Wolves, dogs and humans in regular contact can mutually impact each other’s skin microbiota

Humans have the least and pet dogs have the most diverse skin microbiota

Out of all four groups, species richness and diversity were lowest in human skin microbiota, whereas the pet dog group had the highest species richness and diversity. All four groups differed significantly to each other (Kruskal–Wallis; Chao1, chi-squared = 20.828, df = 3, p < 0.001; Shannon, chi-squared = 23.332, df = 3, p < 0.001). In the following pairwise comparisons all groups differed significantly from each other (p < 0.005), except for the WSC dog group vs. the WSC wolf group, that showed a very similar species richness and diversity (Dunn’s-test for multiple comparisons; Chao1, p = 0.574; Shannon, p = 0.579; Fig. 1.). The low species richness and diversity in the human skin microbiota has also been shown in previous studies^42,43 and might be driven by physiological differences of the skin between humans and canines, such as pH and hair covering, as well as by skin hygiene practices and differences in regular environmental contact^44,45. For humans, protection from invasion by microorganisms is controlled by microbial desiccation, competition with resident microbiota, and an acidic pH. The average reported cutaneous pH of humans is 4.8, while the average cutaneous pH of dogs is 7.4, suggesting that the higher pH might support a more complex skin microbiota composition, as compared to humans⁴⁵. Moreover, Clemente et al.³³ showed that the skin microbiome of uncontacted humans living outdoors is significantly more diverse than that of westernized people, supporting the assumption that modern habits, including personal and environmental hygiene, lead to a decrease in skin microbiota species richness and diversity^33,46. With respect to the high diversity of the pet dogs’ microbiota, we suggest that the pet dog skin was regularly exposed to a diverse set of environmental microbiomes, both indoors and outdoors. The WSC animals (wolves and dogs), in contrast, are restricted to the game park environment.

Humans and pet dogs are less similar compared to WSC dogs and wolves, which are more similar to each other in their skin microbiota composition

The analysis of independent variable influence, i.e. sex (male vs. female), age class (sub adult vs. adult), last antibiotic treatment (early vs. late) and human contact did not reveal significant effects on the canine skin microbiota composition (multivariate PERMANOVA in Adonis; diet, p = 0. 387; age, p = 0.136; sex, p = 0.114; last antibiotic treatment, p = 0.728, human contact, p = 0.539), but the groups differed significantly (multivariate PERMANOVA in Adonis; group, R² = 0.074, F model = 1.622, p = 0.0228). Also in beta diversity significant differences were detected between groups (PERMANOVA; Bray–Curtis dissimilarity; pseudo R² = 0.180; F model = 3.741; p = 0.001). In the pairwise comparison, all groups differed significantly from each other (R² = 0.076–0.209, F model = 1.468–6.598, p = 0.006). In the visual inspection of tSNE plots which were based on Jensen-Shannon divergence and Bray–Curtis dissimilarity (Fig. 2.), humans and pet dogs appear less similar to each other and the other groups, while WSC dogs and wolves appear more similar to each other in their skin microbiota composition. Not surprisingly, among the four groups humans appeared most distinct to all canines that grouped more closely together.

Physiological differences between canine species affecting their microbiota may be relatively small. DeCandia et al. (2019)⁴⁷ found out that coyotes, red and grey foxes living in the wild in North-America responded with a similar microbial community shift to a Sarcoptes scabiei mite infection. However, the environment and living conditions of individuals do lead to a difference in microbial composition within species⁴⁸ suggesting that environmental effects on microbiota are significant. These observations are also consistent with the pairwise alpha diversity comparisons above, where the only non-significant difference between groups was the comparison of WSC dogs to wolves.

While physiological differences of the skin are likely to contribute to the differences observed between humans and canines, in this study mutual contact and living in the same environment have likely reduced difference between the canine groups^44,45. This is probably an important factor that has made the WSC dog and wolf samples most similar to each other (Fig. 2.). This is consistent with the species richness and alpha diversity, and suggests that environmental exposure has a similar or stronger impact on shaping the skin microbiota than the evolutionary segregation of wolves and dogs due to domestication (see also^21,22,35).

It has further been suggested that diet shapes the skin microbiome⁴⁹. Here, diet does not explain the difference between the pet dog group and the WSC animals (PERMANOVA; diet, R² = 0.046, F model = 1.007, p = 0.386). Both dog groups, WSC dogs and pet dogs were fed a similar diet. However, the WSC dog and wolf groups microbial community structure was similar to each other despite being fed different diets (Supplementary Dataset 1).

