Effects of Bacillus subtilis ATCC PTA-122264 on apparent total tract macronutrient digestibility and fecal characteristics, metabolites, and microbiota of healthy adult dogs

Abstract

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.

The objective of this study was to determine the effects of Bacillus subtilis ATCC PTA-122264 on dietary nutrient digestibility and the fecal characteristics, metabolites, and microbiota of healthy adult dogs. Dry matter, organic matter, and energy digestibility tended to be reduced and a few fecal microbiota were affected, but the data demonstrate that daily supplementation of up to 5 × 10⁹ colony-forming units of the Bacillus strain tested is safe and does not affect markers of general health and fecal characteristics of healthy dogs.

Introduction

The gastrointestinal tract harbors a complex microbial community that is crucial for maintaining intestinal and host homeostasis through various mechanisms, including defense against intestinal pathogens, supplying nutrients, facilitating nutrient digestion and absorption, enhancing barrier function, promoting intestinal development, and modulating the immune system (Paone and Cani, 2020; Hill and Round, 2021; De Vos et al., 2022). Disturbances in the gut microbiota, evidenced by reduced diversity, microbiota imbalances, or altered metabolite profiles, can lead to a range of diseases and disorders, including diarrhea, allergies, obesity, stress symptoms, chronic enteropathy, and inflammatory bowel diseases (Lee and Hase, 2014; Pilla and Suchodolski, 2021; Ziese and Suchodolski, 2021).

Because gastrointestinal disorders and stool quality issues are common in companion animals, it is important to identify and test substances and strategies that may support gastrointestinal health, including dietary fibers, prebiotics, and probiotics. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). Incorporating live microorganisms into the diet or through supplementation can promote the growth of beneficial, nonpathogenic bacteria, thereby supporting healthy gut microbiota and its associated metabolites. While bacterial strains, dosages, and delivery format may differ, probiotics have the potential to inhibit the growth of pathogenic bacteria by competing for nutrients and binding sites in the intestinal epithelium, modulating the immune system, producing antimicrobial substances, and acidifying the gastrointestinal tract (Schmitz, 2021; Yang and Wu, 2023). They may also increase short-chain fatty acid (SCFA) production, reduce diarrhea duration, and reduce the concentration of putrefactive compounds that contribute to fecal odor (Schmitz, 2021; Yang and Wu, 2023).

Bacteria of the Bacillus genus, such as the facultative anaerobe Bacillus subtilis, possess the ability to form spores (Biourge et al., 1998). This trait enhances their viability in food and provides resistance to acidic gastric pH (Ngo Thi Hoa et al., 2000; Coppola and Gil-Turnes, 2004; Félix et al., 2010). Spores are dehydrated and, upon exposure to suitable nutrients and moisture in the gastrointestinal tract, germinate and proliferate (Moir, 2006). Studies have demonstrated that B. subtilis can improve fecal odor and consistency, increase fecal dry matter (DM) %, and reduce fecal ammonia concentrations in dogs (Félix et al., 2010; Paap et al., 2016; Rychen et al., 2017; Bastos et al., 2020; Allenspach et al., 2023). Although some probiotics have been shown to increase nutrient digestibility, the supplementation of B. subtilis has not been demonstrated to have that effect in dogs (Félix et al., 2010).

Even though a few studies have tested B. subtilis supplementation in dogs, strains within a given bacterial species are known to differ genotypically and may have different functional capacities, justifying research on each individually. The current study aimed to determine the apparent total tract macronutrient digestibility of diets fed to healthy adult dogs supplemented with B. subtilis ATCC PTA-122264 and evaluate how its consumption affected serum chemistry, hematology, and fecal characteristics, metabolite concentrations, microbiota populations, and immunoglobulin A (IgA) concentrations. We hypothesized that B. subtilis supplementation would beneficially shift the fecal microbiota populations, beneficially alter fecal metabolite concentrations, and increase fecal IgA concentrations without affecting macronutrient digestibility, serum chemistry, or hematology.

Materials and Methods

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

Animals, diets, and experimental timeline

A replicated 3 × 3 Latin square design experiment was conducted. Twelve healthy adult spayed female dogs (6 ± 1.14 yr old; 8.71 ± 0.91 kg; body condition score 5.42 ± 0.40) were used. All dogs were housed in an environmentally controlled facility at the University of Illinois Urbana-Champaign. Dogs had free access to fresh water at all times. On the basis of the maintenance energy requirement for adult dogs and information from previous feeding records, an amount of food to maintain body weight was offered and intake was measured once daily (8:00 to 9:00 a.m.). Dogs were weighed and body condition was assessed (Laflamme, 1997) once a week prior to feeding. A commercial diet containing no probiotics or prebiotics and little fermentable fiber (Best Dog 21/12; Mid-South Feeds Inc., Alma, GA) was fed to all dogs. The diet was formulated to meet all Association of American Feed Control Officials nutrient recommendations for adult dogs at maintenance (AAFCO, 2023). The analyzed nutrient composition of the diet is listed in Table 1. The following treatments were tested: Control [diet + placebo (1.25 g of maltodextrin)]; Low [diet + 1 × 10⁹ colony-forming units (CFU)/d of B. subtilis ATCC PTA-122264 (Kerry, Inc., Beloit, WI)]; and High [diet + 5 × 10⁹ CFU/d of B. subtilis ATCC PTA-122264]. Placebo and B. subtilis treatments were provided prior to each meal with gelatin capsules.

