PFGE was done as previously described [26 (link), 34 (link)–36 (link)]. Genomic DNA from all isolates was embedded in 2% agarose plugs (InCert Agarose, Lonza, Rockland, USA). DNA was digested with the SmaI enzyme (20 U/μl; Fermentas, Lithuania). Electrophoresis of digested fragments was carried in 1% agarose on CHEF DRII system (Bio-Rad Laboratories, Berkeley, CA, USA) using 0.5xTBE at 14°C. The initial and final switch time was 0.5 and 35 s respectively, at 6V/min for 24 h. The DNA banding patterns were analysed with Gel Compar II BioNumerics v. 7.0 software (Applied Maths, Belgium) and cluster analysis was performed by UPGMA based on the Dice similarity coefficient, with optimization and position tolerance set at 1%. EC isolates were clustered using > 80% homology cut-off, above which strains were considered to be closely related and assigned to the same PFGE type. The reference strain E. cecorum ATCC 43198 was used as control.
Free full text: Click here
