Genetic and Phenotypic Characterization of Bacillus velezensis Strain BV379 for Human Probiotic Applications

Abstract

Bacterial spore-forming Bacillaceae species, including Bacillus subtilis and Heyndrickxia coagulans, are increasingly utilized for probiotic dietary supplementation. Bacillus velezensis is a Bacillus species that is frequently used as a direct-fed microbial in animal feed but less so as a probiotic for humans. The objective of this study was to characterize the suitability of the Bacillus velezensis strain BV379 for probiotic applications by (1) in silico screening for both adverse genetic elements and putatively beneficial traits, (2) in vitro evaluation of interactions with human intestinal epithelial cells, and (3) in vitro characterization of BV379 spore viability at various temperatures, pH, and in the presence of bile salt. In silico screening of the BV379 genome revealed few genes encoding Bacillaceae-associated toxins, virulence factors, and enzymes involved in the production of toxins. While BV379 encodes five antimicrobial resistance genes, minimum inhibitory concentration assays determined that BV379 is susceptible to all eight clinically relevant antibiotics tested. Preliminary cell culture experiments showed that BV379 lysates did not adversely impact human intestinal epithelial cell viability and monolayer permeability. It was also determined that BV379 spores can easily tolerate the harsh pH, bile salt, and microaerobic conditions typical of the GI tract. Altogether, the results presented herein support the safety and potential of Bacillus velezensis strain BV379 for use as an oral probiotic.

Keywords: BV379; Bacillus velezensis; endospore; probiotic; safety; toxicology.

Figure 1

Maximum likelihood phylogenetic tree of BV379 and other Bacillus genomes constructed from concatenated genes: 16S rRNA, gyrB, rpoB, tpiA, and purH, (~9300 nt). The bar indicates the nucleotide substitution rate. (reference), reference genome; (probiotic), oral probiotics [29,72,103].

Figure 2

Effect of 48-h BV379 lysate on Caco-2 cell viability based on intracellular ATP concentrations. There were two controls: untreated Caco-2 cells (negative control) and a 100% lysis-positive control. The blank process treatment was uninoculated media processed identically to BV379 lysates. Data are expressed as the means ± standard deviation of technical triplicates. Significant differences (p < 0.05) between samples are denoted by unshared lower-case letters (a, b).

Figure 3

Effects of BV379 lysates on Caco-2 cell monolayer transepithelial electrical resistance (TEER) in duplicate experiments (n = 2, panels (A,B)). TEER was measured at before treatment (0 h) and at 2, 4, 6, 24, and 48 h after treatment. ■, Untreated Caco-2 cells; ◆, “blank”/uninoculated lysate processing control; ●, BV379 lysate treatment; ▲, LPS treatment (positive control). Values on the y-axis are plotted on a logarithmic scale.

Figure 4

BV379 colony growth on tryptic soy agar plates incubated at 35 °C under aerobic, microaerobic, or anaerobic conditions. Experiments were performed with two biological replicates. Data are expressed as the means ± standard deviation of biological duplicates. Significant differences (p < 0.05) between samples are denoted by unshared lower-case letters (a, b).

Figure 5

BV379 spore viability across a broad range of pH. Data are expressed as the means ± standard deviation of biological duplicates.

Figure 6

BV379 spore viability in 0.3% bile salt over 6 h incubation. Data are expressed as the means ± standard deviation of biological duplicates.

Figure 7

BV379 spore viability at different temperatures over time. Data are expressed as the means ± standard deviation of biological duplicates.