Gelcompar 2 5 6.5

Suspected MRSP isolates, based on resistance to oxacillin,²⁰ were confirmed to carry the mecA gene by PCR.¹⁶ (link) All confirmed isolates were stored at −80°C until further studied. To detect clonal clusters, all MRSP isolates stored in 2010–14 were digested by SmaI macrorestriction (New England BioLabs, USA) and separated by PFGE²¹ (link) with modifications.¹⁵ (link) Isolates that were non-typeable by SmaI were digested using AscI (New England BioLabs, USA). PFGE clusters were illustrated with GelCompar II (v. 6.5; Applied Maths NV, Belgium) by UPGMA-based analysis using the Dice similarity coefficient with an 80% similarity cut-off; optimization and position tolerance were set at 1.2% and 1.3%, respectively. At least one isolate from each PFGE cluster was selected for further characterization by MLST and staphylococcal cassette chromosome mec (SCCmec) typing,²² (link)^,23 (link) with modifications.¹⁶ (link) For some isolates, the MLST sequencing result for the tuf gene was poor with published primers.²² (link) Therefore, the primers were modified as follows: tuf 19F, 5′-GTCCAATGCCACAAACTCG-3′; and tuf 19R, 5′-CCAGCTTCAGCGTAGTCTA-3′. MLST results for isolates were extrapolated to all members of the PFGE cluster. Isolates for which data had previously been published were included in the study.¹⁵ (link)^,16 (link)