Diet exerts a significant influence on the gastrointestinal (GI) health of dogs, modulating fecal microbial populations and metabolite concentrations. Abrupt dietary change, which is common in dogs, has been associated with digestive disturbances, GI discomfort, and loose stools (1, 2). Furthermore, such dietary changes can adversely affect intestinal integrity, alter gut microbiota composition, and disrupt the production of fermentation end-products (1, 3–5). In general, significant changes in the gut microbiota occur within a few days following a dietary change. In dogs fed different diets, microbiota shifts have been observed as early as 2 d after an abrupt diet change, with stability typically achieved within 6 to 10 d (5).

Probiotic studies with companion animals have demonstrated their ability to mitigate GI disorders triggered by dietary changes. A study evaluating Bacillus subtilis C-3102 in dogs demonstrated that B. subtilis-treated dogs had firmer feces, higher fecal scores, and lower fecal ammonia concentrations than control dogs (6). Dietary supplementation with B. subtilis C-3102 has also been shown to improve fecal quality, increase nutrient digestibility, and positively shift gut health parameters by reducing ammonia concentrations, increasing fecal SCFA concentrations, and increasing fecal Lactobacillus spp. and enterococci (7). Similarly, Saccharomyces cerevisiae CNCM I-5660 supplementation was shown to reduce fecal pH and biogenic amine, ammonia, and aromatic compound concentrations, while increasing fecal butyrate concentrations and lowering the dysbiosis index (DI) of dogs following an abrupt dietary change when compared with controls (3). Collectively, those studies suggest that the consumption of live microorganisms may enhance intestinal functionality in dogs and may provide benefits during dietary transition.

Although probiotics are recognized for their potential health benefits, there is a lack of studies evaluating the specific effects of B. subtilis ATCC PTA-122264 on gut microbiota, dysbiosis index, and fecal characteristics in dogs subjected to abrupt dietary change. In a study recently conducted by our team, B. subtilis ATCC PTA-122264 supplementation reduced the relative abundances of Streptococcus, Escherichia coli, and Blautia in dogs, indicating its potential to modulate the gut microbiota (8). Those findings indicate that B. subtilis may improve stool quality, reduce fecal odor, and positively modulate the gut microbiota of dogs. Given that B. subtilis-based probiotics may have the capacity to positively influence intestinal and microbial function, they may aid during dietary transition. Therefore, the objective of the current study was to assess the fecal characteristics and microbiota of B. subtilis-supplemented dogs undergoing an abrupt dietary change. We hypothesized that dogs supplemented with B. subtilis would have a more stable gut microbiome and greater ability to adapt to a new diet.

All animal procedures were approved by the University of Illinois Institutional Animal Care and Use Committee prior to experimentation (protocol #22217).

Fecal scores and pH were not affected by B. subtilis supplementation or dietary change (Table 2). However, the abrupt dietary change reduced (p < 0.0001) fecal DM % and increased (p < 0.0001) fecal dysbiosis index (Figure 1). The dietary change also increased (p < 0.0001) fecal Streptococcus, E. coli, and C. hiranonis abundances and decreased (p < 0.01) fecal Faecalibacterium, Turicibacter, Blautia, and Fusobacterium abundances (Figure 1). Diet-induced changes to fecal characteristics, DI, and bacterial abundances were not impacted by treatment (i.e., B. subtilis supplementation) or treatment*day interactions (Figure 1).

Fecal bacterial alpha diversity measures were reduced (p < 0.05) with diet change (day effect) but were not affected by B. subtilis supplementation or treatment*day interactions (Figures 2, 3). Three of the four alpha diversity measures were affected, with observed ASV, Fisher Index, and the Shannon Index being reduced (p < 0.005) by diet change. Beta diversity, as assessed by unweighted and weighted UniFrac distances, was also affected by diet change (Figures 4, 5). The principal coordinate analysis plots of weighted and unweighted UniFrac distances show separation of microbial communities, with samples shifting after dietary change. Samples collected prior to dietary change were different (p < 0.05) than samples collected at all other time points. Moreover, samples collected 2 d after dietary change were different (p < 0.05) than those collected 14 d after dietary change.

