Gel doc camera system

All bacterial isolates were fingerprinted by the PFGE method, using the PulseNet protocol developed by Centers for Diseases Control and Prevention [61 ]. Chromosomal DNA was subjected to restriction analysis with application of XbaI enzyme (Thermo Fisher Scientific, USA). PFGE analysis was conducted with CHEF DR III PFGE apparatus (Bio-Rad, USA). DNA separation was performed with the following parameters: 1% agarose gel (Prona Agarose) on 0.5 M Tris–Borate–EDTA buffer at 14°C for 19 h at 6.0 V/cm (200 V). Pulse time was ranging of 2.2–63.8 s. The gels were stained with SYBR® Safe - DNA Gel Stain (Thermo Fisher Scientific, Germany) and band patterns were visualized under UV light and photographed using a Gel Doc camera system (Bio-Rad, USA). Molecular Weight Marker ProMega-Markers® Lambda Ladders was used for analysis (Promega, USA). PFGE patterns were analyzed via visual assessment and the dendrograms were generated with UPGMA method using on-line software http://insilico.ehu.es/.

The amplified products from all types of the PCR reactions were resolved on a 2% or 0.8% agarose gel (Sigma-Aldrich, USA) and visualized with Midori Green DNA (Nippon Genetics, Germany) under UV light using a Gel Doc camera system (Bio-Rad, USA) and analyzed with Quantity One software (Bio-Rad, USA). PCR assays were repeated twice to confirm the correctness of the assignment of the investigated strains to their respective patterns.

All PCRs reactions were conducted in a DNA Thermal Cycler T100™ (Bio-Rad, Dublin, Ireland). Amplified products were resolved on a 2% agarose gel (Sigma-Aldrich, Wien, Switzerland) and visualized using Midori Green DNA (Nippon Genetics, Dueren, Germany). Band patterns were visualized under UV light and photographed using a Gel Doc camera system (Bio-Rad) and analyzed with Quantity One software (Bio-Rad). For each PCR reaction, 1 µl of sterile water added to the PCR mixture used as a negative control in the experiment. PCR assays were performed twice to ensure that the strains were correctly assigned to their respective patterns.
For the purpose of the next step of the phylogenetic analyses (RAE-PFGE analysis and MLST), eleven of the tested E. coli strains were selected: UPEC 15279.21, 1119 and 15035.8; APEC 1288, 1239, 189A and 60C and RepEC 200E, 209E, 212E and 305C. These strains were selected based on the obtained results of phylogroup assignment and virotyping.