Table 1.

Analyzed chemical and energy composition of the diet¹ fed to dogs

Item
Dry matter, %	88.42
---- Dry matter basis ----
Organic matter	91.55
Ash	8.45
Crude protein	22.95
Acid-hydrolyzed fat	13.81
Total dietary fiber	22.75
Insoluble fiber	20.63
Soluble fiber	2.12
Gross energy, kcal/g	5.00

¹Ingredients: Ground Yellow Corn, Chicken Byproduct Meal, Pork Meat and Bone Meal, Wheat Middlings, Poultry Fat (Preserved with Mixed Tocopherols), Salt, Calcium Propionate, Potassium Chloride, Artificial Garlic Flavoring, Calcium Carbonate, Vitamin E (as D-Alpha Tocophecyl Acetate), Riboflavin Supplement, Niacin Supplement, Biotin, Calcium Pantothenate, Vitamin A Supplement, Menadione Sodium Bisulfite Complex (source of Vitamin K Activity), Thiamin Mononitrate (source of Vitamin B1), Pyridoxine Hydrochloride (source of Vitamin B6), Vitamin B12 Supplement, Vitamin D3 Supplement, Ferrous Sulfate, Zinc Sulfate, Zinc Oxide, Manganese Sulfate, Copper Sulfate, Sodium Selenite, Calcium Iodate, Cobalt Carbonate, Folic Acid, Mineral Oil.

The experiment was composed of 3 28-d periods. Each experimental period included a 22-d diet adaptation phase, 5-fecal collection phase, and 1 d for blood collection.

Fecal collection, scoring, and handling

During the total fecal collection phase, total feces excreted were collected from each dog, weighed, and frozen at −20 °C until analyses. During the collection phase, all fecal samples were scored using the following scale: 1 = hard, dry pellets, small hard mass; 2 = hard, formed, dry stool; remains firm and soft; 3 = soft, formed, and moist stool, retains shape; 4 = soft, unformed stool, assumes the shape of container; and 5 = watery, liquid that can be poured.

During the fecal collection phase, one fresh fecal sample (within 15 min of defecation) was collected for measurement of pH and dry matter content, microbiota populations, metabolite concentrations, and IgA concentrations. Fecal metabolites of interest include SCFA, which serve as an important energy source for colonocytes, and protein fermentative products [ammonia; branched-chain fatty acids (BCFA); phenols; indoles] that are responsible for fecal odor and are associated with gastrointestinal disease. Fecal aliquots for analysis of phenols and indoles were frozen at −20 °C immediately after collection. One aliquot was collected and placed in 2 N hydrochloric acid for ammonia, SCFA, and BCFA analyses. An aliquot of fresh feces was immediately transferred to sterile cryogenic vials (Nalgene, Rochester, NY), placed on dry ice, and then stored at −80 °C until microbiota and IgA analysis. An additional aliquot was used for fecal DM determination.

Blood collection and analysis

Blood samples were immediately transferred to appropriate vacutainer tubes for hematology (#367841 BD Vacutainer Plus plastic whole blood tube—Lavender with K₂EDTA additive) and serum chemistry (#367974 BD Vacutainer Plus plastic serum tube—red/gray with clot activator and gel for serum separation; BD, Franklin Lakes, NJ) measurement. Tubes for serum isolation were centrifuged at 1,300 × g at 4 °C for 10 min (Beckman CS-6R centrifuge; Beckman Coulter Inc., Brea, CA). Once serum was collected, it was transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for serum chemistry analysis. K₂EDTA tubes were cooled (but not frozen) and then transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for hematology analyses.

Chemical analyses

Fecal samples were dried at 55 °C in a forced-air oven and then diet and fecal samples were ground in a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ) through a 2-mm screen. Diet and fecal samples were analyzed for DM and ash according to AOAC (2006; methods 934.01 and 942.05), with organic matter calculated. Crude protein was calculated from Leco (TruMac N, Leco Corporation, St. Joseph, MI) total nitrogen values according to AOAC (2006; method 992.15). Total lipid content (acid-hydrolyzed fat) was determined according to the methods of the American Association of Cereal Chemists (AACC, 2000) and Budde (1952). The total dietary fiber of diets was determined according to Prosky et al. (1992). Gross energy was measured using an oxygen bomb calorimeter (model 6200, Parr Instruments, Moline, IL).

Fecal IgA concentrations

Fecal samples were vortexed with phosphate-buffered saline (10 mg feces; 100 µL phosphate-buffered saline), followed by centrifugation for 20 min (1,000 × g). The supernatants were collected for measurement of IgA using a commercial ELISA kit (#MBS018650, MyBio-Source, San Diego, CA).

Fecal DNA extraction and PacBio sequencing of 16S rRNA gene amplicons

Total DNA from fecal samples was extracted using Mo-Bio PowerSoil kits (MO BIO Laboratories, Inc., Carlsbad, CA). Concentrations of extracted DNA were quantified using a Qubit 3.0 Fluorometer (Life Technologies, Grand Island, NY). The quality of extracted DNA was assessed by electrophoresis using agarose gels (E-Gel EX Gel 1%; Invitrogen, Carlsbad, CA). The Roy J. Carver Biotechnology Center at the University of Illinois performed PacBio sequencing. The 16S rRNA gene amplicons were generated with the barcoded full-length 16S rRNA gene primers from PacBio and the 2× Roche KAPA HiFi Hot Start Ready Mix (Roche, Wilmington, MA). Full-length 16S PacBio (Pacific Biology, Menlo Park, CA) primers (forward: AGRGTTYGATYMTGGCTCAG; reverse: RGYTACCTTGTTACGACTT) were added in accordance with the PacBio protocol. Amplicons were pooled and converted into a library with the SMRT Bell Express Template Prep Kit 3.0.(Pacific Biology, Menlo Park, CA). The library was sequenced on a SMRT cell 8M in the PacBio Sequel IIe using the CCS sequencing mode and a 15-h movie time. Analysis of CCS was done using SMRT Link V11.1.0 using the following parameters: minimum passes 3, and minimum rq 0.999; HiFi presets (minimum score of 80; minimum end score of 50, minimum reference [read] span of 0.75); asymmetric (different, minimum number of scoring barcode regions 2).

Microbial data analysis

PacBio-based FASTQ reads were processed using a Nextflow-based workflow, targeted amplicon diversity analysis (TADA) TADA used DADA2 v1.22 (Callahan et al., 2019) for trimming and denoising reads based on protocols used for PacBio data to generate amplicon sequence variants (ASV). The DADA2 implementation of the Ribosomal Database Project classifier (Lan et al., 2012)) was used to classify reads using the SILVA 138.1 release, with a database formatted for PacBio HiFi read data (https://zenodo.org/record/4587955). Multiple sequence alignment and maximum likelihood phylogenetic analysis were performed using DECIPHER v2.22 (Wright, 2015)) and FastTree v2.1.10 (Price et al., 2009). QIIME 2 (Caporaso et al., 2011) was used to process the resulting sequence data, using only raw sequence amplicons with quality control values ≥20 according to the DADA2 pipeline (Callahan et al., 2016). Samples were rarefied to 49,463 reads. The taxonomic classifications produced by DADA2, as well as its quantifications, were imported into phyloseq (version 1.44.0) in R (version 4.3.1). The rarefied samples were used for alpha diversity analyses, including observed ASV, Shannon Index, Inverse Simpson Index, and Fisher Index. Rarified samples were also used for beta diversity analysis, with principal coordinate analysis performed using weighted and unweighted unique fraction metric (UniFrac) distances. ANOVA-like differential expression version 2 was estimated using the ALDEx2 package (version 1.35.0) to determine specific taxa that were statistically responsible for the observed discrimination between treatments, with Benjamini–Hochberg adjusted P value, and q < 0.05 was accepted as statistically significant. Analysis of compositions of microbiomes with bias correction version 2 (ANCOM-BC2) was also estimated using the ANCOMBC package (version 2.4.0) to determine specific taxa that were statistically responsible for the observed discrimination between treatment, with Benjamini–Hochberg adjusted P value, and q < 0.05 was accepted as statistically significant. Spearman’s rank correlation coefficients (r) were carried out using a microbiome package (version 1.24.0), with Benjamini–Hochberg adjusted P value and q < 0.05 being accepted as statistically significant.

Quantitative polymerase chain reaction (PCR) and dysbiosis index

DNA was extracted from an aliquot of 100 to 120 mg fecal sample using a bead-beating method with a Mo-Bio Powersoil DNA isolation kit. The qPCR assays were applied to quantify total bacteria, Blautia, Clostridium (Peptacetobacter) hiranonis, Escherichia coli, Faecalibacterium, Fusobacterium, Streptococcus, and Turicibacter. The qPCR assays were conducted as described previously (AlShawaqfeh et al., 2017). Both positive and negative controls were included for all qPCR assays to ensure the accuracy and reliability of the results. The DI was calculated based on the results of the qPCR assays using a previously described algorithm (AlShawaqfeh et al., 2017), with a DI < 0 and with all targeted taxa within the reference interval considered normal, a DI < 0 but with any of the targeted taxa outside the reference interval defined as the minor shift in the microbiome, a DI between zero and 2 defined as a mild to moderate microbiome shift, and a DI > 2 classified as significant dysbiosis.