The relative abundances of several bacterial phyla and genera were affected by diet change, but not by B. subtilis supplementation or treatment*day interactions (Figures 6, 7). Using ANCOMBC analysis, the relative abundances of fecal Turicibacter, Prevotella, Lactobacillus, Lachanospiraceae NK4A136 group, Faecalibaculum, Faecalibacterium, Cellulosilyticum, Bifidobacterium, and Anaerofilum were reduced (p < 0.05) with the dietary change. In contrast, the relative abundances of fecal Sutterella, Peptoclostridium, Fusobacterium, Escherichia-Shigella, Collinsella, Ruminococcus gnavus group, and Eubacterium brachy group were increased (p < 0.05) with the dietary change.

Abrupt dietary change is common in dogs and can lead to GI disturbances, including altered fecal consistency and fecal microbiota and metabolite shifts (1–5). While the gut microbiota adapt within a few days following a diet transition, the extent and stability of these changes depend on various factors, including microbiota balance and the presence of beneficial taxa (3, 4). Live microorganisms, such as B. subtilis, have been studied for their role in modulating gut microbiota, enhancing fecal characteristics, and mitigating digestive disturbances (6, 7). However, the specific effects of B. subtilis ATCC PTA-122264 on microbiome stability, dysbiosis index, and fecal characteristics in dogs undergoing abrupt dietary transitions remain unexplored. This study evaluated whether supplementation with B. subtilis ATCC PTA-122264 could promote a more stable gut microbiome and improve fecal characteristics in dogs following an abrupt diet change.

Consistent with previous research, diets with markedly different ingredient compositions, macronutrient profiles, and formats (wet vs. dry) were selected to facilitate shifts in the microbiome. Dogs were moved from a dry extruded kibble diet containing moderate protein and fat concentrations and high dietary fiber concentrations to a canned diet containing higher protein and fat concentrations and lower dietary fiber concentrations. The fiber type was also different with twice the amount of soluble fiber present in the canned diet. Diet transition immediately reduced fecal DM but was stabilized after 6 days. The diet change did not impact fecal scores or pH, however. Previous studies have shown that fecal scores (increased, indicating looser stools) and fecal DM (decreased) shifted and stabilized within 2 days of a dietary change (5), while fecal pH also decreased following the transition and reached stability within the same period (3).

Compared with the control, supplementation with B. subtilis ATCC PTA-122264 did not affect fecal scores, DM, or pH after the dietary change. These results were similar to the supplementation of S. cerevisiae (0.12 g per dog per day) in a previous study, where fecal pH, scores, or DM were similar to control animals following a dietary transition (3). Without the challenge of dietary transition, previous studies have reported that B. subtilis C-3102 supplementation at 1 × 10⁹ CFU/d and 2 × 10⁹ CFU/d led to firmer stools, higher fecal DM, and lower fecal pH than control dogs (7). Another study demonstrated that dietary supplementation with 0.01% B. subtilis C-3102 (1 × 10¹⁰ CFU/g) resulted in drier feces and higher fecal scores than those observed in control dogs (6). In our previous study testing B. subtilis ATCC PTA-122264 at doses of up to 5 × 10⁹ CFU/day, fecal characteristics were not affected but there was a tendency for reduced apparent total tract digestibility of DM, organic matter, and energy (8). While fecal DI tended to be affected, supplementation resulted in lower abundances of Streptococcus, E. coli, and Blautia in dogs receiving the low treatment (1 × 10⁹ CFU/day), suggesting potential microbiota-modulating effects (8). Those findings highlight strain-dependent differences in B. subtilis effects on fecal characteristics and microbiota modulation, suggesting that the abrupt dietary transition in this study may have presented a greater challenge to GI stability than B. subtilis supplementation alone could mitigate.

