The skin microbiome in healthy and allergic dogs

Abstract

Background: Changes in the microbial populations on the skin of animals have traditionally been evaluated using conventional microbiology techniques. The sequencing of bacterial 16S rRNA genes has revealed that the human skin is inhabited by a highly diverse and variable microbiome that had previously not been demonstrated by culture-based methods. The goals of this study were to describe the microbiome inhabiting different areas of the canine skin, and to compare the skin microbiome of healthy and allergic dogs.

Methodology/principal findings: DNA extracted from superficial skin swabs from healthy (n = 12) and allergic dogs (n = 6) from different regions of haired skin and mucosal surfaces were used for 454-pyrosequencing of the 16S rRNA gene. Principal coordinates analysis revealed clustering for the different skin sites across all dogs, with some mucosal sites and the perianal regions clustering separately from the haired skin sites. The rarefaction analysis revealed high individual variability between samples collected from healthy dogs and between the different skin sites. Higher species richness and microbial diversity were observed in the samples from haired skin when compared to mucosal surfaces or mucocutaneous junctions. In all examined regions, the most abundant phylum and family identified in the different regions of skin and mucosal surfaces were Proteobacteria and Oxalobacteriaceae. The skin of allergic dogs had lower species richness when compared to the healthy dogs. The allergic dogs had lower proportions of the Betaproteobacteria Ralstonia spp. when compared to the healthy dogs.

Conclusions/significance: The study demonstrates that the skin of dogs is inhabited by much more rich and diverse microbial communities than previously thought using culture-based methods. Our sequence data reveal high individual variability between samples collected from different patients. Differences in species richness was also seen between healthy and allergic dogs, with allergic dogs having lower species richness when compared to healthy dogs.

Figure 1. Principal coordinates analysis for healthy dogs.

Principal coordinates analysis of unweighted Unifrac distances of 16S rRNA genes clusters samples based on similarities of bacterial molecular phylogenetic trees. (A) No clustering differences are observed in 3 healthy dogs with fleas compared to 9 healthy dogs without fleas, demonstrating that the presence of fleas does not appear to influence the microbial diversity. (B) Similarly, there were no clustering differences between male and female dogs. (C) Clustering differences were seen in the samples collected from mucosal surfaces or mucocutaneous junctions.

Figure 2. Alpha diversity in different skin sites for healthy dogs.

Alpha diversity measures at 1000 sequences per sample in the different sites of canine skin (x axis). The y axis represent the data points for the Chao1 index (species predictor estimator) (A), number of observed species (B) and Shannon diversity index (diversity index that accounts for species abundance and evenness) (C) data points (y axis) for each skin site. Error bars represent the standard deviations. A: Axilla; C: Conjunctiva; CP: Concave pinna, DL: Dorsal lumbar; DN: Dorsal Nose; DP: Dorsal Perianal; E: Ear; G: Groin; I: Interdigital 4&5; LP: Lip commissure; N: Nostril; PO: Periocular.

Figure 3. Rarefaction curves from different skin sites from healthy dogs.

Rarefaction curves of 16S rRNA gene sequences obtained from different skin sites from healthy dogs. The analysis was performed on a randomly selected subset of 1000 and 3000 sequences per sample. Haired skin shows higher Chao1 metric, more observed species, and higher Shannon index compared to the samples from mucosal surfaces, e.g. nostril and conjunctiva. Lines represent average of each skin site, whereas the error bars represent the standard deviations.

Figure 4. Bacterial phyla and families in healthy dogs.

Average of most common bacterial phyla and families identified in different sites in the skin of healthy dogs.

Figure 5. Principal coordinates analysis for allergic versus healthy dogs.

Principal coordinates analysis plot of unweighted Unifrac distances of 16S rRNA genes. No clustering differences are observed between allergic versus healthy dogs in the samples from the nostril, axilla, groin and interdigital skin.

Figure 6. Rarefaction curves of 16S rRNA gene from allergic versus healthy dogs.

Rarefaction curve of 16S rRNA gene sequences obtained from axilla, groin, interdigital skin and nostril mucosa from allergic and healthy dogs. Lines represent average of each group, whereas the error bars represent the standard deviations. The analysis was performed on a randomly selected subset of 1000 sequences per sample.

Figure 7. Bacterial phyla and families in allergic versus healthy dogs Average of most common bacterial phyla and families identified in axilla, groin, interdigital skin and nostril in allergic and healthy dogs.