Gelcompar software

To amplify the ITS region, we used primers forward: 5′-GAAGTCGTAACAAGG-3′ and reverse: 5′-CAAGGCATCCACCGT-3′ as previously described [17] (link). The PCR was performed with 20-μL reaction volumes comprising 10 μL Master mix (Ampliqon, Odense, Denmark), 0.5 μL forward primer 10 pmol (Bioneer, Daejeon, Korea), 0.5 μL reverse primer 10 pmol (Bioneer, Daejeon, Korea), 8.5 μL distilled water and 50 ng bacterial DNA.
The PCR was performed in a thermocycler (PEQLAB, Erlangen, Germany) with an initial denaturation at 95°C for 5 min; and 25 cycles, including denaturation steps at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 5 min. Electrophoresis of the PCR product was performed in an 8% polyacrylamide gel. The gel was stained using 1% silver nitrate and detection used Gel Doc (GVM20 model syngene, Cambridge, UK). The digital image was stored electronically as a TIFF image and analysed with GelCompar software (Applied Maths, Sint-Martens-Latem, Belgium) by using the Dice correlation coefficient and the UPGMA method.

PFGE typing of S aureus strains was performed following the protocol of Yetkin et al. (6 ). Briefly, bacterial cells were embedded into low melting agarose including lysostaphin (5 U/mL). Cells in plugs were digested with proteinase K. After washing the plugs, genomic DNA in the plugs was restricted by 30 U of SmaI (Promega Corporation, Durham, NC, USA) for 24 hours at 25°C in a water bath. DNA fragments were separated on 1% agarose gels run in 0.5 × Tris-borate-ethylenediaminetetraacetic acid buffer using a CHEF-DR III system (Bio-Rad Laboratories, Nazareth, Belgium). The electrophoresis conditions were 14°C at 6 V/cm² for 20 hours. The initial and final switch times were 5.3 and 34.9 seconds, respectively. The gel was stained with ethidium bromide (5 μg/mL) for 20 minutes and destained with distilled water for 30 minutes. The DNA band profiles were visualized under ultraviolet light, photographed using a Gel Logic 2200 Imaging System (Kodak Co, Rochester, NY, USA), and analyzed by GelCompar software (version 7.0; Applied Maths, Sint-Martens-Latem, Belgium). A 1% band tolerance was used for the comparison of DNA profiles. The clonal relationship among isolates was evaluated using the criteria of Tenover et al. (7 (link)).

Molecular epidemiology of selected isolates was assessed by PFGE.¹⁷, ¹⁸ Briefly, bacterial cells embedded in 1.6% low‐melting‐point agarose plugs were lysed with lysozyme and proteinase K and then chromosomal DNA was digested with 40 U Sma I (Fermentas). Fragmented DNA samples were electrophoresed in 1% pulsed‐field certified agarose in 0.5× TBE buffer by the contour‐clamped homogeneous electric field method with a CHEF‐DRII drive module (Bio‐Rad Laboratories Ltd.) with 10‐40 seconds pulse times, for 21 hours at 14°C at 6 V cm. The gels were stained with ethidium bromide to detect the DNA band profiles, and the image was digitized with a Gel Doc 1000 system (Bio‐Rad Laboratories). The DNA band profiles were analyzed with GelCompar software (version 3.0; Applied Maths). Band tolerances of 1.5% and 1% normalization were used for comparison of DNA profiles.