UNVEILING THE CULTURABLE BACILLUS SPP. ISOLATED FROM HONEY IN BURKINA FASO AS A POTENTIAL SOURCE FOR ANTIMICROBIAL PEPTIDES

Sonagnon H.S. Kouhounde1*image, Ulysse Daa Kpode2,3image, Hilarie Bonane1image, Rene Comlan Yaovi4image, Kifouli Adeoti2,3image, Mamoudou H. Dicko5image

1Laboratory of Applied Biological Sciences, New Dawn University, 01 BP 234 Bobo-Dioulasso 01, Burkina Faso. 2Laboratory of Food Microbiology Technology and Phytopathology, University of Abomey-Calavi, Benin. 3Team of Plant Biodiversity and Ecology, University of Abomey-Calavi, Benin. 4Laboratory of Agrosystems Bioresources and Environmental Health, Nazi Boni University, Bobo-Dioulasso, Burkina Faso. 5Laboratory of Biochemistry, Biotechnology, Food Technology, and Nutrition, Joseph KI-ZERBO University, 03 BP 7021 Ouagadougou, Burkina Faso.

 

Abstract

Backgrounds and objectives: Honey is increasingly recognized as a promising reservoir of beneficial microorganisms with activity against plant pathogens. However it is poorly documented that in the literature. This study contributes to explore the diversity of AMPs genes produced by Bacillus spp. isolated from honey in Burkina Faso in order to propose a biological solution against biodiversity loss, water soil pollution and pest resistance caused by chemicals pesticides. 

Methods: Methodology was based on isolation and preliminarily characterization of 40 presumptive Bacillus spp. followed by their survival under thermal stress, salt tolerance and AMPs related genes identification. 

Results: Data from PCR analysis revealed that all isolates belonged to the genus Bacillus. In total, 70% of strains carried at least one AMP gene, in which 50% carried one gene. Regarding AMPs-gene associations, 25% carried two genes, 14.3% carried three genes, 7.1% carried four genes, and 3.6% carried five of the six targeted AMP-genes. 

Conclusions: These data indicate that honey produced in Burkina Faso could be used to isolate Bacillus strains with genetic potential for broad-spectrum antimicrobial activity. This efficacy could be tested in future studies to confirm these findings.

Keywords: Antimicrobial peptides, AMP-genes, Bacillus spp., Burkina Faso, Honey microbiota. 

 

INTRODUCTION

 

Bee-breeding, commonly found in all regions of the globe1, is the science and practice of raising bees to exploit their products. It contributes to improving the incomes of populations in the Global South2. Beyond honey and its multiple virtues, the maintenance of biodiversity and the pollination of flowering plants are undoubtedly the most valuable ecosystem services provided by bees. Bees, therefore, have both ecological and economicstakes3. Although bee breeding contributes to fight against poverty and can help create income-generating jobs4, it is often a secondary activity in Africa.  Indeed, honey, which is a viscous sweetener produced by bees from nectar or secretion of floweringplants5, is a highly valued commodity due to its medicinal and nutritional important values. Its major constituents include 80% of carbohydrates (35% glucose, 40% fructose and 5% sucrose), and water (20%)6, while its minor components include minerals, proteins, vitamins, organic acids, phenolic compounds, enzymes (hydrolases and oxido-reductases) and otherphytochemicals7. 

The nutritional value of honey dependson the floral monofloral (Manuka, acacia, clover, lavender, and eucalyptus) or multifloral (a mix from different local wild flowers, herbs, and flowering bushes) sources, geographical origin, processing and storage conditions8. These factors determine its specific physicochemical composition and bioactive properties that are beneficial to humans9. Several studies reveal that honey display several therapeutic traits10. Furthermore, honey is an important substance in traditional African medicine11 in both human and animal medicine.

Although considered as safe with respect to pathogenic microorganisms, it has been reported that yeasts, molds, and aerobic mesophilic bacteria can survive in its matrix12. Studies report that Cladosporium, Aspergillus spp., Penicillium spp., and Bacillus spp. are the main microbial groups found in honey. These microorganisms can originate from nectar, pollen, and other parts of the plant flower. Their presence in honey has been linked to the water content, which is likely to encourage the growth of existing microorganisms12. Recent studies reported the presence of a diversity of Bacillus species on flowers foraged by bees and even in the produced honey13.

Furthermore, several studies have demonstrated that Bacillus species have attracted considerable interest due to their ability to produce cyclic lipopeptides and their antifungal properties14. Among these interesting molecules are surfactants, fengycins, and iturins. These lipopeptides act as potent biosurfactants known for their antifungal and phytoprotective properties15. 

