Marcela Bucekova

Honeybee glucose oxidase—its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.

Antibiofilm efficacy of honey and bee-derived defensin-1 on multi-species wound biofilm.

Many clinically relevant biofilms are polymicrobial. Examining the effect of antimicrobials in a multispecies biofilm consortium is of great clinical importance. The goal of this study was to investigate the effect of different honey types against bacterial wound pathogens grown in multispecies biofilm and to test the antibiofilm activity of honey defensin-1 (Def-1) in its recombinant form. A modified Lubbock chronic wound biofilm formed by four bacterial species (Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa and Enterococcus faecalis) was used for evaluation of honey and recombinant bee-derived Def-1 antibiofilm efficacy. Recombinant Def-1 was prepared by heterologous expression in Escherichia coli. We showed that different types of honey (manuka and honeydew) were able to significantly reduce the cell viability of wound pathogens (Staphylococcus aureus, Streptococcus agalactiae and Pseudomonas aeruginosa) in mature polymicrobial biofilm. None of the tested honeys showed the ability to eradicate Enterococcus faecalis in biofilm. In addition, recombinant Def-1 successfully reduced the viability of Staphylococcus aureus and Pseudomonas aeruginosa cells within established polymicrobial biofilm after 24 and 48 h of treatment. Interestingly, recombinant Def-1 did not affect the viability of Streptococcus agalactiae cells within the biofilm, whereas both natural honeys significantly reduced the viable bacteria. Although Enterococcus faecalis was highly resistant to Def-1, Def-1 significantly affected the biofilm formation of Enterococcus faecalis and Streptococcus agalactiae after 24 h of treatment, most likely by inhibiting its extracellular polymeric substances production. In conclusion, our study revealed that honey and Def-1 are effective against established multispecies biofilm; however, Enterococcus faecalis grown in multispecies biofilm was resistant to both antimicrobials.

Microwave processing of honey negatively affects honey antibacterial activity by inactivation of bee-derived glucose oxidase and defensin-1

Microwave (MW) thermal heating has been proposed as an efficient method for honey liquefaction, while maintaining honey quality criteria. However, little is known about the effects of MW thermal heating on honey antibacterial activity. In this study, we aimed to determine the effects of MW heating on the antibacterial activity of raw rapeseed honeys against Pseudomonas aeruginosa and Staphylococcus aureus, with a particular focus on two major bee-derived antibacterial components, defensin-1 and hydrogen peroxide (H2O2). Our results demonstrated that MW thermal heating completely abolished honey antibacterial activity whereas conventional thermal treatment at 45 and 55°C did not affect the antibacterial activity of honey samples. A significant decrease in both glucose oxidase activity and H2O2 production as well as defensin-1 amount was observed in MW-treated samples. Given that defensin-1 and H2O2 are regular antibacterial components of all honeys, MW heating may have similar negative effects on every type of crystallized/liquid honey.

Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey

Honeydew honey is increasingly valued due to its pronounced antibacterial potential; however, the underlying mechanism and compounds responsible for the strong antibacterial activity of honeydew honey are still unknown. The aim of this study was to investigate the inhibition of bacterial growth of 23 honeydew honey samples. Activity of bee-derived glucose oxidase (GOX) enzyme, the content of defensin-1 (Def-1) and hydrogen peroxide (H2O2), and total polyphenol content were determined in the 23 honey samples. Our results demonstrated that antibacterial activity of honeydew honey was equivalent to medical-grade manuka and kanuka honey and was abolished by catalase. Although H2O2 is an important factor in the inhibition of bacterial growth, polyphenolic compounds and their interaction with H2O2 are the key factors responsible for high antibacterial activity of honeydew honey. In addition, our results indicated that the antibacterial activity of honeydew honey is not dependent on GOX-mediated production of H2O2 or the presence of Def-1.

Antibiofilm Efficacy of Honeybee Products Against Wound Biofilm

Treatment of bacterial biofilm in the wound is complicated by the mechanisms underlying biofilm growth. Many clinically relevant biofilms are polymicrobial. Furthermore, mixed species biofilms have complementary metabolic strategies for obtaining nutrients and degrading host immune molecules. Therefore, there is an urgent need to introduce novel or reemerging effective approaches to combat bacterial biofilms in chronic wounds.

Effect of thermal liquefying of crystallised honeys on their antibacterial activities

Thermal liquefying of crystallised honey is the most convenient option for beekeepers to fully liquefy honey. A controlled mild thermal treatment is widely used for effective and safe liquefying of crystallised honey. In this study, we demonstrated that thermal liquefying at different temperatures of 45, 55 and 65 °C does not affect the overall honey antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus, and, in some cases, it may even increase the antibacterial potential of honey. A mild thermal treatment of crystallised honey was able to significantly increase its glucose oxidase (GOX) enzyme activity. On the other hand, the increased GOX activity in treated honey samples did not affect levels of accumulated H2O2, suggesting that some phytochemicals may contribute to or modulate antibacterial activity. Taken together, a mild thermal liquefaction of crystallised honey may increase the potential bioactivity of this matrix, which is also affected by the botanical origin.

Honey: an effective regenerative medicine product in wound management

Honey has successfully been used in treatment of a broad spectrum of injuries including burns and non-healing wounds. It acts as antibacterial and anti-biofilm agent with anti/pro-inflammatory properties. However, besides these traditional properties, recent evidence suggests that honey is also an immunomodulator in wound healing and contains several bee and plant-derived components that may speed up the wound healing and tissue regeneration process. Identifying their exact mechanism of action allows better understanding of honey healing properties and promotes its wider translation into clinical practice. This review will discuss the physiological basis for the use of honey in wound management, its current clinical uses, as well as the potential role of honey bioactive compounds in dermal regenerative medicine and tissue re-modelling.