Animals and study design
A total of seven healthy, privately-owned dogs without previous history of infectious or inflammatory skin disease were included in the study (Additional File 1). Their age ranged from 1.5 to 14 years (mean age: 6.3 years). There were 4 intact female dogs and 3 neutered male dogs. The animals were not treated with antibiotics, anti-inflammatory agents, or immunosuppressive drugs for at least four weeks prior to sample collection or with topical treatments or antiseptic bathing during at least two weeks prior to sample collection. The dogs were to undergo different surgical procedures (ovariectomy, splenectomy and resection of abdominal cystic mass, and removal of benign skin tumours) and samples were taken immediately before the interventions. The owners gave written consent to the collection of samples, and the hospital's research committee approved the procedure.
Two sets of samples of healthy abdominal skin were collected from each dog, one for microbiological culture and the other for microbiota sequencing. Each set was composed of swab samples (from environment and skin surface) and one cutaneous biopsy that was divided into epidermis, dermis, and subcutaneous tissue (Fig. 1).
For the traditional bacteriological culture, the skin surface was sampled using a cotton sterile swab (Deltalab; Rubí, Spain), which was deposited on a modified Stuart transport-medium until culture. For microbiota sequencing, we sampled using a sterile nylon swab (Zymo Research, Irvine, CA), which was introduced in DNA/RNA Shield lysis tubes (Zymo Research, Irvine, CA) and kept at 4ºC until sequencing. In both cases, prior to sampling swabs were moistened with sterile 0.9% sodium chloride solution (FisioVet®, B.Braun Melsungen AG; Mesulgen, Germany) and then rubbed for 60 seconds over a 4 cm2 area of skin. Also, sterile swabs were deposited on the sterilized surgical field while the samples were obtained and processed (environmental blank samples).
After collecting the skin surface samples, the skin biopsies were obtained. For surgical skin preparation, hair was removed using a disinfected electric clipper, and, subsequently, skin was rubbed using gauze impregnated in a soap solution based on 4% chlorhexidine digluconate (ClorhexVet®, B.Braun Melsungen AG; Mesulgen, Germany). After that, the skin was scrubbed according to a standardized protocol based on 1% chlorhexidine digluconate aseptic solution scrubs (Desinclor® 1%, Imark; Ajalvir, Madrid, Spain) alternated with 96% alcohol. This procedure was repeated 3 times, with a 30-second contact time in each step.
After surgical skin disinfection, two 6-mm biopsies were obtained under sterile conditions from an area of the abdominal skin close to the site where skin surface swabs were taken. Each skin biopsy was sectioned into epidermis, dermis, and subcutis on a sterile surgical field to avoid microbial contamination during the process. Each sample was dissected using sterile surgical material. To minimize sample cross-contamination, gloves and sets of surgical material were replaced between the processing of each sample.
Specifically, epidermis was obtained by sectioning a layer approximately one millimeter thick from the outermost part of the biopsy sample with a scalpel blade. Then, the outermost 3 millimeters of the sample were dissected and classified as dermis, while the rest of the sample was considered subcutaneous tissue.
To evaluate the skin microbiota in deeper layers, we considered three types of negative controls: i) at sample collection, a swab was placed on the working surface during the whole process to assess the environmental contamination; ii) at DNA extraction, a swab without any sample was processed with the samples to assess the potential contamination of the DNA extraction kit; iii) at the PCR step, a PCR without any sample was processed to assess the potential contamination of the PCR kit.
Bacterial culture
Samples for aerobic bacterial culture (environmental blank, skin surface swab, and epidermal, dermal, and subcutaneous tissue) were sent in transport medium to Leti® Animal Health Laboratories (Barcelona, Spain) and processed within 2 days after collection. Streak plating from each sample on blood agar, MacConkey agar, and Sabouraud Chloramphenicol agar was performed, and then the sample was incubated at 37 °C for 48 hours.
