Gastrointestinal and stool quality issues are common in companion animals. In addition to dietary fibers and prebiotics, the consumption of live microorganisms may be used to support the gastrointestinal health of pets. Spore-forming Bacillus species are gaining interest due to their viability during processing, storage, and within the gastrointestinal tract. The objective of the current study was to determine the effects of B. subtilis ATCC PTA-122264 supplementation on dietary apparent total tract macronutrient digestibility and the fecal characteristics, metabolites, and microbiota of healthy adult dogs. Twelve healthy adult beagle dogs (6 ± 1.14 yr
8.71 ± 0.91 kg body weight) were used in a replicated 3 × 3 Latin square design. Dogs were fed to maintain body weight and allotted to 1 of the 3 treatments each experimental period (n = 12/treatment): Control [kibble diet + placebo (1.25 g of maltodextrin)], Low [kibble diet + 1 × 109 colony-forming units (CFU)/d of B. subtilis], and High (kibble diet + 5 × 109 CFU/d of B. subtilis). Each experimental period was composed of a 22-d adaptation phase, 5-d fecal collection phase, and 1 d for blood collection. Fecal microbiota data were evaluated using QIIME2. All other data were analyzed using the Mixed Models procedure of SAS, with P <
0.05 being considered significant. B. subtilis supplementation tended to decrease (P <
0.10) apparent total tract dry matter, organic matter, and energy digestibilities but did not influence food or energy intake, fecal output, and apparent total tract protein or fat digestibilities. Most serum metabolites, hematology, fecal characteristics, and fecal bacterial alpha and beta diversity indices were not affected. Fecal dysbiosis index tended to be affected and fecal Streptococcus, Escherichia coli, and Blautia abundances were lower (P <
0.05) in dogs allotted to the Low treatment. These data suggest that daily supplementation of up to 5 × 109 CFU/d of B. subtilis ATCC PTA-122264 is safe and does not affect markers of general health and fecal characteristics of healthy dogs, warranting further exploration.