates against toxic oxidative stress.

The properties of bacterial isolates from polluted environments which are characterized by increased levels of oxidative stress do not reflect only the level of contaminants, but also arise as a consequence of many permanently changed conditions. The survival rate of Comamonas terrigena N3H isolates from an environment with elevated levels of H(2)O(2) is correlated with stimulation of catalase. The response of bacterial catalase to the effect of phenol in exogenous conditions was affected by the presence of an additional contaminant, Cd(2+). An isolate of Aspergillus niger selected from river sediment containing 363 mg/kg As, 93 mg/kg Sb at pH 5.2-4.8 grew on Czapek-Dox agar ~1.6 times faster than an isolate of the same species from coal dust sediment with approximately the same level of pollution (400 mg/kg As) but somewhat lower pH (3.3-2.8). It also exhibited differences in the microscopic characteristics of its mycelial structures. Both isolates exhibited a higher tolerance to the exogenic toxic effects of metals (As(5+), Cd(2+), and Cu(2+) at 5, 25, or 50 mg/L) than a control culture, but the differences in tolerance between them were only slight. These laboratory results suggest that there are complicated relationships which may exist in the "in situ" environment.

Isolates of Comamonas spp. exhibiting catalase and peroxidase activities and diversity of their responses to oxidative stress.

For survival isolates of Comamonas testosteroni CCM 1931, C. testosteroni K3, C. terrigena N3H or N1C and C. terrigena CCM 2409, selected largely from polluted environments, the production of catalase and dianisidine-peroxidase activity was important. Electrophoretic resolution of cell-free extracts of aerobically grown strains in Luria-Bertani medium during the exponential phase revealed distinctive expression of catalatic and peroxidatic activities detected with 3,3'-diaminobenzidine tetrahydrochloride (DAB). The protection of isolates from 20 or 40 mM H(2)O(2) stress was characterized with a considerable diversity in catalase and peroxidase responses that resulted from hydroperoxidase's variant of original isolates, indicating also a selective pressure of environment. Results indicate catalase to be important for adaptation of cultures to high concentration of 60mM H(2)O(2). The greatest appreciable differences in sensitivity to toxic effect of H(2)O(2) (20 or 40 mM) treatment between individual isolates and their adapted variants during the growth were observed until the middle of exponential phase. Isolates exhibited diversity in catalases responses to possible contaminants o-or p-phenylenediamine (PDA) as well. Only positional isomer p-PDA (1 or 2mM) stimulated catalase activity unlike from isomer o-PDA in C. terrigena N3H cells. The study can contribute to understanding of bacterial antioxidative enzymatic responses in the presence of possible physiological stress resulting mainly from environmental pollutants.

Screening of bacterial isolates from polluted soils exhibiting catalase and peroxidase activity and diversity of their responses to oxidative stress.

For the survival of individual isolates of gram-negative bacteria Pseudomonas putida, Achromobacter xylosoxidans, and the gram-positive bacterium Bacillus megaterium, in an environment polluted with crude oil products, the production of catalases exhibiting both catalase and dianisidine-peroxidase activity is important. Electrophoretic resolution of cell-free extracts of aerobically grown strains in Luria-Bertani medium during exponential phase revealed distinctive expression of catalatic and peroxidatic activities detected with 3,3'-diaminobenzidine tetrahydrochloride. A considerable diversity in microbial catalase and peroxidase responses to 20 or 40 mM H(2)O(2) stress, resulted from hydroperoxidase's variant of original isolates, indicating an environmental selective pressure. However, catalase was important for the adaptation of cultures to high concentration of 60 mM H(2)O(2). Appreciable differences in the sensitivity to toxic effect of H(2)O(2) (20 or 40 mM) treatment between individual isolates and their adapted variants during growth were observed until the middle of exponential phase, but they were insignificant at the entry to stationary phase. Isolates also exhibited a considerable diversity in catalases responses to phenolic contaminants 1 and 2 mM o- or p-phenylenediamine. Catalase activity of bacterium P. putida was visibly stimulated only by p-phenylenediamine and not by its positional isomer o-PDA. This study contributes to a better understanding of the role catalases play in bacterial responses to a polluted environment.

Molecular diversity of katG genes in the soil bacteria Comamonas.

UNLABELLED: Three complete katG genes coding for bifunctional catalase-peroxidases (KatGs) from the beta-proteobacterium Comamonas terrigena and two related strains of Comamonas testosteroni have been cloned and sequenced. Catalase-peroxidases are unique bifunctional enzymes known to be expressed in these soil bacteria in response to environmental and/or oxidative stress. The evolutionary and structural diversity of these enzymes is investigated based on multiple sequence alignment and comprehensive phylogenetic analysis. The reconstructed phylogenetic tree and well-known structure-function relationships were applied to inspect the conservation of essential residues. Observed diversity is discussed with respect to the fact that KatGs are distinctive gene-duplicated peroxidases comprising a N-terminal (enzymatically active) and a C-terminal (heme-less) domain. The unique promoter motifs regulating katG transcription in four strains of Comamonas were detected and compared with E. coli katG promoter. The relationship between the promoter sequences and the corresponding expression levels was analyzed. A significant difference in heat shock-inducible catalatic and peroxidatic activities between E. coli K12 and Comamonas terrigena & testosteroni strains was observed. The peculiar variability in gene-coding sequences appears to be more significant for such activity output among Comamonas strains than differences in their promoter regions. The functional role of observed increased diversity in the C-terminal domain is discussed with respect to potential modification of catalytic features at the N-terminal domain that could be relevant for these soil bacteria to cope with stressors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00203-009-0541-4) contains supplementary material, which is available to authorized users.