In this respect this study was performed with the main objective to determine the diversity of antifungal peptides genes of the genus Bacillus spp. in honey produced in Hauts Bassins region.

 

MATERIALS AND METHODS

 

Sampling

Sampling was carried out in three provinces (Houet, Tuy, and Kenedougou) based in Hauts Bassins Region. These provinces have been reputed to be among the top five honey-producing provinces in Hauts Bassins Region of Burkina Faso. In detail, four samples were collected from Houet province, two samples from Kenedougou province, and four samples from Tuy province. A total of 10 honey samples were collected from producers in sterile jars and transported in a cooler container from sampling sites to the laboratory.

Isolation and purification of presumptive Bacillus spp.

Honey samples were treated according to the standard procedure16. For each sample, 10 g was taken and diluted into 90 mL of diluent and gently homogenized, then decimal dilutions were obtained. Bacillus spp. were isolated and purified according to method applied by Ouoba and co-workers in which 1 mL of each dilution was plated onto Luria Bertani agar medium (Powder, Liofilchemsrl, ITALY) and plates incubated at 37°C for 24 hours17. B. cereus ATCC 14579 was used in this study as control strain. 

Preliminary characterization of isolates 

The isolates were described for colony shape, size, color, consistency, and outline, and cultured cells were observed under the microscope (B-292, Optika, Italy) for cellmorphology and ability to Gram stain. Catalase test was performed following the conventional procedure, using a few drops of 3% (v/v) hydrogen peroxide. The sporulation test was carried out according to the method described by Gounina-Allouane and co-workers18. If visual growth was observed, the isolate was stained using methylene blue solution at the concentration of 0.5% at pH 12to Schaeffer Fulton method. 

Salt tolerance assay

To determine the salt tolerance, the isolates were cultured on LB broth medium (Powder, Liofilchemsrl, Italy) supplemented with NaCl at different final concentrations (2.5, 5.0, and 10%), and the results were read by measuring bacterial growth after incubation at 37°C/ for 24 hours.

Detection and quantification of genes following the RT-PCR reaction

Fresh bacterial cultures were revived in nutrient broth for 18 hours. Then, 200 μL of chelex (Chelex 100, BIORAD, USA) was dispensed into Eppendorf tubes, and 1 μL of the fresh bacterial culture was added.The cultures were incubated at 56°C for 30 - 60 min. The mixture was vortexed for 10 s and returned to incubation at 90°C for 10 min. before being centrifuged at 13,000 rpm for 5 min. Once the extraction was completed, the reaction medium was prepared for the amplification19. 

Primer pairs (Table 1) targeting genes (16S RNA, ituA, hag, tasA, srf, sps, mrsH, B. cereus) previously described13 as beneficial genes for the prevention and control of fungal contamination were used to characterize the 40 selected isolates. Two different amplification programs were performed (a 32-cycles program followed by a 30-cycles program) using a T100 PCR thermo cycler (My Cycler, Bio-Rad Laboratories).

For amplification, PCR was carried out following a 32-cycles program including one cycle of initial DNA denaturation at 95°C for 10 min followed by a set of 30 cycles program with denaturation for 40s at 95°C, hybridization at 59°C for 40s and elongation for 2 min at 72°C. Then a final elongation (1 cycle) war performed for 6 min at 72°C. The PCR products were run on a 2% agarose gel at 120 volts for 1 hour and visualized on a transilluminator (TM-20 UV, UVP, France). The 100 bp DNA Ladder ready to load (Solis Biodyne, Estonia) was used as a control size.

Data analysis 

The results were analyzed using R software version 4.3.2. A multivariate analysis (based on potential differences and associations between the amplified gene fragments) and hierarchical visualization were performed using several packages. The readxl package was used to import the raw data from an Excel file, while the dendextend package allowed for the manipulation and customization of the dendrograms. Initially, the data were loaded directly from the Excel file containing all the observations. Hierarchical clustering was performed using Ward D2 method based on the size of the amplified fragments associated to the targeted genes. 

 

RESULTS

 

Isolation and phenotypic characterization of bacterial isolates

A total of 40 isolates belonging to the Bacillus genus were obtained. Identification was based on macro-scopic, microscopic, physiological, and molecular characteristics. Macroscopic observation of the bacterial colonies, after 24 hours, revealed two colony types: whitish, creamy colonies with regular outlines and large, sticky, rounded colonies. The isolates exhibited Gram-positive bacillus-shaped cells grouped in chains. All isolates were positive for catalase and able to grow after the heat shock. Results on tolerance to NaCl revealed that all isolates were able to grow in NaCl up to a concentration of 10%.

Amplification of the 16S gene region and genes involved in the synthesis of peptides with antimicrobial peptide

The identification based on the 16S used primer confirmed that all isolates belong to Bacillus genus. Isolated strains can be divided into four (04) classes concerning the presence of genes involved in the production of antimicrobial peptides. Cluster 1 includes 28 strains. This group, within which B. cereus is strongly represented, is characterized by strains presenting ituA, tasA, Srf, and haggenes (from the most extreme to the least extreme). Cluster 2 included strains positive for the presence of tasA, Srf, and ituA genes. Cluster 3 is composed of strains characterized by highituA peptide synthesis values. Cluster 4 is composed of strains characterized by the ability to synthesize antimicrobial peptides, such as tasA, Hag, ituA, and Srf.

The search for genes (Table 3) responsible for the synthesis of bioactive microbial secondary metabolites was identified in our strains. However, a negative PCR result for spaS was obtained in almost all strains. Twelve out of the 40 strains (BH1.0, BH1.1, BH1.2, BH2.1, HT3.0, HT3.1, HT3.2, BT7.0, OK9.0, OK10.0, KK11.2 and KK12.2) did not show antifungal genes.

 

DISCUSSION

 

The genus Bacillus spp., a sporulating, Gram-positive bacterium was detected in honey accordingly to previous findings. Moreover, as already reported, the Bacillus isolated from honey in Asia has been described as able to secrete various peptides and bacteriocins with antifungal activities20. Bacillus spp. devote a large portion of their genome to secondary metabolism, as an adaptive response to environmental competition. Bacillus spores have been detected in soil and the ocean, where complex microbial communities have been described21,22. By producing secondary metabolites that can inhibit closely related species and other microorganisms in the ecological niche, Bacillus spp have gained significant survival advantages.

In the present study, 6 pairs carrying genes, such as ituA (IturinA), hag (Flagellin), tasA (TasA), srf (Surfactin), spaS (Subtilin) and mrsA (Mersacidin), which are related to the production of antifungal peptides, were investigated. Our results are consistent with those of previous studies23 who identified tasA, ituA and hag simultaneously in the same strain of Bacillus sp isolated from honey and demonstrated that these genes conferred properties to control certain fungal species such as Fusarium graminearum, Rhizoctonia solani, and Botrytis cinerea. The association of multiple AMPs exhibit synergistic effects, particularly among lipopeptides such as surfactin, iturin, and fengycin, which collectively enhance antimicrobial potency beyond the activity of individual molecules. This genomic organization reflects the high plasticity of Bacillus genomes, in which horizontal gene transfer and the acquisition of biosynthetic gene clusters contribute to the accumu-lation of multiple AMP determinants. Strains possessing multiple AMP genes are therefore strong candidates for biotechnological applications. 

According to Zhou and co-workers, the first three antifungal peptides have their own characteristics and antifungal spectrum23. The iturin family is a cyclic lipopeptide type containing 7 amino acids, with strong antifungal activity. Iturin's antifungal mechanism places its hydrophobic tail into the plasma membrane of indicator cells and automatically assembles to form an ion channel, thus allowing leakage from the cytoplasm. Additionally, iturin A can release electrolytes and polymer aggregates, increase the electrical conductivity and permeability of the biomass membrane, resulting in surface tension effects on the cell membrane, inhibiting pathogen formation24.

These lipopeptides can modify biofilm formation, motility, and virulence gene expression and one of them (TasA) is involved in sporulation and biofilm formation in Bacillus25.

 

CONCLUSION

 

The objectives that prompted the present study can be considered achieved. First, the microbiological analysis of the honey revealed the presence of Bacillus. Honey produced in Burkina Faso is a valuable source of interesting bacteria with applications in agronomy and the food industry, where an increasing interest in natural, safe, new, and effective solutions against the harmful effects of global warming has been detected. 

 

ACKNOWLEDGEMENT

 

Authors are thankful for the New Dawn University, Burkina Faso to provide necessary facilities during this work. 

 

AUTHOR’S CONTRIBUTIONS

 

Kouhounde SHS: formal analysis, conceptualization, writing original draft. Kpode UD: formal analysis, critical review. Bonane H: conceptualisation. Yaovi RC: critical review, formal analysis. Adeoti K: data organization. Dicko MH: critical review. Final manuscript was checked and approved by all authors.    

 

DATA AVAILABILITY

 

The related author can provide the empirical data supporting the study's conclusions upon request.

 

FUNDING

 

This research has received financial supportfrom the European Union’s Horizon Europe Research, innovation program through URBANE under the grant Agreement 101059232. 

 

CONFLICT OF INTEREST

 

There are no conflicts of interest in regard to this project.

 

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