Seakem gold

The PulseNet protocol, according to Ribot et al. [49 (link)] was used to conduce pulsed-field gel electrophoresis (PFGE). Bacteria grown at 37 °C overnight on TSA (OXOID^®) were suspended in tubes containing 2mL of phosphate-buffered saline (PBS: 0.01 M phosphate buffer; pH 7.2; 0.85% NaCl). After agarose blocking, genomic DNA digestion was performed with 30 U of XbaI enzyme (Invitrogen^®) for 2 h at 25 °C.The DNA fragments were separated on 1% agarose gel (SeaKem Gold^®) in 0.5X TBE buffer in CHEF DRIII (Bio-Rad^®, Hercules, CA, USA) for 18h with the following parameters: 200 V, 120° angle, 6 V/cm gradient, and 14 °C buffer temperature. The comparison of the band patterns was performed by the UPGMA analysis method, using the Dice similarity coefficient with a tolerance of 1.5% in the comparison of the position of the bands.

To verify the genetic relatedness of isolates, pulsed-field gel electrophoresis (PFGE) analysis was executed using the PulseNet protocol, as previously recommended (CDC, 2017 ). Bacteria grown at 37°C overnight on Tryptic Soy Agar (TSA) (OXOID^®) were suspended in tubes containing 2 mL of phosphate buffered saline (PBS: 0.01 M phosphate buffer; pH 7.2; 0.85% NaCl). After agarose blocking, gDNA digestion was performed with 30 U of Xba1 enzyme (Invitrogen^®) for 2 h at 37°C.
The DNA fragments were separated on 1% agarose gel (SeaKem Gold^®) in 0.5X TBE buffer in CHEF DRIII (Bio-Rad^®, California, United States) for 18 h with the following parameters: 200 V, 120° angle, 6 V/cm gradient and 14°C buffer temperature. The gels were stained with ethidium bromide, photographed under UV light in a transilluminator (Loccus Biotechnology^®) and evaluated using the BioNumerics program.