Gelcompar 2 software version 6

The PFGE method was used to determine the genetic relationship among the investigated S. aureus isolates, following the previously described method with modifications [20 (link)]. The electrophoresis was performed using a CHEF-DR^® II PFGE apparatus (Bio-Rad, Hercules, CA, USA).
The similarity of the PFGE profiles was compared according to the percentage similarity of the profiles estimated by the Dice coefficient and grouped by UPGMA (unweighted pair group arithmetic mean method) using GelCompar II software version 6.5 (Applied Maths). The analysis used S. aureus ATCC^® 25923™ as a reference strain.

ITS1-5.8S-ITS2 was amplified from the cell-free DNA lysate using primers ITS1 and ITS4 mentioned elsewhere. The amplification was carried out in a 25 μL final reaction volume containing 50 ng of the genomic DNA as previously described [41 (link)]. The amplified ITS fragment was analyzed by 2.0% (w/v) agarose gel electrophoresis at 80 V in 0.5× TBE (45 mM Tris-borate, 1 mM EDTA, pH 8.0) buffer to check its intactness and absence of non-specific amplification. The PCR product (4 μL) was digested with 5 U of TaqI (Promega, Madison, USA) in a 10 μL reaction volume at 65°C as per manufacturer’s instructions. The restriction patterns were analyzed by electrophoresis of the 10 μL reaction volume on 2.0% (w/v) agarose gel in parallel with PCR 100 bp Low DNA ladder (Sigma-Aldrich) as molecular size standard. The electrophoresis was run at 80 V for 2 h in 0.5× TBE buffer. The gel was then stained in 0.5 μg/mL ethidium bromide solution for 30 min with rocking at 15 rpm on a platform rocker (Tarsons, Kolkata, India). After destaining for 30 min in sterile deionized water, the gel was photographed using ChemiDoc MP gel documentation system (Bio Rad, Hercules, USA). The restriction fingerprints were analysed for the absence or presence of discriminating fragments using GelCompar II software, version 6.5 (Applied Maths, Sint-Martens-Latem, Belgium).

Of the total of 139 ESBL-E isolates, 118 isolates from 84 horses were available for further investigation. PFGE typing was performed for the 118 isolates according to the PulseNet O157 protocol [21 ]. Clonal similarity of the strains was determined [22 (link)] and analysed by UPGMA-cluster analysis with 0.5% optimization and 1% Dice band matching tolerance with a ≥ 85% similarity cut off value using GelCompar II software version 6.5 (Applied Maths NV, Belgium).