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, 5 (3), e9768

Phylogenetic Characterization of Fecal Microbial Communities of Dogs Fed Diets With or Without Supplemental Dietary Fiber Using 454 Pyrosequencing

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Phylogenetic Characterization of Fecal Microbial Communities of Dogs Fed Diets With or Without Supplemental Dietary Fiber Using 454 Pyrosequencing

Ingmar S Middelbos et al. PLoS One.

Abstract

Background: 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.

Principal findings: 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.

Conclusions: 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Rarefaction analysis of V3 16S data from canine fecal samples.
(A) Dogs fed a low-fiber control diet. (B) Dogs fed a diet supplemented with beet pulp fiber. Each line represents a single animal or a pooled sample. Analysis was performed on a random 2,000- sequence subset from each sample. Operational Taxonomical Units (OTU) in this analysis were defined at 96% similarity.
Figure 2
Figure 2. Phylum assignment of V3 16S sequences from dogs fed diets with or without supplemental fiber.
Assignment according to the Ribosomal Database Project classifier (v10.2; ≥80% confidence). (A) Means of all individual fecal DNA samples. abColumns within phylum not sharing letters are different (P<0.05). (B) Observed values for single fecal DNA samples pooled by diet. (C) Phylum assignment of V3 16S sequences from fecal samples from individual dogs fed diets with (BP) and without (C) supplemental fiber, according to the Ribosomal Database Project classifier (v10.2; ≥80% confidence). The changes that occur in individual animals may be lost when DNA samples are pooled, or when population means are calculated.
Figure 3
Figure 3. Changes within Firmicutes in fecal samples of dogs fed diets with and without supplemental fiber.
Class assignments according to the Ribosomal Database Project classifier (v10.2; ≥80% confidence). abColumns within class not sharing letters are different (P<0.05).
Figure 4
Figure 4. UniFrac analysis of V3 16S sequences from canine fecal samples.
(A) Principal Component Analysis scatter plot of individual samples by dietary treatment (control  =  red circles; beet pulp supplemented  =  blue squares). (B) Principal Component Analysis scatter plot of individual samples combined with pooled DNA samples (pooled control  =  green triangle; pooled beet pulp  =  gold triangle). (C) A jackknifed clustering of the environments in the UniFrac dataset (100 permutations). The numbers next to the nodes represent the number of times that particular node was observed (out of 100) in a random sampling from the whole dataset.
Figure 5
Figure 5. UniFrac analysis of V3 16S sequences (Firmicutes and Fusobacteria only) from canine fecal samples.
Principal Component Analysis scatter plots of individual samples (control  =  red circles; beet pulp  =  blue squares) combined with pooled samples (pooled control  =  green triangle; pooled beet pulp  =  gold triangle). (A) Clustering within the phylum Firmicutes. (B) Clustering within the phylum Fusobacteria.

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