Ingmar S Middelbos’s research while affiliated with University of Illinois, Urbana-Champaign and other places

Four healthy adult cats were used in a crossover design to determine phylogeny and metabolic functional capacity of the cat's gastrointestinal microbiota using a metagenomic approach. Healthy adult cats (1.7 y old) were fed diets containing 4% cellulose, fructooligosaccharides (FOS), or pectin for 30 d, at which time fresh fecal samples were collected. Fecal DNA samples from each cat consuming each diet were subjected to 454 pyrosequencing. Dominant phyla determined using two independent databases (MG-RAST and IMG/M) included Firmicutes (mean= 36.3 and 49.8%, respectively), Bacteroidetes (mean= 36.1 and 24.1%, respectively), and Proteobacteria (mean= 12.4 and 11.1%, respectively). Primary functional categories as determined by KEGG were associated with carbohydrates, clustering-based subsystems, protein metabolism, and amino acids and derivatives. Primary functional categories as determined by COG were associated with amino acid metabolism and transport, general function prediction only, and carbohydrate transport and metabolism. Analysis of carbohydrate-active enzymes revealed modifications in several glycoside hydrolases, glycosyl transferases, and carbohydrate-binding molecules with FOS and pectin consumption. While the cat is an obligate carnivore, its gut microbiome is similar as regards microbial phylogeny and gene content to omnivores.

Nine young adult (1.73 ± 0.03 yr) male cats were used to determine the effects of microbial adaptation to select dietary fiber sources on changes in pH in vitro and on total and hydrogen gas, short-chain fatty acid (SCFA), and branched-chain fatty acid (BCFA) production. Cats were adapted to diets containing 4% cellulose, fructooligosaccharides (FOS), or pectin for 30 d before fecal sampling. Each cat was used as a single donor, and fecal inoculum was reacted with each of the aforementioned fiber substrates. Adaptation to dietary FOS resulted in a greater change in pH when exposed to FOS than pectin (adaptation × substrate, P < 0.001). When exposed to the FOS substrate, adaptation to dietary FOS or pectin increased hydrogen gas production (adaptation × substrate, P = 0.021). Adaptation to dietary FOS increased acetate and total SCFA production when exposed to FOS substrate in vitro (adaptation × substrate, P = 0.001). When exposed to the FOS substrate, propionate production tended to increase with adaptation to dietary cellulose (adaptation × substrate, P = 0.060). The BCFA + valerate tended to decrease with adaptation to dietary FOS when exposed to FOS substrate in vitro (adaptation × substrate, P = 0.092). Fructooligosaccharides resulted in the greatest change in pH and production of total gas (P < 0.001), hydrogen gas (P < 0.001), acetate (P < 0.001), propionate (P < 0.001), butyrate (P < 0.001), total SCFA (P < 0.001), and total BCFA + valerate production (P < 0.001). Adaptation to the FOS or pectin diet increased production of hydrogen gas with FOS and pectin substrates. Adaptation to pectin increased (P = 0.033) total gas production with FOS and pectin substrates. Overall, adaptation to either FOS or pectin led to greater SCFA and gas production, but adaptation to FOS resulted in the greatest effect overall.

A galactoglucomannan oligosaccharide (GGMO) obtained from fiberboard production was evaluated as a dietary supplement for dogs. The GGMO substrate contained increased concentrations of oligosaccharides containing mannose, xylose, and glucose, with the mannose component accounting for 35% of DM. Adult dogs assigned to a 6 × 6 Latin square design were fed 6 diets, each containing a different concentration of supplemental GGMO (0, 0.5, 1, 2, 4, and 8%) that replaced dietary cellulose. Total tract DM and OM apparent digestibilities increased (P < 0.001) linearly, whereas total tract CP apparent digestibility decreased (P < 0.001) linearly as dietary GGMO substrate concentration increased. Fecal concentrations of acetate, propionate, and total short-chain fatty acids increased (P ≤ 0.001) linearly, whereas butyrate concentration decreased (P ≤ 0.001) linearly with increasing dietary concentrations of GGMO. Fecal pH decreased (P ≤ 0.001) linearly as dietary GGMO substrate concentration increased, whereas fecal score increased quadratically (P ≤ 0.001). Fecal phenol (P ≤ 0.05) and indole (P ≤ 0.01) concentrations decreased linearly with GGMO supplementation. Fecal biogenic amine concentrations were not different among treatments except for phenylethylamine, which decreased (P < 0.001) linearly as dietary GGMO substrate concentration increased. Fecal microbial concentrations of Escherichia coli, Lactobacillus spp., and Clostridium perfringens were not different among treatments. A quadratic increase (P ≤ 0.01) was noted for Bifidobacterium spp. as dietary GGMO substrate concentration increased. The data suggest positive nutritional properties of supplemental GGMO when incorporated in a good-quality dog food.

This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP). Pooled fecal DNA samples from each treatment were subjected to 454 pyrosequencing, generating 503,280 (K9C) and 505,061 (K9BP) sequences. Dominant bacterial phyla included the Bacteroidetes/Chlorobi group and Firmicutes, both of which comprised ∼35% of all sequences, followed by Proteobacteria (13-15%) and Fusobacteria (7-8%). K9C had a greater percentage of Bacteroidetes, Fusobacteria and Proteobacteria, whereas K9BP had greater proportions of the Bacteroidetes/Chlorobi group and Firmicutes. Archaea were not altered by diet and represented ∼1% of all sequences. All archaea were members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in K9C, but none in K9BP. Less than 0.4% of sequences were of viral origin, with >99% of them associated with bacteriophages. Primary functional categories were not significantly affected by diet and were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of several gastrointestinal metagenomes demonstrated phylogenetic and metabolic similarity between dogs, humans and mice. More research is required to provide deeper coverage of the canine microbiome, evaluate effects of age, genetics or environment on its composition and activity, and identify its role in gastrointestinal disease.

