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, 10 (5), e0127259
eCollection

Characterization of Microbial Dysbiosis and Metabolomic Changes in Dogs With Acute Diarrhea

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Characterization of Microbial Dysbiosis and Metabolomic Changes in Dogs With Acute Diarrhea

Blake C Guard et al. PLoS One.

Abstract

Limited information is available regarding the metabolic consequences of intestinal dysbiosis in dogs with acute onset of diarrhea. The aim of this study was to evaluate the fecal microbiome, fecal concentrations of short-chain fatty acids (SCFAs), as well as serum and urine metabolites in healthy dogs (n=13) and dogs with acute diarrhea (n=13). The fecal microbiome, SCFAs, and serum/urine metabolite profiles were characterized by 454-pyrosequencing of the 16S rRNA genes, GC/MS, and untargeted and targeted metabolomics approach using UPLC/MS and HPLC/MS, respectively. Significantly lower bacterial diversity was observed in dogs with acute diarrhea in regards to species richness, chao1, and Shannon index (p=0.0218, 0.0176, and 0.0033; respectively). Dogs with acute diarrhea had significantly different microbial communities compared to healthy dogs (unweighted Unifrac distances, ANOSIM p=0.0040). While Bacteroidetes, Faecalibacterium, and an unclassified genus within Ruminococcaceae were underrepresented, the genus Clostridium was overrepresented in dogs with acute diarrhea. Concentrations of fecal propionic acid were significantly decreased in acute diarrhea (p=0.0033), and were correlated to a decrease in Faecalibacterium (ρ=0.6725, p=0.0332). The predicted functional gene content of the microbiome (PICRUSt) revealed overrepresentations of genes for transposase enzymes as well as methyl accepting chemotaxis proteins in acute diarrhea. Serum concentrations of kynurenic acid and urine concentrations of 2-methyl-1H-indole and 5-Methoxy-1H-indole-3-carbaldehyde were significantly decreased in acute diarrhea (p=0.0048, 0.0185, and 0.0330, respectively). These results demonstrate that the fecal dysbiosis present in acute diarrhea is associated with altered systemic metabolic states.

Conflict of interest statement

Competing Interests: Co-author Jan Suchodolski is a PLOS ONE Editorial Board member and this does not alter our adherence to PLOS ONE Editorial policies and criteria.

Figures

Fig 1
Fig 1. Rarefaction analysis of 16S rRNA gene sequences obtained from canine fecal samples.
Lines represent the mean and error bars represent standard deviations. The analysis was performed on a randomly selected subset of 6,900 sequences per sample. NHD = acute non-hemorrhagic diarrhea; AHD = acute hemorrhagic diarrhea.
Fig 2
Fig 2. Principal Coordinate Analysis (PCoA) of unweighted UniFrac distances of 16S rRNA genes.
Blue squares = healthy dogs, green circles = acute non-hemorrhagic diarrhea (NHD), and red triangles = acute hemorrhagic diarrhea (AHD); ANOSIM for healthy dogs vs. dogs with AD (NHD and AHD combined), p = 0.0040; and ANOSIM for NHD or AHD vs. healthy dogs, p = 0.0020 for both.
Fig 3
Fig 3. Differentially abundant bacterial groups.
Groups differentially abundant between healthy dogs and dogs with acute diarrhea. Red bars represent bacterial groups associated with dogs with acute diarrhea, while green bars represent bacterial groups associated with healthy dogs.
Fig 4
Fig 4. Heatmap illustrating the relative abundance of predominant bacterial genera in fecal samples.
Healthy = healthy dogs; NHD = acute non-hemorrhagic diarrhea; AHD = acute hemorrhagic diarrhea. “Unclass.” denotes an unclassified genus within the respective taxa.
Fig 5
Fig 5. Groups of interest based on A) Sequencing (% of sequences) and B) quantitative PCR results. The qPCR data was expressed as log amount of DNA for each particular bacterial group per 10 ng of isolated total DNA.
Acute Diarrhea = both groups combined (NHD and AHD). Bars represent the median value for each group. P-values adjusted based on the Benjamini and Hochberg false discovery rate.
Fig 6
Fig 6. A) Differentially abundant gene families identified within healthy dogs and dogs with acute diarrhea. B) Taxa contributing to the MAC protein gene and Tranposase enzyme gene (K03406 and K07483, respectively) (Table 3) in all dogs.
H.__ = Healthy, A.__ = acute non-hemorrhagic diarrhea, R.__ acute hemorrhagic diarrhea.
Fig 7
Fig 7. Proportion of total fecal SCFA concentrations.
Circles = healthy dogs; Squares = acute diarrhea both groups combined (NHD and AHD). Bars represent the median value for each group. P-values adjusted based on the Benjamini and Hochberg false discovery rate.
Fig 8
Fig 8. Correlations between SCFAs and bacterial groups.
A) Correlations between butyric acid and select bacterial groups. B) Correlations between propionic acid and select bacterial groups.
Fig 9
Fig 9. Analysis of serum metabolites.
A) Principle component analysis plot where red dots represent dogs with acute diarrhea while green dots represent healthy dogs. Ellipses show the 95% confidence distribution for each group. B) Relative concentration of kynurenic acid, tryptophan, and their ratio to one another in serum (p = 0.0048, 0.8282, and 0.0036; respectively).
Fig 10
Fig 10. Analysis of urine metabolites.
A) Principle component analysis plot where red dots represent dogs with acute diarrhea while green dots represent healthy dogs. Ellipses show the 95% confidence distribution for each group. B) Relative concentration of 2-methylindole and 5-methoxy-1H-indole-3-carbaldehyde in serum (p = 0.0185 and 0.0330, respectively).

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"The author(s) received no specific funding for this work."
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