DNA extraction
Skin swab and biopsy samples were collected and kept at 4ºC in RNA/DNA Shield lysis tubes (Zymo Research, Irvine CA) until the DNA extraction. Bacterial DNA was extracted using the ZymoBIOMICS™ DNA Microprep Kit (Zymo Research, Irvine CA) following the manufacturer’s instructions. DNA samples (20 μl) were stored at −20 °C until further processing. To assess for contamination from the laboratory or reagents, a blank sample with a sterile swab tip was processed for each extraction batch. Total DNA was quantified using Nanodrop.
V4 16S PCR amplification and massive sequencing
The hypervariable V4 region of 16S rRNA gene was amplified for each sample and negative control. A negative template control (NTC) for the PCR was also included. The forward primer was composed of the adapter linker, the key, the barcode (different for each sample), and the forward primer 515F (5’-GTGYCAGCMGCCGCGGTAA-3’). The reverse primer was composed of the adapter linker and the R806 reverse primer (5’-GGACTACNVGGGTWTCTAAT-3’). The Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA) was used to amplify the V4 16S rRNA gene. PCR mixture (25 μl) contained 2.5 μl of DNA template, 5 μl of 5× Phusion® High Fidelity Buffer, 2.5 μl of dNTPs (2 mM), 0.5 μM of each primer, and 0.5 U of Phusion® Hot Start II Taq Polymerase. The thermal profile consisted of an initial denaturation of 30 sec at 98°C, followed by 30 cycles of 15 sec at °98 C, 15 sec at 55°C, 20 sec at 72°C, and a final extension of 7 min at 72°C. After DNA purification using an Agencourt AMPure XP kit (Beckman Coulter, Brea, CA) with a ratio 1:1, quality and quantity of PCR products were determined using Agilent Bioanalyser 2100 (Agilent, Santa Clara, CA) and Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA).
The pool containing barcoded samples and negative controls was sequenced on an Ion Torrent Personal Genome Machine (PGM) with the Ion 318 Chip Kit v2 (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions.
Quality control of the sequences and ASV filtering
Raw reads were demultiplexed and imported into the Quantitative Insight Into Microbial Ecology 2 (QIIME 2) software program[31], which was used to analyze the whole dataset. DADA2 was used as quality filtering method in order to denoise, dereplicate single-end sequences, and remove chimeras[32]. The forward primer was removed, and the sequences were truncated at a length of 220 bases. Amplicon Sequence Variants (ASVs) were used to classify the sequences and assign taxonomy using SILVA release 132[33] at 99% of similarity and trimmed to the V4 region with the primers used, as reference database.
With the initial ASV table, we removed chloroplasts and mitochondria sequences since they are not bacterial members of the microbiota. Moreover, Shewanellaceae and Halomonadaceae families were also removed, since they presented the highest abundance in the NTC sample (Additional file 6).
After this first filtering step, we applied decontam R package [34] to remove other contaminants. Since these are low-biomass samples, we applied the prevalence method as recommended by the authors. Since different environments need to be decontaminated differently (especially when there are differences on initial biomass) [34], we applied the software three times: i) to skin swab samples and blanks, ii) to the biopsies and blanks, and iii) to the whole dataset. Contaminants that appeared TRUE in the biopsies approach or in two out of the three approaches were removed from the final ASV table (Additional File 7).
Taxonomy and diversity analysis
For carry out diversity analyses, we worked at the sequencing depth of 6,000 sequences/sample. With the final filtered ASV table (Additional File 5), alpha diversity was calculated using observed ASVs and the Shannon Index. Beta diversity was assessed with Bray-Curtis, Weighted UniFrac and Unweighted UniFrac.
Statistical significance of the alpha diversity values between skin samples groups (skin surface, epidermis, dermis, subcutaneous tissue, and environmental blank) was assessed using a pairwise Wilcoxon test (paired) in R, and p-values were corrected for FDR. Statistical significance of the beta diversity clustering by skin region was assessed using PERMANOVA.