Statistical analysis

Data were analyzed using the Mixed Models procedure of SAS (SAS Institute, Inc., Cary, NC). The fixed effect of treatment was tested. Dogs were considered a random effect for all analyses. Data were tested for normality using the UNIVARIATE procedure of SAS. Differences between treatments were determined using a Fisher-protected least significant difference, with a Tukey adjustment to control for experiment-wise error. A probability of P < 0.05 was accepted as being statistically significant and P < 0.10 being trends. Reported pooled standard errors of the means were determined according to the Mixed Models procedure of SAS.

Results

The body condition score, food and energy intake, fecal output, and fecal characteristics of dogs were not affected by B. subtilis supplementation (Table 2). Body weight tended to be lower (P = 0.0717) with B. subtilis supplementation. The apparent total tract digestibilities of DM, organic matter, and energy tended to be reduced by B. subtilis supplementation. The digestibilities of protein and fat were not affected by treatment.

Table 2.

Apparent total tract macronutrient and energy digestibilities and body weight, body condition score, food and energy intake, fecal output, and fecal characteristics of dogs supplemented with B. subtilis

Item	Control	Low	High	SEM1	P value
Body weight, kg	8.78	8.73	8.53	0.36	0.0717
Body condition score2	5.29	5.42	5.38	0.12	0.3200
Food intake
g food/d as-is	174.7	172.4	169.4	14.34	0.8250
g food/d DM	154.5	152.5	149.7	12.68	0.8187
kcal/d	772.6	762.3	748.7	63.38	0.8316
Fecal output
g/d as-is	107.6	114.9	118.5	14.26	0.3368
g/d DM	37.08	41.56	40.63	2.55	0.4338
Fecal score3	2.56	2.51	2.62	0.11	0.2631
Fecal DM4	34.59	35.76	35.22	0.69	0.3681
Digestibility, %
Dry matter	75.97	73.38	72.41	1.19	0.0716
Organic matter	79.88	77.75	76.87	1.02	0.0811
Protein	80.66	78.30	78.15	1.40	0.2801
Fat	82.37	79.84	79.48	2.29	0.6269
Energy	80.79	78.93	77.84	0.98	0.0608

¹SEM = pooled standard error of the means.

²Nine-point body condition scoring system was used (Laflamme, 1997).

³Fecal score: 1 = hard, dry pellets, small hard mass; 2 = hard formed, dry stool, remains firm and soft; 3 = soft, formed and moist stool, retains shape; 4 = soft, unformed stool, assumes shape of container; 5 = watery, liquid that can be poured. Values reported are 5-d averages during the total fecal collection period.

⁴Values reported are 5-d averages during the total fecal collection period.

The serum albumin:globulin ratio was higher (P < 0.05) in dogs allotted to the Control treatment than those allotted to the High treatment (Table 3). Serum phosphorus, sodium, and blood urea nitrogen:creatinine ratio tended to be affected (P < 0.10) by B. subtilis supplementation, but all remained within the reference ranges for adult dogs. All other serum metabolite concentrations and hematological outcomes were unaffected by B. subtilis supplementation (Table 3 and Table 4).

Table 3.

Serum metabolite concentrations of dogs supplemented with B. subtilis

Item	Reference ranges1	Control	Low	High	SEM2	P value
		--- mg/dL ---
Creatinine	0.5 to 1.5	0.65	0.67	0.67	0.02	0.2267
Blood urea nitrogen	6 to 30	14.00	13.67	13.33	0.53	0.2860
Calcium	7.6 to 11.4	9.78	9.82	9.82	0.12	0.8500
Phosphorus	2.7 to 5.2	3.77	3.23	3.33	0.18	0.0929
Glucose	68 to 126	86.83	86.83	85.83	2.72	0.9098
Total bilirubin	0.1 to 0.3	0.25	0.26	0.24	0.02	0.6856
Cholesterol	129 to 297	202.75	205.00	204.75	12.34	0.8736
Triglycerides	32 to 154	58.83	52.33	51.00	4.55	0.1221
		--- g/dL ---
Total protein	5.1 to 7.0	5.73	5.76	5.75	0.09	0.9512
Albumin	2.5 to 3.8	3.13	3.15	3.12	0.06	0.7261
Globulin	2.7 to 4.4	2.60	2.61	2.63	0.07	0.7647
		--- mmolL ---
Sodium	141 to 152	145.42	146.17	146.00	0.46	0.0791
Potassium	3.9 to 5.5	4.28	4.36	4.23	0.08	0.2311
Chloride	107 to 118	113.50	113.75	113.50	0.62	0.7677
Bicarbonate	---	19.58	20.00	20.08	0.74	0.7229
		--- U/L ---
Alkaline phosphatase	7 to 92	45.67	39.50	41.75	4.39	0.6645
Corticosteroid-induced alkaline phosphatase	0 to 40	3.33	3.08	3.25	1.45	0.4850
Alanine aminotransferase	8 to 65	23.92	23.25	24.92	2.72	0.3927
Creatine phosphokinase	26 to 310	100.50	102.25	94.50	14.85	0.3769
Anion gap	8 to 25	16.58	16.83	16.58	0.62	0.8744
Blood urea nitrogen:creatinine	---	21.77	20.63	20.22	0.89	0.0940
Albumin:globulin ratio	0.6 to 1.1	1.22a	1.21ab	1.18b	0.04	0.0435
Sodium:potassium ratio	28 to 36	34.08	33.83	34.67	0.65	0.3589

