Evaluation of Probiotic Properties of Lactic Acid Bacteria Isolated from the Gut of Honeybees Apis mellifera

European honey bees (Apis mellifera) are the most important pollinator insects that play vital role in maintenance of all most all life forms on earth. However, over the last decade major concerns have raised due to decline in the population of these insect species. A variety of factors have been responsible for these concerns of which the most important is honey bee bacterial diseases like Nosemosis (Nosema), American and European foulbrood diseases. While, overuse of antibiotics utilized for the treatment and control of these diseases has resulted in the emergence of antibiotic resistant strains of these pathogens. Probiotics have been considered a suitable substitute of antibiotics in human and animals. In last several years, lactobacillus species isolated from honeybees have been considered of significance in enhancing the life span of honeybees by reducing the incidence of bacterial and viral infections in these tiny insects. Among the isolated microbes in the gut of honeybees, Lactic Acid Bacteria (LAB) are of utmost importance showing direct impacts on the health of their host by modulating the gut microbial flora and are termed as Probiotic bacteria.The objective of this research was to isolate and identify LAB from different parts of the intestinal tract of honeybees Apis mellifera and to characterize their probiotic properties.

Twenty-four honeybees collected from the hives located in the city of karaj were analyzed for the presence of LAB species. The stomach contents of honeybees were inoculated into MRS broth, incubated at 37ºCfor 48 hrs. The obtained colonies were purified and identified to species levels phenotypically and geno-typically. Hemolytic activity and sugar fermentation reactions of the isolates were recorded and later subjected 16SrRNA sequencing using a pair of universal primers. The identified isolates were evaluated for their viability in acidic conditions at pH 2.5, 3.0, 4.0 and 6.5 at different time intervals. Bile resistance of the isolates was tested by culturing the isolates in the presence of different concentrations of the said salts (0.5, 0.7 and 1%). Survival of LAB isolates in simulated gastric and intestinal conditions containing different enzymes and bile salts, antibacterial spectrum against a number of gram positive and gram-negative pathogens by agar well diffusion assay, and their in vitro colonization ability (aggregation, co-aggregation and hydrophobicity percentages) were evaluated. The results were analyzed statistically.

Twenty nine gram positive, catalase negative and non-hemolytic colonies were isolated from 24 honeybee samples. Among these, only 7 colonies showed enhanced antibacterial activity and were selected for further studies. Based on phenotypic characteristics, sugar fermentation reactions and 16S rRNA sequencing the isolates were identified as Lactobacillus acidophilus (1), Lacticaseiobacillus casei, Lactiplantibacillus plantarum (2), Lactobacillus apis (1), Enterococcus faecium (1) and Pediococcus acidilactici (1). During probiotic characterizations, the identified isolates were shown to retain their viability in acidic conditions and resisted pH 2.5, 3 and 4 for more than 4 hrs. However, slight decrease in viability at pH 2.5 and 3.0 was observed, compared to pH 4.0 and above. All isolates appeared bile resistant and tolerated all used concentrations of bile salts during 8 hrs of incubation. Survival rate of the isolates in simulated intestinal conditions was significantly (p˂0.05) greater compared to simulated gastric conditions indicating greater stability of the isolates to alkaline conditions rather than to acidic conditions. L.acidophilus and E.faecium showed least resistance in gastric conditions and their growth rate was decreased more than 50% under said conditions. In contrast, the growth rate of these two isolates was highest in simulated intestinal conditions as they resisted these conditions for more than 24 hours.  The isolates demonstrated antibacterial affect against a number of tested pathogens including Listeria monocytogenes, Escherichia coli, Enterococcus faecalis and Streprtococcus mutans. The auto-aggregation, and cell surface hydrophobicity percentages of L.casei appeared highest compared to other tested Lactic Acid bacteria in study (p˂0.05), while, L.apis showed the highest co-aggregation with S.typhi strain. P.acidilactici possessed the least auto-aggregation (46%), co-aggregation (10%) and hydrophobicity (43%) percentage. Auto-aggregation ability appeared directly related to hydrophobicity percentages and isolates showing high aggregation ability also showed high hydrophobicity %.

In conclusion, honeybee gut appeared a reservoir of LAB with probiotic potential. It is suggested that further studies should be conducted in order to determine the health benefits of these LABs in honeybees, with especial emphasis on their ability to prevent honeybee diseases.