García-Fonticoba et al. Animal Microbiome https://doi.org/10.1186/s42523-020-00050-8 (2020) 2:34 Animal Microbiome RESEARCH ARTICLE Open Access The microbiota of the surface, dermis and subcutaneous tissue of dog skin Rocío García-Fonticoba1, Lluís Ferrer1,2, Olga Francino3 and Anna Cuscó4* Abstract Background: Studies using highly sensitive molecular techniques have detected bacterial communities below the human epidermis. Depending on their abundance and composition, this finding could be clinically relevant. The aim of this study was to determine if bacteria can be detected in the dermis and subcutaneous tissue of dogs without cutaneous disease using two different approaches: traditional cultures and DNA sequencing of the V4 region of bacterial 16S rRNA gene using next-generation sequencing (NGS). Results: Seven healthy dogs were included in the study, and two sets of samples were collected from each subject. Sample sets were composed of a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, a skin surface swab, and an environmental blank sample for contamination control. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA amplification and sequencing. Five different bacterial genera (Staphylococcus, Bacillus, Corynebacterium, Streptococcus, and Enterococcus) were isolated in five out of the seven skin surface swab samples with aerobic microbiological culture methods, while no growth was obtained from the other two samples. Although some DNA could be amplified from epidermal, dermal, and subcutaneous tissue samples, the results of the NGS were similar to those of the blanks. Conclusion: When investigated with aerobic microbiological culture methods, the dermis and subcutaneous tissue of dogs are sterile. NGS techniques lead to the detection of some bacterial DNA, similar to the signal detected in blanks, which does not support the presence of a microbiota in dermis or subcutaneous tissue. Keywords: Skin microbiota, Epidermis, Dermis, Subcutaneous tissue, Next-generation sequencing, Dog Background microorganisms in the pathogenesis of dermatological The study of the cutaneous microbiota has aroused enor- conditions such as atopic dermatitis [8–10]. mous interest in the last decade [1–3]. Numerous studies Culture-based techniques have been useful to detect have revealed the complexity of the microbial skin com- bacterial populations in the epidermal surface but have munities of humans, dogs, and cats [4–6]. Multiple inter- failed to detect microorganisms in the dermis and sub- actions between the skin microbiota and the immune cutaneous tissue [11, 12]. This explains why skin micro- system have also been demonstrated, so that a diverse and biota has been traditionally assumed to be restricted to balanced microbiota is considered an essential component the epidermal surface and appendageal structures, while of a healthy skin [4, 7, 8]. Furthermore, identifying and dermis and subcutaneous tissue have been considered characterizing the cutaneous microbiota can help us to sterile. However, some organisms may not be easily de- understand how microbial diversity may contribute to cu- tectable with conventional culture methods due to many taneous diseases, and to elucidate the roles of different factors [13]. The use of next-generation sequencing (NGS) techniques to characterize microbial communities * Correspondence: anna.cusco@vetgenomics.com has increased in recent years, opening the possibility of 4 Vetgenomics, Ed. Eureka, PRUAB, Campus UAB, Bellaterra, 08193 Barcelona, readily identifying bacteria that may be elusive to Spain culture-based studies [14, 15]. Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 2 of 9 Using NGS and other molecular approaches, the group submitted for bacterial culture and the other for bacter- of Nakatsuji changed the paradigm of a sterile dermis ial DNA amplification and sequencing (Fig. 1). and subcutaneous tissues [16]. These authors were able to identify a small bacterial community in the dermis Bacterial cultures and superficial subcutaneous adipose tissue of healthy Bacterial cultures from swabs of abdominal skin surface humans. A subsequent study confirmed the existence of yielded 11 isolates from 5 of the 7 dogs, while no bacter- a bacterial community in the dermis and revealed signifi- ial growth was obtained from skin surface samples of the cant differences in microbial composition when compar- other two dogs. Bacterial isolates included Streptococcus ing epidermal and dermal compartments [11]. sp., Staphylococcus sp., Corynebacterium sp., Bacillus ce- The existence of dermal microbiota is of doubtless reus, and Enterococcus sp. (Table 1). physiological importance, because these microorganisms Cultures of samples from epidermis, dermis, and sub- can play a role in the regulation of the delicate immune cutaneous tissue were negative in all