Pulsed-Field Gel Electrophoresis of UPEC O25b Strains

PFGE tests were performed according to Ochoa et al. [15 (link)]. First, the UPEC O25b strains were embedded in low-melting-point (LMP) agarose blocks from Promega Corporation (Madison, WI, USA). Next, digestion of the samples was performed using 20 U of the restriction enzyme Xba1 (New England Biolabs, Ipswich, MA, USA) at 37 °C for 20 h. The PFGE shift was performed for 24 h at 200 V (7 v/cm) to an angle of 120° at 14 °C, with an initial pulse of 2.16 s and a final pulse of 13.58 s, in a CHEF Mapper system (Bio-Rad Life Science Research, Hercules, CA, USA). The macrorestriction products in the PFGE gels were stained with GelRed^® Nucleic Acid Stain (Biotium, Fremont, CA, USA), visualized with UV light, and digitized using the CCD Camera Documenting System BK04S-3 (Biobase, Mexico City, Mexico). PFGE pulsotypes were analyzed using NTSYSpc v2.02j and PAST v4.03 software, and the clonality degree was estimated according to the criteria described by Tenover et al. [22 (link)]. A lambda ladder PFGE marker (New England Biolabs, Hertfordshire, England, UK) was used as a molecular weight marker in the PFGE assay.