¹Reference ranges provided by the University of Illinois Veterinary Diagnostic Laboratory.

²SEM = pooled standard error of the means.

^abMean values within the same row with unlike superscript letters differ (P < 0.05).

Table 4.

Hematology of dogs supplemented with B. subtilis

Item	Reference range1	Control	Low	High	SEM2	P value
Red blood cell, 106/µL	5.5 to 8.5	7.29	7.20	7.30	0.17	0.8136
Reticulocyte count, %	---	0.59	0.69	0.48	0.11	0.1058
Reticulocyte count, µL	---	44220	49537	35150	8575	0.1491
Hemoglobin, g/dL	12 to 18	16.01	15.93	16.04	0.37	0.9627
Hematocrit, %	35 to 52	48.14	47.74	48.11	1.05	0.9296
Mean cell volume, fl	58 to 76	66.11	66.38	65.92	0.52	0.1918
Mean corpuscular hemoglobin, pg	20 to 25	21.97	22.14	21.97	0.20	0.3376
Mean corpuscular hemoglobin concentration, g/dL	33 to 38.6	33.23	33.36	33.33	0.13	0.6255
Platelets, 103/µL	200 to 700	281.81	232.08	248.74	35.36	0.4480
Mean platelet volume, fl	---	10.65	10.87	10.72	0.18	0.1207
White blood cell count, 103/µL	6 to 17	5.58	5.31	5.70	0.44	0.5878
Neutrophils, %	---	66.00	65.89	66.68	1.74	0.8706
Lymphocytes, %	---	25.74	25.36	24.38	1.79	0.7909
Monocytes, %	---	3.64	3.98	3.25	0.48	0.5404
Eosinophils, %	---	4.65	4.70	4.31	0.96	0.5301
Basophils, %	---	0.26	0.35	0.29	0.07	0.6986
Neutrophils, 103/µL	3 to 11.5	3.75	3.39	3.81	0.42	0.6436
Lymphocytes, 103/µL	1 to 4.8	1.40	1.34	1.36	0.12	0.8648
Monocytes, 103/µL	0.2 to 1.4	0.20	0.22	0.19	0.03	0.7673
Eosinophils, 103/µL	0.1 to 1	0.24	0.24	0.24	0.05	0.9960
Basophils, 103/µL	0 to 2	0.01	0.02	0.02	0.00	0.5176

¹Reference ranges provided by the University of Illinois Veterinary Diagnostic Laboratory.

²SEM = pooled standard error of the means.

Fecal characteristics, metabolite concentrations, and IgA concentrations were not affected by B. subtilis supplementation (Table 5). Fecal bacterial alpha diversity indices (Figure 1) and beta diversity, as assessed by unweighted and weighted UniFrac distances (Figure 2), were not affected by B. subtilis supplementation. Fecal dysbiosis index tended to be lower (P = 0.0755) with B. subtilis supplementation, but all remained within the normal range (Table 6). Fecal Streptococcus and Blautia abundances were greater (P < 0.05) in dogs fed the High B. subtilis dose than those fed the Low B. subtilis dose (Table 6). Also, fecal E. coli abundance was lower (P < 0.05) in dogs fed the Low B. subtilis dose than in control dogs and dogs fed the High dose of B. subtilis (Table 6).

Table 5.