cases. These sam- responses of the skin. Moreover, depending on their ples were taken from abdominal skin, following surgical abundance and composition, the presence of a dermal or skin disinfection and under sterile conditions. An envir- subcutaneous microbiota could also be clinically relevant onmental blank sample was also cultured during each [11, 16]. In animals with cutaneous lesions, skin biopsies biopsy procedure, in all cases negative. are commonly investigated with cultures and molecular techniques to detect and identify microorganisms that Microbiota analysis could have an etiologic role [17–20]. An initial assump- All the samples, including the blanks, presented DNA tion in these investigations is that the dermis and sub- amplification and were sequenced, even when the bac- cutaneous tissues of healthy animals are sterile. terial cultures were negative. We evaluated microbiota Although some authors have investigated the dermal diversity and composition in the skin surface swabs, the and subcutaneous microbiota of dogs with pyogranulo- environmental blank, and the biopsy samples, obtained matous dermatitis and panniculitis [21] and in dogs with after surgical skin disinfection, corresponding to epider- mast cell tumors [22], the composition of the microbiota mis, dermis, and subcutaneous tissue. After V4 16S of the different layers of the skin (epidermis, dermis, and rRNA amplification, skin surface samples presented the subcutaneous tissue) of dogs without cutaneous disease largest quantity of DNA when compared to inner skin has not been studied. layers and blank samples (Additional file 2). The primary aim of this study was to determine if bac- teria can be detected below the canine epidermis (dermis Microbiota diversity and subcutaneous tissue) using two different approaches: To evaluate the overall microbial community structure aerobic microbiological cultures and microbiota profiling and similarity between samples (beta diversity) in terms using DNA sequencing of the V4 region of the bacterial of abundance and composition, we used a Bray-Curtis 16S rRNA gene. Studies in low-biomass samples have to distance matrix. The associated Principal Coordinates be performed very carefully since the samples are prone to Analysis (PCoA) plot shows that skin surface samples contamination with exogenous DNA, leading to false clustered separately from the rest of the samples (Fig. 2). results [23–25]. To address this risk, we included three We observed similar trends, when analyzing Unweighted different blank samples to detect any potential environ- and Weighted UniFrac PCoA plots (Additional file 3). mental or kit contamination. The secondary aim of this Confirming this observation, PERMANOVA pairwise study was to compare the microbial profiles obtained comparisons were significant between skin surface and all using aerobic microbiological culture methods with those the skin layers and blanks (p-value < 0.05). Moreover, ex- obtained with NGS in skin samples of healthy dogs. traction blanks –but not environmental blanks– were sta- tistically different from epidermis (Unweighted UniFrac), Results dermis (Weighted UniFrac), and subcutaneous tissue A total of seven healthy, privately-owned dogs (4 intact (Bray Curtis and Weighted UniFrac) (Additional file 4). females and 3 neutered males) without previous history This implies that, in qualitative and quantitative terms, of skin disease were included in the study. Their ages the skin surface samples, obtained before surgical scrub- ranged from 1.5 to 14 years (mean age: 6.3 years) (Add- bing, have a microbiota composition very different from itional file 1). Two sets of samples were collected from the epidermis, dermis, and subcutaneous tissue, the envir- each subject. Sample sets were composed of a skin sur- onmental blank, and the extraction blank. face swab taken prior to surgical skin disinfection, a 6- To evaluate microbiota diversity within samples, we mm abdominal skin biopsy, including epidermis, dermis used two alpha diversity metrics: observed ASVs (rich- and subcutis, and an environmental (operating room) ness) and Shannon diversity index (richness and even- blank sample. One set of samples from each dog was ness) (Fig. 3). For both indexes, samples from skin García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 3 of 9 Fig. 1 Collection of samples. Seven dogs were included in the study. Two sets of samples were collected from each subject. Samples sets were composed of a skin surface swab taken prior to surgical skin disinfection, a 6-mm abdominal skin biopsy, including epidermis, dermis, and subcutis, and an environmental (operating room) blank sample. One set of samples from each dog was submitted for bacterial culture and the other one for bacterial DNA amplification and sequencing. See “Animals and study design” of “Methods” section