Gene navigator

Isolates of interest were analysed using pulsed-field gel electrophor esis (PFGE), as described by the Centers for Disease Control and Prevention, with the following changes: cultures were grown on BHI medium, at 37°C, aerobically overnight, and cell densities were standardised to an OD 610nm ≈1.0, using a spectrophotometer (BioDrop, UK). [9] Cells were embedded in a 1.0% SeaKem Gold agarose gel (Lonza, Switzerland) and were digested for 2 hours at 25°C using SmaI (Thermo Scientific, USA) restriction endonuclease and ApaI as a secondary enzyme (2 hours at 37°C). Digested plugs were electrophoresed through a 1.0% SeaKem Gold agarose gel (Lonza, Switzerland) in 0.5% TBE buffer (14 °C), using the Gene Navigator (Amersham Biosciences, UK) at 200 V, with a ramped switching time of 3.5 -23.5 seconds over 18 hours. The resultant gel was visualised using ethidium bromide staining, and the gel image analysed and dendrogram generated using Gel Compare II (BioNumerics v.6.6, Applied-Maths, USA), with an optimisation of 1.0 and a band tolerance of 1.5. The dendrogram was constructed using the pairwise average from the SmaI and ApaI results, with the UPGMA (unweighted pair group method with arithmetical mean) method and branch quality was calculated using the cophenetic correlation. E. faecalis ATCC 51299 was used as a control and for normalisation.