In Silico Safety Assessment of Bacillus Isolated from Polish Bee Pollen and Bee Bread as Novel Probiotic Candidates

Abstract

Bacillus species isolated from Polish bee pollen (BP) and bee bread (BB) were characterized for in silico probiotic and safety attributes. A probiogenomics approach was used, and in-depth genomic analysis was performed using a wide array of bioinformatics tools to investigate the presence of virulence and antibiotic resistance properties, mobile genetic elements, and secondary metabolites. Functional annotation and Carbohydrate-Active enZYmes (CAZYme) profiling revealed the presence of genes and a repertoire of probiotics properties promoting enzymes. The isolates BB10.1, BP20.15 (isolated from bee bread), and PY2.3 (isolated from bee pollen) genome mining revealed the presence of several genes encoding acid, heat, cold, and other stress tolerance mechanisms, adhesion proteins required to survive and colonize harsh gastrointestinal environments, enzymes involved in the metabolism of dietary molecules, antioxidant activity, and genes associated with the synthesis of vitamins. In addition, genes responsible for the production of biogenic amines (BAs) and D-/L-lactate, hemolytic activity, and other toxic compounds were also analyzed. Pan-genome analyses were performed with 180 Bacillus subtilis and 204 Bacillus velezensis genomes to mine for any novel genes present in the genomes of our isolates. Moreover, all three isolates also consisted of gene clusters encoding secondary metabolites.

Keywords: Bacillus spp.; bee bread; bee pollen; pan-genome; probiotics; safety evaluation.

Figure 1

Synteny analysis of (a) BB10.1, (b) BP20.15, and (c) PY2.3. Pairwise genome alignment for the selected strains is shown in the dot plots generated by D-GENIES tool.

Figure 2

Representation of (a) BB10.1 and BP20.15 pan-genome and (b) PY2.3 gene content (extrapolated median-based line) according to how the pan-genome varies as genomes are added in random order to the analysis. The blue line represents unique genes; the red line represents new genes. The heatmap (c) represents the presence and absence of particular genes in BB10.1 and BP20.15 pan-genome. The X-axis denotes the particular species name. The yellow color indicates gene presence; the red color indicates gene absence. The Y-axis represents individual gene clustering, while the topmost axis represents genomes clustering (high-quality figure in Supplementary File).

Figure 3

Distribution of cluster of orthologous group (COG) functional categories to the proteins of isolate (a) BB10.1; (b) BP20.15; (c) PY2.3. The X-axis denotes the number of proteins and Y-axis denotes COG categories. Each alphabet represents a unique COG category. A—RNA processing and modification, C—energy production and conversion, D—cell cycle control, cell division, and chromosome partitioning; E—amino acid transport and metabolism; F—nucleotide transport and metabolism; G—carbohydrate transport and metabolism; H—coenzyme transport and metabolism; I—lipid transport and metabolism; J—translation, ribosomal structure, and biogenesis; K—transcription; L—replication, recombination, and repair; M—cell wall/membrane/envelope biogenesis; N—cell motility; O—post-translational modification, protein turnover, chaperones; P—inorganic ion transport and metabolism; Q—secondary metabolites biosynthesis, transport, and catabolism; S—function unknown; T—signal transduction mechanisms; U—intracellular trafficking, secretion, and vesicular transport; and V—defense mechanisms.

Figure 4

The percent distribution of CAZymes classes. (a). BB10.1, (b). BP20.15, (c). PY2.3. The color scheme at the bottom represents different CAZyme classes: red, CBM—carbohydrate-binding module; green, CE carbohydrate esterases; blue, GH—glycoside hydrolases; cyan, GT—glycosyl transferases; pink, PL—polysaccharide lyases; and yellow, AA—auxiliary activity.

Figure 5

The predicted probability of probiotic genes in the genomes of isolates (a) BB10.1, (b) BP20.15, and (c) PY2.3. The green color indicates probiotic genes, and the red color indicates non-probiotic genes. The x-axis values (0–100) represent the probability of a gene being a probiotic gene. The y-axis represents the contig number in the genome.