Responses in the mycelial growth of Aspergillus niger isolates to arsenic contaminated environments and their resistance to exogenic metal stress.

Isolates of Aspergillus niger, selected from coal dust sediment of a mine containing As (400 mg/kg), pH 3.3-2.8, and from river sediment found near the mine (As, 363 mg/kg, Sb, 93 mg/kg), pH 5.2-4.8, growing on Czapek-Dox agar exhibited distinct responses in the mycelial growth in arsenic contaminated environments. The radial growth of the isolate from the coal dust in comparison to the control strain from an environment without pollution was reduced approximately to one-half. It formed black, very small compact colonies, with dense sporulation. The opposite, the strain from the river sediment, grew better in Czapek-Dox agar like the control. It formed larger colonies with dense centre and strong sporulation. Also, the culture from river sediment developed faster than the coal dust isolate and control strain. Differences were also recorded in size and thickness of conidia heads, phialide, metulae, and conidiophores. Both isolates from contaminated localities exhibited higher tolerance to exogenic toxic effects of As5+, Cd2+ and Cu2+ (5, 25 or 50 mg/l) than the control culture. Tolerance was monitored using the growth of biomass in liquid Czapek-Dox medium. We confirmed the morphological identification of our isolates to A. niger species with the PCR method. The results refer to complicated relations between biotic and abiotic effects that may directly affect the processes observed in the in situ environment.

The expression and diversity of catalases in isolates of genus Comamonas in response to the oxidative stress of a polluted environment.

We have evaluated the role of monofunctional heme-containing catalase encoded by cat-1 gene from the soil bacterium Comamonas terrigena N3H in the response to various forms of oxidative stress. Our results indicate that this constitutively expressed catalase represents the major source for the defence of Comamonas terrigena cells against toxic peroxides but the cells can express also a second form of catalase that is bigger and its regulation is probably more complicated. The sequence analysis confirmed the presence of highly conserved catalase sequence motifs in two environmental strains of Comamonas terrigena but in those strains that were not exposed to oxidative stress, no such sequence motif could be detected. The results obtained underline the importance of catalase expression in the defence mechanism against oxidative stress in bacterial cells.

Production of catalases by Aspergillus niger isolates as a response to pollutant stress by heavy metals.

Isolates of Aspergillus niger, selected from the coal dust of a mine containing arsenic (As; 400 mg/kg) and from the river sediment of mine surroundings (As, 1651 mg/kg, Sb, 362 mg/kg), growing in minimal nitrate medium in the phase of hyphal development and spore formation, exhibited much higher levels of total catalase activity than the same species from the culture collection or a culture adapted to soil contaminated with As (5 mg/L). Electrophoretic resolution of catalases in cell-free extracts revealed three isozymes of catalases and production of individual isozymes was not significantly affected by stress environments. Exogenously added stressors (As(5+), Cd(2+), Cu(2+)) at final concentrations of 25 and 50 mg/L and H(2)O(2) (20 or 40 mM) mostly stimulated production of catalases only in isolates from mines surroundings, and H(2)O(2) and Hg(2+) caused the disappearance of the smallest catalase I. Isolates exhibited a higher tolerance of the toxic effects of heavy metals and H(2)O(2), as monitored by growth, than did the strain from the culture collection.

Lag period of 14CO2 evolution from dioctyl sulpho[2,3-14C]succinate in relation to adaptation of bacterium, Comamonas terrigena, to dialkyl esters of sulphosuccinate.

Comamonas terrigena, strain N3H, which was isolated from soil polluted with crude oil products, degraded dioctyl sulphosuccinate, a synthetic commercial surfactant. The primary degradation of this compound, the cleavage of ester bonds between octyl groups and sulphosuccinate, lasted significantly shorter time than the subsequent breakdown of the sulphosuccinate moiety of dioctyl sulpho[2,3-(14)C]succinate. (14)CO(2) evolution had a significant shorter lag period with cells in Tris/phosphate medium, without inorganic sulphate and adapted to surfactant, than unadapted cells. The acceleration of the primary degradation by adapted cells also suggest that some enzymes involved in surfactant degradation are inducible. The bacterium may be useful for bioremediation.

Expression, purification, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H.

Catalases are essential components of the cellular equipment to cope with oxidative stress. We have purified and characterize herein the most abundant heme-containing catalase-1 from the soil bacterium Comamonas terrigena N3H. This oxidative stress-induced enzyme was isolated from exponential phase cells grown in the presence of peroxyacetic acid. We have used consecutive steps of hydrophobic, molecular sieve, and ion exchange chromatography to achieve a high state of purity for this metalloenzyme. The purified sample of catalase exhibited a specific catalytic activity of 55,900 U/mg, allosteric behavior in peroxidic reaction, a broad pH optimum, and a rather atypical electronic spectrum. The sample of highest purity was subjected to mass spectrometry analysis. The molecular weight of the subunit of this homodimeric protein was determined as 55,417 Da. The Qq-TOF mass analysis method allowed us to sequence short tryptic fragments of this catalase. Five such fragments with a total length of 57 amino acids together with several enzymatic properties allowed the classification of this hydroperoxidase as belonging to clade III of monofunctional catalases. The highest sequence similarity is with the catalase from Vibrio fischeri. The presented results imply the significance of this inducible enzyme in the prevention of toxic effects of oxidative stress for bacterial cells.