The hologenome theory of evolution supports the idea that specific groups or species evolved together with their microbiomes, and that this symbiosis greatly affects their health status^50,51. A loss of microbiome diversity can be caused by several factors and may impact health. For example, a decrease in species richness and diversity can be caused by specific living conditions over several generations, as seen in both humans and canines living in or close to urban environments, as compared to populations living under natural conditions³³. Outside of these long-term shifts in the skin microbiomes of certain groups, similar changes in diversity that do not necessarily impact health, can also be caused within shorter time spans, as represented in our canine groups. The pet dogs within the current study were similar to each other in terms of beta diversity, but distinct from the WSC animals (Fig. 2). Of the pet dogs, four out of twelve were originally born at the WSC and later on adopted by the human caretakers, while remaining on the same diet; their origin however apparently did not leave remaining track in their microbiome as they were not more similar to the WSC dogs than the other pet dogs. Lastly, three of the four humans, that were a priory classified as having close physical interactions with the WSC dogs and wolves were more similar to the WSC animals than the other human participants with less animal contact (PERMANOVA; R² = 0.167, F = 1.967, p = 0.077; Fig. 2). This was also found when conducting a PCoA (Supplementary Fig. 1). Based on this finding, a new group named “human close” was used to label downstream analyses and included the three human samples that clustered closer to the animals.

The skin microbiota of humans with close contact to WSC animals is more similar to the microbiota of the WSC animals

LEfSe analysis revealed an enrichment of Acidobacteria, Verrucomicrobia and Proteobacteria (in particular Alphaproteobacteria and Gammaproteobacteria) in wolves, and Bacteroidetes and Proteobacteria (in particular Betaproteobacteria) in WSC dogs, whereas Firmicutes were enriched in human samples (Fig. 3A). Interestingly, not only the three canine groups, but also the humans with close contact to the WSC animals, had a microbiome that showed higher proportions of Proteobacteria, Acidobacteria, Verrucomicrobia, and Bacteroidetes (Fig. 4). The proportions of Firmicutes and Actinobacteria were lower in these groups compared to the humans without close contact to the WSC animals. This suggests that contact with the animals increased the ratio of gram negative to gram positive microorganisms on the skin, and the phylum level diversity. The Proteobacteria appeared to be particularly shared between humans with close animal contact and pet dogs (Fig. 4). The patterns observed at phylum level could also be seen on lower taxonomic levels in the LEfSe analysis, with a significant enrichment of f.e. Sphingomonadaceae in the wolf group (Fig. 3B), and f.e. Pseudomonadaceae in the WSC dog group (Fig. 3C). Consistent with this observation, humans with close contact to the WSC animals had microbial community shifts mainly caused by an increase in Pseudomonadaceae, Sphingomonadaceae and Flavobacteriaceae abundance and a decrease of Staphylococcacceae and Corynebacteriaceae abundance (Supplementary Fig. 2).

Lehtimaki et al.³⁹ found that intensive contact to forest and arable land (which was the case for our canine groups compared to the human individuals) correlated with a higher diversity of soil-based Proteobacteria and other soil-related taxa on skin, while in urban areas, skin-based Actinobacteria were more abundant. This was also the case in our human samples, with the exception of those from humans with very close contact to WSC animals. This supports the importance of human-animal interactions in providing exposure to environmental microbes and affecting the skin microbiome composition^2,23. These findings also indicate that not only the pet dog skin microbiota seems to be greatly affected by their surroundings, but that the human skin microbiota can also show strong shifts when being exposed to canine animals and their environment. Coelho et al.⁵³ recently indicated that the similarities in the gut microbiome of dogs and humans might not be solely explained by direct transmission, but rather a function of similar physiology and lifestyle⁵³. Lehtimaki et al.⁵⁴ also examined that a shared living environment as well as lifestyle patterns correlate with microbial similarities in dog-owner pairs and that they influence the structure of skin microbiota in both species. In our study, direct transmission might have also contributed to the similarities observed, as the skin was not cleaned before sampling thereby washing off allochthonous microorganisms, as applied in skin microbiota sampling of amphibians^43,55. However, different to former studies on pets^2,38, we found no evidence that owners and pet dogs living in the same household would have more similar skin microbiota to each other than to other pet dogs or other owners, respectively.

A rural environment or countryside lifestyle are known to boost protection against allergic diseases³⁹. This protection has suggested to be mediated by acquiring a more diverse microbiome or by exposure to environmental microbes that support immune tolerance³⁹. In this study, the three humans with close contact to the WSC animals had a microbiota structure with altered composition (PERMANOVA; R² = 0.167, F = 1.967, p = 0.077) but without higher species richness and/or diversity. Whether these changes might benefit their hosts in terms of immune responses or allergies, as suggested previously^39,46,54, needs further investigation.