for more detailed information surface presented the greatest diversity and were signifi- other (Fig. 4). Some of the microbial families were found cantly different from the skin layers (epidermis, dermis, ubiquitously in both skin samples and environmental subcutaneous tissue) and blanks. Epidermis, dermis, and blanks. subcutaneous tissue shared similar values and were not The most abundant phyla identified in samples from significantly different from each other. The skin layers skin surface were Proteobacteria, followed by Actinobac- presented significantly higher numbers of observed teria, Bacteroidetes, and Firmicutes. Moraxellaceae was ASVs than blanks, but the difference could not be con- the most common family, followed by Nocardioidaceae, firmed using the Shannon Index. Xanthomonadaceae, and Sphingomonadaceae. On samples from epidermis, dermis, and subcutaneous Microbiota composition tissue, the most abundant phyla were Firmicutes, Proteo- The samples from skin surface swabs showed a high de- bacteria, Actinobacteria, and Bacteroidetes. Similar find- gree of variability between dogs in terms of abundance ings were obtained when sequencing environmental of bacterial taxa. In contrast, samples from all other skin blanks, where Firmicutes and Proteobacteria were the compartments and blanks were very similar to each predominant phyla. Table 1 Number of isolated strains of bacteria from skin surface samples BACTERIAL NUMBER OF COLONIES ISOLATES Dog1 Dog 2 Dog 3 Dog 4 Dog 5 Dog 6 Dog 7 Streptococcus sp 0 4 0 0 0 0 2 Staphylococcus sp 0 1 13 0 2 0 0 Corynebacterium sp. 0 0 0 25 3 0 0 Bacillus cereus 0 0 4 1 0 0 4 Enterococcus sp 0 0 7 0 0 0 0 García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 4 of 9 Fig. 2 Principal coordinate analysis plots of Bray-Curtis distance matrices. Clustering of skin surface samples displaying similarity in abundance and composition of bacterial 16S rRNA gene sequences contained in these samples. Clustering of cutaneous skin layers and blanks reveals similarities in their microbial communities Additional file 5 shows the final ASV table for all the seen, skin swabs were the samples that presented samples, after removal of contaminants (see Methods). more DNA amplification and positive microbiological cultures, and present lower Halomonadaceae and Microbiota negative controls Shewanellaceae values (Additional file 6). Decontam R When evaluating the negative controls, some trends package allowed us to detect extra contaminants can be elucidated. We identified two clear PCR con- representing lower relative abundance such as several taminants: the families Halomonadaceae and Shewa- genera of the Burkholderiaceae family (Variovax, nellaceae. The higher the input DNA mass, the lower Ralstonia, Achromobacter, Aquabacterium), among others the concentration of the contaminants. As previously (Additional file 7). Fig. 3 Alpha diversity analysis of the skin surface, skin layers, and environmental blanks. Observed ASV (A) and Shannon index (B) were evaluated. Boxplots represent alpha diversity values at 6000 sequences/sample. Statistical comparisons were performed at this depth using a paired Wilcoxon test. P-value < 0.05 is considered significant García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 5 of 9 Fig. 4 Relative abundance of most abundant bacterial taxa per dog and type of sample. High variability is observed between dogs, with samples from skin being the most variable. Despite individual variability, samples from all skin compartments are similar to each other, and also in comparison to environmental blank samples. Families with less than 5% relative abundance are represented in grey (Other families) Discussion aerobic cultures incubated for 48 h at 37 °C. Nor were In this study, traditional culture-based methods and V4 enriched media employed; under such specific condi- 16S rRNA NGS were used to investigate the presence of tions, selection and growth pressure exist for certain a microbiota in the dermis and subcutaneous tissue of bacteria to the detriment of others. dogs with no cutaneous disease. In general, the cultured bacteria from the skin swabs Bacterial growth was obtained from samples of the were also identified in the NGS microbiota analysis, which skin surface in five of the seven dogs, while no growth agrees with previous studies [14, 27]. One exception was was observed from epidermis, dermis, subcutaneous tis- the Enterococcus sp. that was isolated from skin surface sue, or environmental blanks. These results are clinically samples of dog 3 but was not detected in the microbiota relevant, as they indicate that, when investigated with analysis. Failure of NGS to identify culturable bacteria, al- aerobic microbiological culture methods, the dermis and though previously reported [14], is challenging to justify. subcutaneous