Transitioning from the high-fiber kibble diet to the high-protein, high-fat canned diet led to a reduction in SCFA-producing bacteria (e.g., Faecalibacterium, Turicibacter, Blautia, Fusobacterium) and an increase in potentially pathogenic bacteria (e.g., E. coli, C. hiranonis, and Streptococcus), resulting in a higher DI. Similarly, dogs fed a high-protein (44%, DM basis), high-fat (28%, DM basis) bones and raw food diet had a greater abundance of E. coli, Streptococcus, and C. perfringens, as well as a higher DI, than dogs fed commercially available kibble (31% crude protein and 18% fat, DM basis). Additionally, fecal Faecalibacterium abundance was lower in dogs consuming the bones and raw food diet than those fed kibble (16). In a previous study evaluating S. cerevisiae during an abrupt dietary change, dogs in the treatment group demonstrated a lower DI, lower E. coli abundance, and higher Turicibacter abundance than control animals, regardless of the day. Furthermore, transitioning from a low-protein (21%, DM basis), low-fiber (6%, DM basis) diet to a high-protein (28%, DM basis), high-fiber (27%, DM basis) diet improved (reduced) the DI, increased fecal Fusobacterium abundance, and decreased fecal C. hiranonis and Streptococcus abundances (3). Those findings suggest that dietary fiber content plays a significant role in microbiota modulation and that specific probiotic strains may offer benefits during dietary transition.

The ANCOMBC analysis in the current study revealed a reduction in SCFA-producing genera (e.g., Turicibacter, Prevotella, Lactobacillus, Lachanospiraceae NK4A136 group, Faecalibaculum, Faecalibacterium, Cellulosilyticum, Bifidobacterium, Anaerofilum) and an increase in potentially pathogenic bacteria (e.g., Peptoclostridium, Escherichia-Shigella, Ruminococcus gnavus group) following the abrupt dietary change. These findings are consistent with prior research showing that fiber intake in dogs promotes the growth of beneficial SCFA-producing bacteria, such as Turicibacter, Lactobacillus, Lachnospira, Faecalibacterium, Bifidobacterium and decrease Escherichia and Peptoclostridium (17–19). SCFA-producing genera, including Bifidobacterium, Lactobacillus, Faecalibacterium, Anaerofilum, Prevotella, Lachnospiraceae NK4A136 group, and Faecalibaculum utilize dietary fibers as substrates, contributing to GI health (19–25). Additionally, Cellulosilyticum has been identified as a bacterium capable of breaking down both fiber and protein, highlighting its potential role in microbial metabolism (26). High-protein diets often increase fecal Peptoclostridium, E. coli, and Streptococcus, decrease fecal Bifidobacterium and Faecalibacterium, increase concentrations of fecal protein catabolites (e.g., phenols, indoles, and branched-chain fatty acids), and reduce fecal concentrations of SCFA (16, 27–31). Moreover, C. perfringens, E. coli, and Streptococcus are recognized as potential pathogens (32–34), while Ruminococcus gnavus group has been positively associated with parvovirus GI infections in dogs (35).

Abrupt dietary changes in dogs, particularly those involving significant shifts in fiber, protein, and fat content, can lead to notable alterations in GI function, including changes in fecal characteristics and disruptions to the gut microbiota. While the gut microbiota has the capacity to adapt to dietary transitions, the extent of this adaptability can vary based on factors such as the presence of beneficial microbes. In this study, B. subtilis ATCC PTA-122264 supplementation was not able to attenuate the changes to fecal characteristics or microbiota. Other studies have shown positive effects of different probiotic strains, including Bacillus species, under stable conditions, suggesting that further exploration of different doses or strains may reveal more pronounced benefits. Nevertheless, the dietary change in this study led to an increase in potentially pathogenic bacteria and a decrease in beneficial SCFA-producing genera, which is consistent with previous research. This highlights the complexity of the relationship between diet and the gut microbiome and suggests that further investigation into the effects of different Bacillus strains and dietary interventions is necessary to better understand their role in maintaining GI health during abrupt diet changes in dogs.