Fresh fecal sample characteristics and metabolite concentrations of dogs supplemented with B. subtilis

Item	Control	Low	High	SEM1	P value
Fecal score2	3.00	2.63	2.75	0.21	0.1461
pH	7.11	7.12	6.99	0.15	0.7966
Dry matter, DM, %	30.27	31.13	31.68	0.83	0.2091
	--- (μmol/g DM) ---
Total SCFA3	472	460	468	23.70	0.8369
Acetate	286	277	285	11.20	0.6319
Propionate	115	115	116	12.72	0.9863
Butyrate	71.1	68.7	67.0	10.70	0.8411
Total BCFA3	23.6	17.4	19.3	3.508	0.5200
Isobutyrate	8.35	7.35	7.70	0.896	0.7512
Isovalerate	10.40	7.81	8.63	1.155	0.2191
Valerate	4.83	2.20	2.96	1.687	0.6788
Total phenols + indoles	11.4	12.1	12.7	0.733	0.3429
Total phenols	3.06	2.97	3.10	0.173	0.7967
Phenol	0.47	0.37	0.37	0.074	0.9585
4-methylphenol	0.22	0.21	0.24	0.022	0.4160
4-ethylphenol	2.37	2.39	2.49	0.113	0.6788
Total indoles	8.38	9.11	9.63	0.592	0.2113
Indole	4.25	4.65	4.88	0.324	0.2430
7-methylindole	1.15	1.19	1.30	0.129	0.3200
2-methylindole	2.98	3.28	3.46	0.260	0.2887
Ammonia	81.9	74.6	73.0	4.476	0.3414
Immunoglobulin A, mg/g DM	6.72	6.95	7.05	0.35	0.7656

¹SEM = pooled standard error of the means.

²Fecal score: 1 = hard, dry pellets, small hard mass; 2 = hard formed, dry stool, remains firm and soft; 3 = soft, formed and moist stool, retains shape; 4 = soft, unformed stool, assumes shape of container; 5 = watery, liquid that can be poured.

³Total SCFA: short-chain fatty acids (acetate + propionate + butyrate); total BCFA: branched-chain fatty acids (isobutyrate + isovalerate + valerate).

Figure 1.

Bacterial alpha diversity measures (Observed ASV, Shannon Index, Inverse Simpson Index, Fisher index) of fecal samples from dogs supplemented with B. subtilis.

Figure 2.

Bacterial beta diversity indices of fecal samples from dogs supplemented with B. subtilis, as assessed by unweighted and weighted UniFrac distances.

Table 6.

Fecal bacterial abundance (log DNA/gram feces) and dysbiosis index of dogs supplemented with B. subtilis

Item	Reference range	Control	Low	High	SEM	P value
Dysbiosis index	<0	−3.69	−4.14	−3.40	0.82	0.0755
		--- Log DNA ---
Total bacteria	---	11.09	11.03	11.08	0.05	0.3158
Faecalibacterium	3.4 to 8.0	5.33	5.45	5.49	0.13	0.6096
Turicibacter	4.6 to 8.1	7.99	7.74	7.88	0.23	0.2841
Streptococcus	1.9 to 8.0	5.20ab	4.74b	5.31a	0.56	0.0366
Escherichia coli	0.9 to 8.0	4.97a	4.23b	4.97a	0.30	0.0108
Blautia	9.5 to 11.0	9.95ab	9.78b	10.02a	0.13	0.0321
Fusobacterium	7.0 to 11.3	9.12	9.08	9.06	0.14	0.8152
Clostridium hiranonis	5.1 to 7.1	6.24	6.00	6.18	0.12	0.1936

^abMean values within the same row with unlike superscript letters differ (P < 0.05).

Using 16S rRNA gene sequencing, the relative abundances of fecal microbiota were not affected by B. subtilis supplementation (Figure 3). The most abundant bacterial phyla were Firmicutes (recently renamed Bacillota; Control: 69.3% ± 4.2%, Low: 67.9% ± 3.4%, High: 66.8% ± 3.7%), Bacteroidota (Control: 17.1% ± 3.1%, Low: 18.7% ± 2.2%, High: 19.9% ± 2.8%), and Fusobacteriota (Control: 8.5% ± 1.2%, Low: 8.4% ± 1.2%, High: 8.3% ± 1.4%). The most prevalent genera were Lactobacillus (Control: 14.0% ± 3.2%, Low: 13.8% ± 2.8%, High: 14.7% ± 3.4%), Allobaculum (Control: 10.9% ± 3.2%, Low: 13.2% ± 2.1%, High: 11.1% ± 3.7%), and Bacteroides (Control: 7.7% ± 1.9%, Low: 8.4% ± 2.8%, High: 8.3% ± 1.7%).

Figure 3.

Relative abundances of the predominant bacterial phyla and genera of fecal samples from dogs supplemented with B. subtilis.

