Effect of the Probiotic Bacillus subtilis DE-CA9TM on Fecal Scores, Serum Oxidative Stress Markers and Fecal and Serum Metabolome in Healthy Dogs

Abstract

Background: There is increasing interest in the use of Bacillus species as probiotics since their spore-forming ability favors their survival in the acidic gastric environment over other probiotic species. The subsequent germination of B. subtilis to their vegetative form allows for their growth in the small intestine and may increase their beneficial effect on the host. B. subtilis strains have also previously been shown to have beneficial effects in humans and production animals, however, no reports are available so far on their use in companion animals.

Study design: The goal of this study was therefore to investigate the daily administration of 1 × 10⁹ cfu DE-CA9^TM orally per day versus placebo on health parameters, fecal scores, fecal microbiome, fecal metabolomics, as well as serum metabolomics and oxidative stress markers in ten healthy Beagle dogs in a parallel, randomized, prospective, placebo-controlled design over a period of 45 days.

Results: DE-CA9^TM decreased the oxidative status compared to controls for advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS) and reactive oxygen metabolites (d-ROMS), suggesting an antioxidant effect of the treatment. Fecal metabolomics revealed a significant reduction in metabolites associated with tryptophan metabolism in the DE-CA9^TM-treated group. DE-CA9^TM also significantly decreased phenylalanine and homocysteine and increased homoserine and threonine levels. Amino acid metabolism was also affected in the serum metabolome, with increased levels of urea and cadaverine, and reductions in N-acetylornithine in DE-CA9^TM compared to controls. Similarly, changes in essential amino acids were observed, with a significant increase in tryptophan and lysine levels and a decrease in homocysteine. An increase in serum guanine and deoxyuridine was also detected, with a decrease in beta-alanine in the animals that ingested DE-CA9^TM.

Conclusions: Data generated throughout this study suggest that the daily administration of 1 × 10⁹ cfu of DE-CA9^TM in healthy Beagle dogs is safe and does not affect markers of general health and fecal scores. Furthermore, DE-CA9^TM administration had a potential positive effect on some serum markers of oxidative stress, and protein and lipid metabolism in serum and feces.

Keywords: Bacillus subtilis; metabolome; microbiome; oxidative stress; probiotics.

Figure 1

Fecal scores (scored using the Purina 7–point scoring scheme) over the treatment period (day 0–45) for placebo and DE-CA9^TM Treatment. All fecal scores were observed to be at scores 2 and 3, with only 2 outliers above 3 observed randomly over time. While there was an effect of time (F(8, 72) = 4.806, p < 0.0001), there was no effect of treatment (F(1, 72) = 0.7750, p = 0.3816) nor an interaction effect of time X treatment (F(8, 72) = 0.4132, p = 0.9094).

Figure 2

Total Bacteria, Turicibacter, Streptococcus, E. coli, Blautia, Fusobacteria, and Clostridium hiranonis were assessed. There was an effect of time for Total Bacteria (p = 0.0069) and Turicibacter (p = 0.0168), and an effect of DE-CA9^TM supplementation for universal log DNA (p = 0.0069), Faecalibacteria (p = 0.04), and Turicibacter (p = 0.0216), but there were no interaction effects, and no post hoc significant differences were observed within or between groups at any timepoint. Columns represent individual pens of dogs (2 dogs per pen for combined fecal samples).

Figure 3

DE-CA9^TM significantly decreased levels of d-ROMS, TBARS, and AOPP in the serum compared to baseline, whereas no such decrease was seen in the placebo group. There was a significant effect of time for FOX (p = 0.0262), dROMS (p = 0.0144), CUPRAC (p = 0.0004), FRAP (p = 0.0125), TEAC (p < 0.0001), Uric acid (p = 0.0294), AOPP (p = 0.0003), and TOS (p < 0.0001). There was no effect of treatment per se for any group, and an interaction effect was observed for TBARS (p = 0.002). Black bars represent the DE-CA9^TM group. + represents p < 0.05 compared to the placebo baseline; * represents p < 0.05 compared to the DE-CA9^TM baseline.

Figure 4

DE-CA9^TM decreased several metabolites associated with amino acid metabolism, lipid and fatty acid metabolism and tryptophan metabolism in feces from the canines. Yellow shading represents an overall significant effect of treatment between DE-CA9^TM and the placebo. * represents p < 0.05 between DE-CA9^TM and placebo at this respective timepoint.

Figure 5

DE-CA9^TM decreased several metabolites associated with amino acid metabolism, lipid and fatty acid metabolism, and tryptophan metabolism in serum from the canines. Levels of 2-aminobutyric acid, cadaverine, urea, lysine, and tryptophan were significantly increased in the DE-CA9^TM group compared to the controls. Yellow shading represents an overall significant effect of treatment between DE-CA9^TM and the placebo. * represents p < 0.05 between DE-CA9^TM and placebo at this respective timepoint.