Cleancut agarose

PFGE was performed as described by Vela et al. (2003) [24 (link)] with some changes. Briefly, the overnight cultures of Streptococcus spp. isolates on CAB were suspended in saline to obtain a density of 4 according to the McFarland standard. An equal volume of bacterial suspension and 2% CleanCut Agarose (Bio-Rad, Hercules, CA, USA) were mixed to prepare agarose discs. The agarose discs were incubated overnight at 37 °C with 1 mg/mL of lysozyme (Sigma-Aldrich, Steinheim am Albuch, Baden-Württemberg, Germany) and then incubated for another 24 h at 50 °C with 500 μg/mL of proteinase K (A&A Biotechnology, Gdańsk, Poland). To perform the restriction digest, a solution with 20 U/μL of SmaI (Thermo Fisher Scientific, Waltham, MA, USA) was prepared and incubated overnight at 25 °C. A total of 1.1% (w/v) agarose gel was used for the electrophoresis with the following parameters: a running time of 21 h, a temperature of 14 °C, a voltage gradient of 6 V/cm, an initial pulse time of 1 s and a final pulse time of 30 s. The reference strain S. dysgalactiae subsp. equisimilis ATCC^®12394 was also used in this study.

DNA plugs for pulse-field gel electrophoresis were prepared using CHEF Genomic DNA Plug Kits (Bio-Rad Laboratories Inc., Hercules, CA, USA) as follows. DOA9 was cultured for 6 d at 28°C in peptone salts yeast extract medium [16 (link)]. Bacterial cells were collected by centrifugation, washed twice with 0.85% NaCl, and resuspended with 0.85% NaCl to an OD₆₀₀ of 5. The cell suspension (0.5 mL) was centrifuged at 8,000 × g for 5 min at 10°C, then resuspended with 0.5 mL of cell suspension buffer and mixed thoroughly with 0.5 mL of 2% CleanCut agarose (Bio-Rad Laboratories Inc., Hercules, CA, USA). The mixture was transferred to plug molds and solidified at 4°C for 30 min. The solidified agarose plugs were treated with lysozyme (1 mg/mL) at 37°C for 4 h and with proteinase K (30 U/mL) at 50°C overnight. Fragments of 225–6,000 kb and 225–2,200 kb were separated on 0.8% certified megabase agarose (Bio-Rad Laboratories Inc., Hercules, CA, USA) in TAE buffer or 1% certified megabase agarose in 0.5xTBE buffer, respectively. Contour-clamped homogeneous electric field (CHEF) electrophoresis was conducted at 14°C in a temperature-controlled cooling unit using the autoalgorithm mode in the CHEF Mapper gel electrophoresis system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The gel was stained with 0.5 mg mL⁻¹ ethidium bromide for 1 h and then destained in H₂O for 1 h.

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.