Lysis solution

Total DNA of the S. epidermidis isolates was extracted with the ZR Fungal/Bacterial DNA MiniPrep™ kit (Zymo Research, USA), with some modifications to the manufacturer's instructions. A volume of 2 mL of overnight Brain Heart Infusion broth (Merck, Germany) was used as starting material for the extractions. Beta-mercaptoethanol [0.5% (v/v)] (Merck, Germany) was added to the DNA binding buffer (Zymo Research, USA). A volume of 600 μL of Lysis solution (Zymo Research, USA), instead of the recommended 750 μL of Lysis solution (Zymo Research, USA), was used in the ZR BashingBead™ Lysis Tubes (Zymo Research, USA). The DNA obtained was used in all downstream PCR applications.

Colostrum and milk samples collected from 54 multiparous cows were subjected to genomic DNA extraction using the ZR-96 Fungal/Bacterial DNA Kit (Zymo Research, Irvine, CA, USA) following modified protocols of the manufacturer as follows: 1.5 mL of each sample was centrifuged at 12,000×g for 20 min at 4 °C. Supernatants were carefully removed and 200 μL of TE buffer and 300 μL of 0.5 M EDTA (pH = 8) were added to the pellet. The mixture was incubated for 20 min at room temperature and again centrifuged at 12,000×g for 10 min. Supernatants were removed and pellets were resuspended by adding 200 μL of PBS buffer and vortexing for 30 s. Next, 1 g of autoclaved 0.5 mm silica beads, 400 μL of Lysis Solution (Zymo Research, CA, USA), and 18 μL of 20 mg/mL Proteinase K (Zymo Research, CA, USA) were added to each tube and incubated in a heated shaker at 45 °C for 45 min. Tubes were then vortexed for 2 min using a 2010 GenoGrinder (SPEX SamplePrep, NJ, USA) at 1700 stroke per minutes. Then, 400 μL of the resulting mixture was then transferred to the deep-well plate of the Fungal/Bacterial DNA Kit and extraction process continued following manufacturer protocol. Negative controls (n = 3) were included in the extraction process by replacing 1 mL of sterile water instead of colostrum/milk samples.

In order to isolate DNA, down and feather (50 mg) samples were homogenized in 750 µL of Lysis Solution (Zymo Research, Irvine, CA, USA), using a Bead Mill 4 (Thermo Fisher Scientific, Waltham, MA, USA). The DNA was isolated, using ZymoBIOMICS DNA Mini Kit (Zymo Research) according to the manufacturer’s protocol. The isolated DNA was quantified, using a Qubit DNA HS Assay kit (Thermo Fisher Scientific). To isolate DNA from the rinse water, the bacteria in the water was collected using a 0.2 µm filter, and the DNA was isolated from the crashed filter (5 m/s, 300 s, using Beat Mill 4) using the ZymoBIOMICS DNA Mini Kit. 16S rRNA sequencing was performed using an Oxford Nanopore MinION (Oxford Nanopore Technologies, Oxford, UK). Full-length 16S rRNA genes were amplified with 16S_barcode_27f primer (5′-TTT CTG TTG GTG CTG ATA TTG CAG RGT TTG ATC MTG GCT CAG-3′) and 16S_barcode_1492r primer (5′-ACT TGC CTG TCG CTC TAT CTT CGG YTA CCT TGT TAC GAC TT-3′). A DNA library was constructed with PCR Barcoding Kit and Ligation Sequencing Kit (Oxford Nanopore Technologies), according to the manufacturer instruction. The DNA library was applied into MinION, and the output data were analyzed by using EPI2ME software (Oxford Nanopore Technologies).