tissue of healthy dogs are considered The growth obtained using traditional culture methods sterile. may be a false positive due to contamination [28] or the Eleven isolates from five bacterial genera grew from consequence of low specificity of the V4 primer [29]. the skin surface samples, including Streptococcus sp., The NGS approach made it possible to characterize Staphylococcus sp., Corynebacterium sp., Enterococcus the skin surface microbiota and compare it with the sp., and Bacillus cereus. This contrasts with the number amplification and sequencing of the epidermis, dermis, of families detected in these samples using V4 16S rRNA and subcutaneous samples, as well as the environmental gene sequencing. Furthermore, the isolated bacteria were blank, for each dog. The skin surface had a rich and di- not necessarily the ones with a greater abundance in the verse microbiota. It was found that Proteobacteria, Acti- NGS analysis. Thus, culture-based methods overrepre- nobacteria, Bacteroidetes, and Firmicutes were the most sent the relative abundance of some easily culturable common phyla observed in the skin surface samples, species while underestimating the real microbiota diver- concordant with previous studies in healthy dogs [5, 9]. sity detected using NGS. A delicate balance among In contrast, samples from the epidermis, dermis, and temperature, nutrients, incubation time, and atmosphere subcutaneous layers showed less richness and diversity determines the success of bacterial cultures [26]. The and were more similar to the blanks. These results sug- standard culture methods present the ideal conditions gest that most of the taxa identified in epidermis, der- just for a minority of the bacteria that compose the cuta- mis, and subcutaneous tissue are probably contaminants. neous microbiota [13]. In this study, we performed only However, further investigation, including more animals, García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 6 of 9 will be needed to determine whether any low-abundance analyses are needed to determine whether some specific taxa could be inhabiting the inner layers, as suggested by low-abundant bacteria may inhabit these regions. the significant differences in observed ASVs and by the non-significant differences in Shannon index when com- paring skin layers and environmental blanks. Methods It is essential to underline that the inner layers of the Animals and study design skin are low-biomass samples, and prone to exogenous A total of seven privately-owned dogs without previous bacterial DNA contamination, which can significantly in- history of infectious or inflammatory skin disease were fluence the resulting microbial community profile [23, 24]. included in the study (Additional file 1). Their age Sample contamination can have several sources, either ranged from 1.5 to 14 years (mean age: 6.3 years). There during sample collection or during sample processing, mi- were 4 intact female dogs and 3 neutered male dogs. crobial DNA extraction, PCR amplification, and sequen- The animals were not treated with antibiotics, anti- cing [24]. In the present study, three negative controls inflammatory agents, or immunosuppressive drugs for at were processed with the samples to assess contamination least 4 weeks prior to sample collection or with topical during sample collection (environmental blank), DNA ex- treatments or antiseptic bathing during at least 2 weeks traction (extraction blank), and PCR amplification (NTC). prior to sample collection. The dogs were to undergo There are some limitations to this study. First of all, different surgical procedures (ovariectomy, splenectomy the number of dogs included in the study. Considering and resection of abdominal cystic mass, and removal of that the skin microbiota is highly variable, we may have benign skin tumours) and samples were taken immedi- missed significant differences by looking at this number ately before the interventions. The owners gave written of animals. Larger cohorts are needed to evaluate if any consent to the collection of samples, and the hospital’s low-abundant bacteria may inhabit in the skin layers. research committee approved the procedure. Secondly, the separation of the skin into its three com- Two sets of samples of healthy abdominal skin were partments (epidermis, dermis, and subcutaneous tissue) collected from each dog, one for microbiological culture was done by naked-eye dissection, without the help of and the other for microbiota sequencing. Each set was magnification instruments, as in the study of Nakatsuji composed of swab samples (from environment and skin el al. It was done in this way because this is the most surface) and one cutaneous biopsy that was divided into commonly used method in clinical dermatology when epidermis, dermis, and subcutaneous tissue (Fig. 1). investigating