Modulation of Canine Gut Microbiota by Prebiotic and Probiotic Supplements: A Long-Term In Vitro Study Using a Novel Colonic Fermentation Model

Abstract

The gut microbiota plays a crucial role in dogs' health, influencing immune function, digestion, and protection against pathogens. This study evaluates the effects of three canine dietary supplements-Microbiotal (prebiotic), Lactobacillus reuteri (probiotic), and a combination of both-on the gut microbiota composition of a healthy canine donor using an in vitro colonic fermentation model. The SCIME™ platform, adapted to simulate the canine gastrointestinal tract, was used to monitor microbial shifts in the luminal and mucosal environments of the proximal and distal colon over a 2-week treatment period. The microbial communities were analyzed using 16S rRNA sequencing to assess changes at various taxonomic levels. Alpha- and beta-diversity indices were calculated, while LEfSe and treeclimbR were employed to identify taxa-driving microbial shifts. Results indicated that all treatments led to significant modulations in key microbial groups, with enrichment of Limosilactobacillus, Bifidobacterium, Prevotella, and Faecalibacterium. These changes suggest improved saccharolytic fermentation and butyrate production, particularly when prebiotics and probiotics were co-administered. This study highlights the promising benefits of combined prebiotic and probiotic supplementation in promoting gut health and microbial diversity, providing a basis for future studies targeting the metabolic activity of the gut microbiota using the same supplements and technology.

Keywords: canine gut microbiota; colonic fermentation model; microbial diversity; microbial shifts; prebiotic; probiotic.

Figure 1

Standard setup of the Simulator of the Canine Intestinal Microbial Ecosystem (SCIME™), consisting of four sequential reactors, simulating the different canine gastrointestinal tract regions.

Figure 2

Modified version of the SCIME into a Triple-M-SCIME used for the current study. St + SI: vessel serving as stomach and small intestine, PC: proximal colon, and DC: distal colon.

Figure 3

Effect of treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) on alpha diversity as calculated using four different measures (observed (count of unique taxa in each sample), Chao1, Shannon, and Simpson) in the luminal proximal colon (PC) at the end of the control (CTRL) and treatment (TR) period. Three samples (A, B, C) were collected during each period, represented by different colors.

Figure 4

Effect of treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) on alpha diversity as calculated using four different measures (observed (count of unique taxa in each sample), Chao1, Shannon, and Simpson) in the luminal distal colon (DC) at the end of the control (CTRL) and treatment (TR) period. Three samples (A, B, C) were collected during each period, represented by different colors.

Figure 5

Effect of treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) on alpha diversity as calculated using four different measures (observed (count of unique taxa in each sample), Chao1, Shannon, and Simpson) in the mucosal proximal colon (PC) at the end of the control (CTRL) and treatment (TR) period. Three samples (A, B, C) were collected during each period, represented by different colors.

Figure 6

Effect of treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) on alpha diversity as calculated using four different measures (observed (count of unique taxa in each sample), Chao1, Shannon, and Simpson) in the mucosal distal colon (DC) at the end of the control (CTRL) and treatment (TR) period. Three samples (A, B, C) were collected during each period, represented by different colors.

Figure 7

Discriminant analysis of principal components (DAPC) to show differences in community composition (beta diversity) in the luminal proximal colon (PC) at the end of the control (CTRL) and treatment (TR) period following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P). Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3). Each dot represents one sample.

Figure 8

Discriminant analysis of principal components (DAPC) to show differences in community composition (beta diversity) in the luminal distal colon (DC) at the end of the control (CTRL) and treatment (TR) period following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P). Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3). Each dot represents one sample.

Figure 9

Discriminant analysis of principal components (DAPC) to show differences in community composition (beta diversity) in the mucosal proximal colon (PC) at the end of the control (CTRL) and treatment (TR) period following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P). Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3). Each dot represents one sample.

Figure 10

Discriminant analysis of principal components (DAPC) to show differences in community composition (beta diversity) in the mucosal distal colon (DC) at the end of the control (CTRL) and treatment (TR) period following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P). Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3). Each dot represents one sample.

Figure 11

Jitter plots showing abundances of different phyla in the luminal proximal colon (PC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on absolute levels. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 12

Jitter plots showing abundances of different phyla in the luminal distal colon (DC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on absolute levels. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 13

Jitter plots showing abundances of different phyla in the mucosal proximal colon (PC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on relative abundances. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 14

Jitter plots showing abundances of different phyla in the mucosal distal colon (DC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on relative abundances. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 15

Jitter plots showing abundances of the 20 most abundant families in the luminal proximal colon (PC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on absolute levels. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 16

Jitter plots showing abundances of the 20 most abundant families in the luminal distal colon (DC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on absolute levels. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 17

Jitter plots showing abundances of the 20 most abundant families in the mucosal proximal colon (PC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on relative abundances. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 18

Jitter plots showing abundances of the 20 most abundant families in the mucosal distal colon (DC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on relative abundances. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 19

Jitter plots showing abundances of the 20 most abundant genera in the luminal proximal colon (PC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on absolute levels. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 20

Jitter plots showing abundances of the 20 most abundant genera in the luminal distal colon (DC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on absolute levels. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 21

Jitter plots showing abundances of the 20 most abundant genera in the mucosal proximal colon (PC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on relative abundances. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).

Figure 22

Jitter plots showing abundances of the 20 most abundant genera in the mucosal distal colon (DC) following treatment with the different test products (Microbiotal, M; probiotic, P; and their combination, M + P) at the end of the control (CTRL) and treatment (TR) period based on relative abundances. Each color represents one of six categories (groups), i.e., CTRL_M (n = 3), TR_M (n = 3), CTRL_P (n = 3), TR_P (n = 3), CTRL_M + P (n = 3), and TR_M + P (n = 3).