Fpquest

Plasmid DNA was isolated from the wild-type strains and the TCs by both the Kado–Liu method (35 (link)) and Qiagen Plasmid Midikit (Qiagen, Germany). The extracted plasmid DNA was electrophoresed in 0.8% agarose gel at 70 V using 1× TBE running buffer and stained with ethidium bromide (0.5 g/mL). Plasmid sizing was done by FP-Quest (Bio-Rad, USA) software using E. coli V517 and Shigella flexneri YSH6000 as molecular weight markers. Replicon-typing PCR elucidated the plasmid incompatibility types (36 (link)).

PFGE analysis of S. aureus isolates was performed according to the PulseNet protocol [43 (link)] with the smaI enzyme (New England Biolabs, Beverly, MA, USA) by using a CHEF-DR III apparatus (Bio-Rad Laboratories Inc., Hercules, CA, USA) for the separation of DNA fragments. XbaI-digested DNA from Salmonella enterica serotype Braenderup H9812 was used as a reference size standard, while PFGE patterns were digitally analyzed using the FPQuest (Bio-Rad Laboratories Pty Ltd. Hercules, CA, USA) software package. PFGE profiles were compared using the Dice correlation coefficient, with a maximum position tolerance of 1.5% and optimization of 1.5%. Similarity, clustering analysis was performed using the unweighted pair group method using averages (UPGMA), and a dendrogram was generated. Two PFGE profiles were classified as indistinguishable if the DNA fragment patterns matched each other completely, while clusters were selected using a cutoff at the 75% level of genetic similarity. The discriminatory power of PFGE analysis was determined using Simpson’s index of discrimination (D), as previously described [44 (link)]. Values for D ranged between 0 and 1, with a value of 1 indicating the most discriminatory method.