the dermis and subcutaneous tissue micro- For the traditional bacteriological culture, the skin sur- biologically [30]. Additionally, it is necessary to highlight face was sampled using a cotton sterile swab (Deltalab; that in the present study the existence of a microbiota Rubí, Spain), which was deposited on a modified Stuart below the epidermis has been investigated using only transport-medium until culture. For microbiota sequen- aerobic cultures and NGS. Perhaps using other highly cing, we sampled using a sterile nylon swab (Zymo Re- sensitive techniques, such as in situ fluorescence used by search, Irvine, CA), which was introduced in DNA/RNA Nakatsuji et al. [16] would have increased the probability Shield lysis tubes (Zymo Research, Irvine, CA) and kept of detecting bacterial populations that were found in at 4 °C until sequencing. In both cases, prior to sampling very small numbers. Finally, only abdominal skin sam- swabs were moistened with sterile 0.9% sodium chloride ples were included to ensure homogeneity of the sam- solution (FisioVet®, B.Braun Melsungen AG; Mesulgen, ples in the comparison. Different body regions have Germany) and then rubbed for 60 s over a 4 cm2 area of variations in anatomy and physiology, including different skin. Also, sterile swabs were deposited on the sterilized number of hair follicles and various types and abun- surgical field while the samples were obtained and proc- dance of glands. It is possible that these differences essed (environmental blank samples). could modify the ability to sustain microbial life. Further After collecting the skin surface samples, the skin bi- research would be needed to asses this point. opsies were obtained. For surgical skin preparation, hair was removed using a disinfected electric clipper, and, Conclusions subsequently, skin was rubbed using gauze impregnated In the present study, no bacteria grew from the epider- in a soap solution based on 4% chlorhexidine digluco- mis, dermis, and subcutaneous tissue of dogs when using nate (ClorhexVet®, B.Braun Melsungen AG; Mesulgen, aerobic microbiological culture methods. Conversely, Germany). After that, the skin was scrubbed according some DNA in an amount similar to the signal detected to a standardized protocol based on 1% chlorhexidine in blanks, could be amplified and sequenced from the digluconate aseptic solution scrubs (Desinclor® 1%, inner skin layers in the NGS approach. All together, Imark; Ajalvir, Madrid, Spain) alternated with 96% alco- these results reveal the absence of a clearly defined hol. This procedure was repeated 3 times, with a 30-s microbiota in the inner layers of dog skin. Further contact time in each step. García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 7 of 9 After surgical skin disinfection, two 6-mm biopsies sample), and the forward primer 515F (5′-GTGYCAGC were obtained under sterile conditions from an area of MGCCGCGGTAA-3′). The reverse primer was com- the abdominal skin close to the site where skin surface posed of the adapter linker and the R806 reverse primer swabs were taken. Each skin biopsy was sectioned into (5′-GGACTACNVGGGTWTCTAAT-3′). The Phusion epidermis, dermis, and subcutis on a sterile surgical field Hot Start II High-Fidelity DNA polymerase (Thermo to avoid microbial contamination during the process. Fisher Scientific, Waltham, MA) was used to amplify the Each sample was dissected using sterile surgical material. V4 16S rRNA gene. PCR mixture (25 μl) contained 2.5 μl To minimize sample cross-contamination, gloves and of DNA template, 5 μl of 5× Phusion® High Fidelity Buf- sets of surgical material were replaced between the pro- fer, 2.5 μl of dNTPs (2 mM), 0.5 μM of each primer, and cessing of each sample. 0.5 U of Phusion® Hot Start II Taq Polymerase. The ther- Specifically, epidermis was obtained by sectioning a mal profile consisted of an initial denaturation of 30 s at layer approximately one millimeter thick from the outer- 98 °C, followed by 30 cycles of 15 s at °98 C, 15 s at most part of the biopsy sample with a scalpel blade. 55 °C, 20 s at 72 °C, and a final extension of 7 min at Then, the outermost 3 mm of the sample were dissected 72 °C. After DNA purification using an Agencourt and classified as dermis, while the rest of the sample was AMPure XP kit (Beckman Coulter, Brea, CA) with a ra- considered subcutaneous tissue. tio 1:1, quality and quantity of PCR products were deter- To evaluate the skin microbiota in deeper layers, we mined using Agilent Bioanalyser 2100 (Agilent, Santa considered three types of negative controls: i) at sample Clara, CA) and Qubit fluorometer (Thermo Fisher Sci- collection, a swab was placed on the working surface entific, Waltham, MA). during the whole process to assess the environmental The pool containing barcoded samples and negative contamination; ii) at DNA extraction, a