Lactic acid bacteria (LAB) naturally inhabiting the digestive tract of honeybees are known for their ability to detoxify xenobiotics. The effect of chlorpyrifos, coumaphos, and imidacloprid on the growth of LAB strains was tested. All strains showed high resistance to these insecticides. Subsequently, the insecticide binding ability of LAB was investigated. Coumaphos and chlorpyrifos were bound to the greatest extent (up to approx. 64%), and imidacloprid to a much weaker extent (up to approx. 36%). The insecticides were detected in extra- and intracellular extracts of the bacterial cell wall. The ability of selected LAB to reduce the cyto- and genotoxicity of insecticides was tested on two normal (ovarian insect Sf-9 and rat intestinal IEC-6) cell lines and one cancer (human intestinal Caco-2) cell line. All strains exhibited various levels of reduction in the cyto- and genotoxicity of tested insecticides. It seems that coumaphos was detoxified most potently. The detoxification abilities depended on the insecticide, LAB strain, and cell line. The detoxification of insecticides in the organisms of honeybees may reduce the likelihood of the penetration of these toxins into honeybee products consumed by humans and may contribute to the improvement of the condition in apiaries and honeybee health.
Lactic acid bacteria (LAB) are an essential part of the microbiota of the digestive tract of honeybees (Apis mellifera L.). Antagonistic activity of 103 LAB strains (isolates from different environments) against 21 honeybee pathogens/opportunistic pathogens (with agar slab method) was screened. The growth of Paenibacillus genus was inhibited to the most extent. The highest antagonistic activity was demonstrated by Lacticaseibacillus casei 12AN, while the lowest by Apilactobacillus kunkeei DSM 12361, a species naturally inhabiting the honeybee gut. LAB isolated from the honeybee environment demonstrated stronger antagonism against pathogens than collection strains. The antagonistic activity of cell-free supernatants (CFSs) from 24 LAB strains against 7 honeybee pathogens was additionally assessed at physiological pH with the microtitration method. The same was determined for selected CFSs at neutralized pH. CFSs with physiological pH showed significantly stronger antibacterial activity than CFSs with neutralized pH. The results confirmed that the mechanism of antimicrobial activity of LAB is acidification of the environment. The obtained results may, in the future, contribute to a better understanding of the antagonistic properties of LAB and the construction of a probiotic preparation to increase the viability of honeybee colonies.
In light of the phenomenon of colony collapse disorder, there has been a growing interest in finding natural and ecological ways for improving honeybee health. The aim of this scientific research was the isolation and characterization of LAB, which in the future could show the potential to construct a protective preparation for honeybees. After performing MALDI-TOF analysis, of a total of 76 bacterial strains isolated from flowers and honeybee products, 31 were identified as Pediococcus pentosaceus, 26 as Pediococcus acidilactici, and 19 as Lactiplantibacillus plantarum. The characterization of the isolated LAB displayed that CO2 production was present in 52 strains. The highest biomass productivity was observed in the case of strain 9/1 isolated from red clover (Trifolium pratense L.) with biomass productivity equal to 2.100. All isolated bacterial strains showed the ability to produce lactic acid. The strain 13/3 isolated from small-leaved lime (Tilia cordata L.) displayed the highest lactic acid production capacity in 100 mL of culture, i.e., 1.903 g of lactic acid. The carbohydrate assimilation pattern was examined using API 50 CH tests. All isolated strains were able to utilize esculin, D-ribose, D-galactose, D-glucose D-fructose, and D-mannose. It was also noted that the reduction of sugars is a strain-dependent ability and is specific for individual strains.
Lactic acid bacteria (LAB) naturally inhabits the organisms of honeybees and can exhibit adhesive properties that protect these insects against various pathogenic microorganisms. Thus, cell surface (auto-aggregation, co-aggregation, hydrophobicity) and adhesive properties of LAB to two abiotic (polystyrene and glass) and four biotic (collagen, gelatin, mucus, and intestinal Caco-2 cells) surfaces were investigated. Additionally, anti-adhesion activity and the eradication of honeybee pathogen biofilms by LAB metabolites (culture supernatants) were determined. The highest hydrophobicity was demonstrated by Pediococcus pentosaceus 19/1 (63.16%) and auto-aggregation by Lactiplantibacillus plantarum 18/1 (71.91%). All LAB showed a broad spectrum of adhesion to the tested surfaces. The strongest adhesion was noted for glass. The ability to co-aggregate with pathogens was tested for the three most potently adherent LAB strains. All showed various levels of co-aggregation depending on the pathogen. The eradication of mature pathogen biofilms by LAB metabolites appeared to be weaker than their anti-adhesive properties against pathogens. The most potent anti-adhesion activity was observed for L. plantarum 18/1 (98.80%) against Paenibacillus apiarius DSM 5582, while the strongest biofilm eradication was demonstrated by the same LAB strain against Melissococcus plutonius DSM 29964 (19.87%). The adhesive and anti-adhesive activity demonstrated by LAB can contribute to increasing the viability of honeybee colonies and improving the conditions in apiaries.
Lactic acid bacteria (LAB) are widely used probiotics and offer promising prospects for increasing the viability of honeybees. Thus, the probiotic potential of 10 LAB strains was determined, which in our previous studies showed the most potent protective abilities. In the current study, we investigated various properties of probiotic candidates. The tested LAB strains varied in susceptibility to tested antibiotics. Isolates showed high viability in sugar syrups and gastrointestinal conditions. None of the LAB strains exhibited β-hemolytic activity, mutual antagonism, mucin degradation, hydrogen peroxide production capacity, or bile salt hydrolase (BSH) activity. Additionally, the cytotoxicity of LAB cell-free supernatants (CFS) was assessed, as well as the effect of CFS from P. pentosaceus 14/1 on the cytotoxicity of coumaphos and chlorpyrifos in the Caco-2 cell line. The viability of Caco-2 cells reached up to 89.81% in the presence of the highest concentration of CFS. Furthermore, LAB metabolites decreased the cytotoxicity of insecticides (up to 19.32%) thus demonstrating cytoprotective activity. All tested LAB strains produced lactic, acetic, and malonic acids. This research allowed the selection of the most effective LAB strains, in terms of probiosis, for future in vivo studies aimed at developing an ecologically protective biopreparation for honeybees.
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