Fecal bacterial genera were significantly correlated with fecal metabolite concentrations (Figure 4). Fecal Prevotella and Fournierella relative abundances were positively (P < 0.05) correlated with fecal total SCFA concentrations. Fecal Holdemanella relative abundance was positively (P < 0.05) correlated with fecal total phenol concentrations. Fecal Terrisporobacter relative abundance was negatively (P < 0.05) correlated with fecal total phenol concentrations. Fecal UCG-002, Olsenella, Dubosiella, Cetobacterium, and Allobacullum relative abundances were positively (P < 0.05) correlated with fecal valerate and butyrate concentrations. Fecal Blautia, Megamonas, Slackia, and Streptococcus relative abundances were negatively (P < 0.05) correlated with fecal valerate and butyrate concentrations. Fecal Holdemanella, Slackia, and Sutterella relative abundances were positively (P < 0.05) correlated with fecal acetate and phenol concentrations. Fecal Epulopsicium, Terrisporobacter, UCG-002, Olsenella, Dubosiella, and Parasuterella relative abundances were negatively (P < 0.05) correlated with fecal acetate and phenol concentrations. Fecal Anaerobiospirillum relative abundance was negatively (P < 0.05) correlated with fecal score. Finally, fecal Sellimonas relative abundance was positively (P < 0.05) correlated with fecal DM percentage.

Figure 4.

Heatmap of significant correlation values (r) between fecal microbial relative abundances and metabolite concentrations. Significant correlations (q < 0.05) are indicated by ‘ + ’.

Discussion

In the current experiment, B. subtilis supplementation was safe and well tolerated by all dogs on trial, without negatively affecting serum metabolites, hematology, or body condition score. There were slight reductions in the apparent total tract DM, organic matter, and energy digestibilities, but no adverse effects on food and energy intake, fecal characteristics and output, fecal metabolites, or the apparent total tract digestibilities of protein and fat. The lack of change to protein and fat digestibility aligns with findings from earlier studies testing other Bacillus strains. Studies testing Bacillus cereus CIP 5832 (7.5 × 10⁶ CFU/d), B. subtilis C-3102 (1 × 10¹⁰ CFU/g supplement used in diet at 0.01%), or a combination of B. subtilis (3.66 × 10⁷ CFU/kg of diet) and B. licheniformis (3.66 × 10⁷ CFU/kg of diet) in dogs also did not observe effects on nutrient or energy digestibilities (Biourge et al., 1998; Félix et al., 2010; Bastos et al., 2020). The trends toward reduced DM, organic matter, and energy digestibilities in dogs receiving the high dose of B. subtilis in the current study suggest a potential benefit in those requiring weight loss or with diabetes whereby controlled nutrient absorption may be desirable. Further research would be necessary to validate these potential benefits.

B. subtilis strains have been reported to impact fecal characteristics and metabolite concentrations, with supplementation being shown to improve fecal scores and/or reduce fecal odor without affecting fecal pH or output (Félix et al., 2010; Paap et al., 2016; Rychen et al., 2017; Bastos et al., 2020; Allenspach et al., 2023). The impacts that B. subtilis supplementation may have on other fecal characteristics and metabolite concentrations have varied across studies. B. subtilis was reported to increase fecal DM content and reduce fecal ammonia concentrations in 7 to 8-mo-old puppies (Félix et al., 2010), but reduce biogenic amine, phenol, and quinoline concentrations in adult dogs (4 yr old) without influencing fecal DM or ammonia, SCFA, and BCFA concentrations (Bastos et al., 2020).

The Safety and Efficacy report from the EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP) highlighted 4 studies testing the effects of B. subtilis in dogs (Rychen et al., 2017). Study #1 was the study conducted by Félix et al. (2010) that is described above. In Study #2, B. subtilis was reported to increase fecal DM % in adult dogs (4- to 8 yr old) after 7 and 14 d of supplementation, but without any differences after 4 wk of consumption and no changes in fecal ammonia concentrations. In Study #3, fecal DM % was reported to increase after 33 d of supplementation without impacting fecal ammonia concentration in the same dogs. In Study #4, the researchers reported no change to fecal ammonia concentrations in 8-wk-old puppies (Rychen et al., 2017).

The absence of observed effects on fecal pH and SCFA concentrations from B. subtilis supplementation may partly be attributed to the type of samples collected (i.e., feces) and their inability to accurately assess microbial activity in the small intestine and proximal large intestine. It is well known that SCFA are rapidly absorbed through the gut lining or used by colonocytes, resulting in very low fecal concentrations relative to that of the colonic digesta (Herschel et al., 1981; Von Engelhardt et al., 1989). A lack of change to fecal pH in dogs fed Bacillus spp. may also relate to the low capacity that many Bacillus strains have for lactate production relative to that of Lactobacillus spp. and other lactic acid bacteria (Félix et al., 2010).

