Molecular Typing of S. aureus Isolates

To demonstrate the clonal diversity of the analyzed S. aureus strains, they were subjected to molecular typing by pulsed-field gel electrophoresis (PFGE), using CHEF Bacterial Genomic DNA Plug Kits (Bio-Rad, Marnes-la-Coquette, France) according to the procedure described by Masiuk et al. [15 (link)]. Briefly, digestion of whole-genomic DNA was performed using the SmaI enzyme (MBI Fermentas, Ontario, Canada) according to the manufacturer’s protocol. PFGE was conducted using a CHEF DR III apparatus (Bio-Rad, Marnes-la-Coquette, France) in 2% agarose gel (DNA Gdansk, Gdansk, Poland) and 1×TBE (Tris-borate-EDTA) buffer with specific parameters of electrophoresis described by Masiuk et al. [15 (link)]. After electrophoresis, the gel was stained with ethidium bromide in a covered container for 30 min, visualized and photographed using a GelDoc-It2 Imager gel imaging system (Analytik Jena US LLC, Upland, CA, USA). S. aureus ATCC 25904 and S. aureus ATCC 6538 strains were included in the study.
The PFGE macrorestriction profiles were analyzed using FPQuest software (Bio-Rad, Marnes-la-Coquette, France). Classification of individual restriction patterns for each genetic profile was conducted using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Dice’s coefficient (2.0%). PFGE results were presented as a dendrogram.