swab without controls was sequenced on an Ion Torrent Personal any sample was processed with the samples to assess the Genome Machine (PGM) with the Ion 318 Chip Kit v2 potential contamination of the DNA extraction kit; iii) at (Thermo Fisher Scientific, Waltham, MA) following the the PCR step, a PCR without any sample was processed manufacturer’s instructions. to assess the potential contamination of the PCR kit. Quality control of the sequences and ASV filtering Bacterial culture Raw reads were demultiplexed and imported into the Samples for aerobic bacterial culture (environmental Quantitative Insight Into Microbial Ecology 2 (QIIME 2) blank, skin surface swab, and epidermal, dermal, and software program [31], which was used to analyze the subcutaneous tissue) were sent in transport medium to whole dataset. DADA2 was used as quality filtering Leti® Animal Health Laboratories (Barcelona, Spain) and method in order to denoise, dereplicate single-end se- processed within 2 days after collection. Streak plating quences, and remove chimeras [32]. The forward primer from each sample on blood agar, MacConkey agar, and was removed, and the sequences were truncated at a Sabouraud Chloramphenicol agar was performed, and length of 220 bases. Amplicon Sequence Variants (ASVs) then the sample was incubated at 37 °C for 48 h. were used to classify the sequences and assign taxonomy using SILVA release 132 [33] at 99% of similarity and DNA extraction trimmed to the V4 region with the primers used, as ref- Skin swab and biopsy samples were collected and kept at erence database. 4 °C in RNA/DNA Shield lysis tubes (Zymo Research, Ir- With the initial ASV table, we removed chloroplasts vine CA) until the DNA extraction. Bacterial DNA was and mitochondria sequences since they are not bacterial extracted using the ZymoBIOMICS™ DNA Microprep members of the microbiota. Moreover, Shewanellaceae Kit (Zymo Research, Irvine CA) following the manufac- and Halomonadaceae families were also removed, since turer’s instructions. DNA samples (20 μl) were stored at they presented the highest abundance in the NTC sam- − 20 °C until further processing. To assess for contamin- ple (Additional file 6). ation from the laboratory or reagents, a blank sample After this first filtering step, we applied decontam R with a sterile swab tip was processed for each extraction package [34] to remove other contaminants. Since these batch. Total DNA was quantified using Nanodrop. are low-biomass samples, we applied the prevalence method as recommended by the authors. Since different V4 16S PCR amplification and sequencing environments need to be decontaminated differently (es- The hypervariable V4 region of 16S rRNA gene was pecially when there are differences on initial biomass) amplified for each sample and negative control. A nega- [34], we applied the software three times: i) to skin swab tive template control (NTC) for the PCR was also in- samples and blanks, ii) to the biopsies and blanks, and cluded. The forward primer was composed of the iii) to the whole dataset. Contaminants that appeared adapter linker, the key, the barcode (different for each TRUE in the biopsies approach or in two out of the García-Fonticoba et al. Animal Microbiome (2020) 2:34 Page 8 of 9 three approaches were removed from the final ASV table Author details 1 (Additional file 7). Fundació Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. 2Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Taxonomy and diversity analysis Spain. 3Servei Veterinari de Genètica Molecular, Departament de Ciencia For carry out diversity analyses, we worked at the se- Animal i dels Aliments, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. 4Vetgenomics, Ed. Eureka, PRUAB, Campus UAB, Bellaterra, quencing depth of 6000 sequences/sample. With the 08193 Barcelona, Spain. final filtered ASV table (Additional file 5), alpha diversity was calculated using observed ASVs and the Shannon Received: 9 July 2020 Accepted: 4 September 2020 Index. Beta diversity was assessed with Bray-Curtis, Weighted UniFrac and Unweighted UniFrac. Statistical significance of the alpha diversity values be- References 1. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC, et al. tween skin samples groups (skin surface, epidermis, der- Topographical and temporal diversity of the human skin microbiome. mis, subcutaneous tissue, and environmental blank) was Science. 2009;324:1190–2. assessed using a pairwise Wilcoxon test (paired) in R, 2. Grice EA, Kong HH, Renaud G, Young AC, Bouffard GG, Blakesley RW, et al. A diversity profile of the human skin microbiota. Genome Res. 2008;18:1043– and p-values were corrected for FDR. Statistical signifi- 50. cance of the beta diversity clustering by skin region was 3. Lederberg J. Infectious history. 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