Abstract
Honeybee venom (Apitoxin, BV), a secretion substance expelled from the venom gland of bees, has being reported as antimicrobial against various bacterial species; however, the mechanism of action remains uncharacterized. In this study, the antibacterial activity of BV was investigated on hygiene indicator Escherichia coli and the environmental pathogen and spoilage bacterial species, Pseudomonas putida and Pseudomonas fluorescens. An array of methods was combined to elucidate the mode of action of BV. Viability by culture on media was combined with assessing cell injury with flow cytometry analysis. ATP depletion was monitored as an indicator to metabolic activity of cells, by varying BV concentration (75, 225and 500 µg/mL), temperature (25 ^circ complement and 37 ^circ complement ), and time of exposure (0 to 24 h). Venom presented moderate inhibitory effect on E. coli by viability assay, caused high membrane permeability and significant ATP loss where the effect was increased by increased concentration. The viability of P. putida was reduced to a greater extent than other tested bacteria at comparable venom concentrations and was dictated by exposure time. On the contrary, P. fluorescens appeared less affected by venom based on viability; however, flow cytometry and ATP analysis highlighted concentration- and time-dependent effect of venom. According to Transmission Electron Microscopy results, the deformation of the cell wall was evident for all species. This implies a common mechanism of action of the BV which is as follows: the cell wall destruction, change of membrane permeability, leakage of cell contents, inactivation of metabolic activity and finally cell death.
Highlights
Honeybee venom (BV, Apitoxin) is secreted from venom gland of worker honeybees and it is one of the products of apiculture among others such as honey, propolis, bee wax (Bogdanov 2017; Massaro et al 2015)
Variation between BV samples can be explained by qualitative and quantitative differences in composition recorded by HPLC profiles of aqueous solutions of BV-1 and BV-2 (150 μg/mL) at 220 nm (Additional file 1: Figure S1), For example, the 1.3-fold higher concentration of melittin in solution of BV-2 compared with that in solution of BV-1 (62 vs 47.5 μg/mL ) could greatly affect their bactericidal activity
The variation in the number of viable cells treated with BV was found to be primarily driven by bacterial species
Summary
Honeybee venom (BV, Apitoxin) is secreted from venom gland of worker honeybees and it is one of the products of apiculture among others such as honey, propolis, bee wax (Bogdanov 2017; Massaro et al 2015). There is ongoing research on medical applications of BV for asthma, arthritis, Parkinson’s disease, Alzheimer’s disease (Ali 2012; Socarras et al 2017; Fratini et al 2017) and treatment of human cancer cells (Hu et al 2006; Ip et al 2012; Jo et al 2012; Jang et al 2003; Liu et al 2013). Despite concerns related to allergenicity and biogenic amine content (Additional file 1: Table S1), there are commercially available products for antiwrinkle facial treatment formulated with BV (e.g., Apiven (France), Manuka Doctor (New Zealand), Rodial (UK)). BV biological activity has attracted interest in medical and cosmetic applications, use in food is considerably less than other bee-products such as honey, bee pollen and propolis and was limited to use as a nutrient ingredient, for example in honey. Concerning previous studies, BV presents the potential to act as a natural antimicrobial in food applications
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