Fecal IgA, the predominant immunoglobulin isotype in the gastrointestinal tract, is produced through both T-dependent and T-independent immune responses. Immunoglobulin A plays a crucial role in mediating protective immunity against enteric pathogens, including viruses, bacteria, and toxins, and contributes to maintaining intestinal homeostasis (Bunker et al., 2015). In a previous study focused on the health of puppies given 3−7 × 10⁸ CFU/d of B. subtilis DSM 15544 (Calsporin^®, Calpis Co, Ltd., Tokyo, Japan), greater fecal IgA concentrations were observed after 43 d in the supplemented group (6.76 mg/g wet feces) than controls (3.48 mg/g wet feces; Rychen et al., 2017). In the present study whereby, healthy adult dogs were tested; however, fecal IgA concentrations were not different among those consuming B. subtilis (6.95 to 7.05 mg/g dry feces) and controls (6.72 mg/g dry feces). The fecal DM % was not reported in the previous study so a direct comparison of the values cannot be performed. It is unknown whether variations in fecal moisture content affected the results of the previous study.

Supplementation with B. subtilis DE-CA9TM (1 × 10⁹ CFU/d) in healthy adult dogs has been reported to reduce the abundance of fecal Faecalibacteria and Turicibacter, which are SCFA producers (Allenspach et al., 2023). The dysbiosis index remained within the normal reference range for all dogs throughout that study, however, so these changes did not appear to be negative (Allenspach et al., 2023). The current study generated similar findings, showing that the low dose of B. subtilis mildly influenced the fecal microbiota without affecting the dysbiosis index. Even though the dysbiosis index was normal for all dogs, the low dose of B. subtilis reduced fecal Streptococcus spp. and E. coli abundances. Because these bacterial taxa are associated with dysbiosis and play the role of potential pathogens (Minamoto et al., 2014; Vázquez-Baeza et al., 2016; AlShawaqfeh et al., 2017; White et al., 2017), the mild reduction in their abundance would be viewed as positive. The high dose of B. subtilis increased Blautia spp. abundance in the current study. Although the increase was small, the bacterial taxa are known to produce SCFA and its abundance is often lower in dogs with chronic enteropathies or diarrhea (Minamoto et al., 2014; Vázquez-Baeza et al., 2016; AlShawaqfeh et al., 2017). In the present study, fecal Blautia spp. abundance was negatively correlated with fecal valerate and butyrate concentrations, but positively correlated with fecal acetate concentrations. Given that Blautia spp. is a primary producer of acetate (Hosomi et al., 2022), this positive correlation was anticipated. Despite this correlation, however, increased fecal acetate concentrations were not observed in any treatments.

In a previous study, Bacillus spp. including most strains of B subtilis were shown to secrete lipopeptide molecules that inhibit quorum sensing and intestinal colonization by Staphylococcus aureus in mice (Piewngam et al., 2018). Piewngam et al. (2023) reported a similar inhibition of S. aureus colonization in the gastrointestinal tract and nasal cavity of humans receiving an oral supplementation of B. subtilis. In 3-wk-old piglets, B. subtilis supplementation has been shown to reduce hepatic and splenic Listeria monocytogenes counts following a challenge of the foodborne pathogen. Collectively, the results of the current study suggest that daily supplementation of B. subtilis may also have the capability of reducing potentially pathogenic bacteria in dogs, but more research is required for validation. The gastrointestinal microbiota populations are difficult to change in healthy adults. In future studies, it may be of value to evaluate B. subtilis supplementation in animals that are in a compromised state (e.g., dysbiosis), under stress (e.g., travel, separation from others), or with active gastrointestinal disease to test potential benefits under those conditions.

In conclusion, the data from the current study suggests that daily administration of up to 5 × 10⁹ CFU/d of B. subtilis ATCC PTA-122264 is safe and does not negatively impact general health, nutrient and energy digestibilities, fecal characteristics, or fecal metabolite concentrations in healthy adult dogs. Additionally, the low dose (1 × 10⁹ CFU/d) may have beneficial effects on the fecal microbiota, namely a reduction in the abundance of potentially pathogenic bacteria (Streptococcus spp. and E. coli).

Glossary

Abbreviations

ANCOMBC

analysis of compositions of microbiomes with bias correction

AOAC

Association of Official Analytical Chemists

BCFA

branched-chain fatty acids

DM

dry matter

SCFA

short-chain fatty acids

UniFrac

unique fraction metric

Conflict of interest statement

J. F. Menton, E. Vinay, and M. Millette are employees of Kerry Group and M. R. Kelly is a private consultant for Kerry Group. All other authors have no conflict of interest.

Author contributions

Patrícia Oba (Data curation, Formal analysis, Investigation, Writing—original draft, Writing—review & editing), Olivia Swanson (Data curation, Formal analysis, Investigation), Yifei Kang (Formal analysis), Julio Mioto (Data curation, Formal analysis, Investigation), John Menton (Resources, Writing—review & editing), Elena Vinay (Resources, Writing—review & editing), Mathieu Millette (Conceptualization, Resources, Writing—review & editing), Melissa Kelly (Conceptualization, Resources, Writing—review & editing), and Kelly Swanson (Conceptualization, Funding acquisition, Project administration, Supervision, Writing—review & editing)