Twelve young adult (1.7 +/- 0.1 yr) male cats were used in a replicated 3 x 3 Latin square design to determine the effects of fiber type on nutrient digestibility, fermentative end products, and fecal microbial populations. Three diets containing 4% cellulose, fructooligosaccharides (FOS), or pectin were evaluated. Feces were scored based on the 5-point system: 1 being hard, dry pellets, and 5 being watery liquid that can be poured. No differences were observed (P > 0.100) in intake of DM, OM, CP, or acid-hydrolyzed fat; DM or OM digestibility; or fecal pH, DM%, output on an as-is or DM basis, or concentrations of histamine or phenylalanine. Crude protein and fat digestibility decreased (P = 0.079 and 0.001, respectively) in response to supplementation with pectin compared with cellulose. Both FOS and pectin supplementation resulted in increased fecal scores (P < 0.001) and concentrations of ammonia (P = 0.003) and 4-methyl phenol (P = 0.003). Fecal indole concentrations increased (P = 0.049) when cats were supplemented with FOS. Fecal acetate (P = 0.030), propionate (P = 0.035), and total short-chain fatty acid (P = 0.016) concentrations increased in pectin-supplemented cats. Fecal butyrate (P = 0.010), isobutyrate (P = 0.011), isovalerate (P = 0.012), valerate (P = 0.026), and total branched-chain fatty acids + valerate (P = 0.008) concentrations increased with supplementation of FOS and pectin. Fecal cadaverine (P < 0.001) and tryptamine (P < 0.001) concentrations increased with supplementation of FOS and pectin. Fecal tyramine concentrations decreased (P = 0.039) in FOS-supplemented cats, whereas spermidine concentrations increased (P < 0.001) in pectin-supplemented cats. Whereas fecal concentrations of putrescine (P < 0.001) and total biogenic amines (P < 0.001) increased with FOS and pectin, the concentrations of these compounds were increased (P < 0.001) in cats supplemented with pectin. Fecal Bifidobacterium spp. concentrations increased (P = 0.006) and Escherichia coli concentrations decreased (P < 0.001) in FOS-supplemented cats. Fecal concentrations of Clostridium perfringens (P < 0.001), E. coli (P < 0.001), and Lactobacillus spp. (P = 0.030) also increased in pectin-supplemented cats. In addition to increasing populations of protein-fermenting microbiota, pectin increased production of fermentative end products associated with carbohydrate compared with protein fermentation. Pectin and FOS may be useful fiber sources in promoting intestinal health of the cat.

Nine young adult male cats were used in a 3 × 3 factorial design to determine the effects of microbial adaptation to fiber type on changes in pH, total and hydrogen gas, short‐chain fatty acid (SCFA), and branched‐chain fatty acid (BCFA) production. Cats were adapted to a diet containing 4% cellulose, fructooligosaccharides (FOS), or pectin for 30 d prior to fecal sampling. Each cat was used as a single donor, and fecal inoculum was subjected to each of the aforementioned fiber substrates. Fructooligosaccharides produced the greatest changes in pH, total gas, hydrogen gas, acetate, propionate, butyrate, total SCFA, valerate, and total BCFA production ( P <0.001) regardless of adaptation to diet. Adaptation to the FOS or pectin diets increased production of total ( P =0.043) and hydrogen ( P =0.008) gas. Greater changes in pH ( P <0.001), acetate ( P =0.001), and total SCFA ( P =0.003) were observed for FOS and pectin substrates with adaptation to these respective substrates. Adaptation to the cellulose diet increased production of propionate ( P =0.045) when exposed to the FOS or pectin substrates. Adaptation to the pectin diet increased production of valerate and total BCFA ( P =0.039 and 0.022, respectively) when exposed to the cellulose or pectin substrates. Overall, adaptation to either FOS or pectin appear to increase SCFA and gas production.

Our objective was to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gut microbiome. Six healthy adult dogs were used in a crossover design and fed diets containing 0% (K9C) or 7.5% beet pulp (K9BP). Pooled fecal DNA samples from dogs fed each diet were subjected to 454 pyrosequencing. Dominant phyla included Bacteroides/Chlorobi and Firmicutes, both of which comprised ~35% of sequences, followed by Proteobacteria (13–15%) and Fusobacteria (7–8%). K9C had greater Bacteroidetes, Fusobacteria, and Proteobacteria, while K9BP had greater Bacteroidetes/Chlorobi and Firmicutes. Archaea, all Crenarchaeota and Euryarhaeota, represented ~1% of all sequences. Three fungal phylotypes, all Dikarya, were present in K9C, but none in K9BP. Viruses represented < 0.4% of sequences, with >99% associated with bacteriophage. Primary functional categories were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups, and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of our data and that of other gut metagenomes demonstrated high phylogenetic and metabolic similarity between dogs, humans, and mice. More research is needed to provide deeper coverage of the canine microbiome and identify its role in gastrointestinal disease.

Sequences derived from dogs fed the Control diet, individual animals (5) and a pooled DNA sample. (0.73 MB ZIP)

Phylogenetic tree constructed from sequences from one dog (G). The tree displays phylogenetic relationships between the microbiome structures when the dog was fed two different diets. Sequences marked red are from the control diet, sequences in black are from the beet pulp-supplemented diet. (0.08 MB PDF)

Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure. Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5%) diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Fusobacteria (23-40% of reads), Firmicutes (14-28% of reads) and Bacteroidetes (31-34% of reads) being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering. Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases.