Rnase

The pulsed-field gel electrophoresis (PFGE) procedure was adapted from previous research [65 (link),66 (link)] with some modifications. The S. aureus isolates collected from a 24 h culture on CAB were suspended in saline to achieve a density of 3.5 on the McFarland standard. The bacterial suspension was mixed with 2% CleanCut Agarose (Bio-Rad, Hercules, CA, USA). The obtained agarose discs were incubated for 18 h at 37 °C in a lysis solution with 2 mg/mL of lysozyme (Sigma-Aldrich, Steinheim am Albuch, Baden-Württemberg, Germany), 5 µg/mL of RNase (A&A Biotechnology, Gdańsk, Poland) and 50 µg/mL of lysostaphin (A&A, Gdańsk, Biotechnology). After the indicated time, the discs were transferred to the solution with 1 mg/mL of proteinase K (A&A Biotechnology, Gdańsk, Poland), where they were incubated for 24 h at 50 °C. The agarose discs were digested with SmaI (20 U/µL) (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 25 °C. The restriction fragments were separated in 1.2% agarose gel (w/v). The separation program was a running time of 20, a temperature of 14 °C, a voltage gradient of 6 V/cm, an initial pulse time of 5 s and a final pulse time of 30 s. The reference strain S. aureus ATCC^®25923 was also used in this study.

Bacterial DNA was isolated from 1.5 mL of the overnight culture. Cells were pelleted by centrifugation at 13,400 rcf for 5 min (centrifuge 5415 D, Eppendorf). The pellet was suspended in 100 μL of lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 1.2% Triton X-100) and 10 μL of lysostaphin (0.4 U/μL, A&A Biotechnology, Poland). For a more efficient isolation procedure, 120 mg of glass beads was added (Glasperlen, 0.10 to 0.11 mm, Sartorius StedimBiotech GmbH, Goettingen, Germany). The mixture, after mixing well by vortexing for 1 min, was incubated at 37°C for 40 min. During the incubation process, every 10 to 15 min, the mixture was briefly vortexed (10 to 15 s). The remaining DNA isolation steps were carried out using a genomic minikit (A&A Biotechnology, Poland) according to the manufacturer’s instructions with the addition of an RNA digestion step (RNase, 10 mg/mL, A&A Biotechnology, Poland). The DNA concentrations were measured using a NanoDrop ND-1000 (Thermo Scientific, USA).