The human skin microbiota

A total of 15,181 ASVs were detected in all skin samples from the four groups. In the human group, 4,676 ASVs were found, of which 581 (12%) were shared with the pet dogs. Firmicutes, in particular Staphylococcaceae were identified as significantly enriched in humans in the LefSe analysis (Fig. 3A,D). The six most abundant ASVs in the human group were classified as Staphylococcus with relative abundances from 7.22 to 2.39%. The dominance of Staphylococcaceae is in line with previous literature⁵⁶. Staphylococcus species are known to especially inhabit the human skin as commensals and opportunistic pathogens⁵⁷. Other highly abundant phylotypes detected in the current dataset have also been described as normal inhabitants of the human skin, such as Corynebacteria, that show a higher sensitivity to environmental factors compared to staphylococci⁵⁸. Cutibacterium has also been found on human skin samples. Cutibacterium acne is seen as an important species within this genus, which is linked to acne and balanced by specific Staphylococcus strains⁵⁹. Several ASVs among the 50 most abundant, which were classified as Staphylococcus, Corynebacterium and Cutibacterium were significantly higher abundant in human samples in the pairwise comparison with canine skin samples (Table 1).

The canine skin microbiota

This is the first study to describe the bacterial composition on the skin of wolves. Overall, the wolf skin was inhabited by similar abundant phylotypes compared to the dog skin. The most abundant ASVs in the three canine groups were classified as Pseudomonas, Rhodococcus, Staphylococcus, Micrococcus and Sphingomonas with relative abundances between 1.26 to 0.60% (Table 1, Supplementary Dataset 2). Several phylotypes were enriched in the wolf group in the LEfse analysis, among them Alpha- and Gammaproteobacteria, Sphingomonadaceae and Pseudomonadales. In the WSC dog group, Bacteroidetes, Betaproteobacteria and Flavobacteriaceae were enriched, while in the pet dog group Actinobacteria, Pseudomonadaceae and Sphingobacteria showed significant enrichment (Fig. 3). Only one of the 50 most abundant ASVs that differed significantly between the WSC wolf and WSC dog group (Sphingomonas ASV 48, enriched in WSC wolf skin samples compared to WSC dog skin samples), while seven ASVs differed significantly between the WSC wolf and pet dog groups (three Staphylococcus-ASVs, Streptococcus, Pedobacter, unclassified Actinomycetales and Sphingosinicella, Table 1). Except of Sphingosinicella all of these ASVs were enriched in the pet dog skin samples, compared to the WSC wolf skin samples. All highly abundant phylotypes have been found on the skin of healthy dogs before². Overall, these findings indicate that the wolf skin microbiota is similar to the dog skin microbiota, but several phylotypes are more dominant in the WSC animals. This might be largely affected by living environment²¹, but whether and to what extent the similar skin microbiota is due to the wolf and dog subjects of the current study sharing the same environment is difficult to tell. A former study on red wolves has shown that living in a captive environment significantly affects the gut microbiota of the animals, and this effect is apparent even if the animals are fed with a diet reflective of their natural environment⁶⁰. Given that our wolves also live in captivity and do share some of the facilities with the dogs, it is not surprising that the highly abundant ASVs on the WSC wolf skin were also present on the WSC dog skin. However, the fact that they were detected also on the skin of the pet dogs in this study suggests fundamental similarities in the skin microbiota of wolves and dogs, although the abundances of these phylotypes differed among groups. In this study, the shared environment of the wolves and WSC dogs seem to have impacted at least the profile of highly abundant phylotypes of the animals’ skin microbiota (Table 1, just 1 ASV was significantly different between WSC dogs and wolves) and the WSC groups had a similar alpha diversity (WSC dog group vs. the wolf group; Dunn’s-test for multiple comparisons; Chao1, p = 0.574; Shannon, p = 0.579). The shared environment might have had more impact on the alpha diversity than their differential diet and their divergent evolution, as also indicated by the lower similarity between the wolf and pet dog group (Dunn’s test; Chao1, p = 0.025; Shannon, p = 0.041) compared to the wolf and WSC dog group (Dunn’s test; Chao1, p = 0.574; Shannon, p = 0.579). The number of shared ASVs within the groups is shown in Supplementary Fig. 3. A huge amount of low abundant organisms was uniquely found in each canine group. The number of unique ASV was more than double in canine groups compared to the humans. The importance of a high skin microbiota diversity in canines, the function of unique low abundant phylotypes, as well as the importance of specific microbial enrichments in groups has not been investigated until now and has to be examined in future research.

Source: Ecology - nature.com