Smai restriction endonuclease

The clonality of MRSA and meropenem-resistant isolates of Klebsiella pneumoniae detected at the Department of Anesthesiology and Intensive Care Medicine was assessed with pulsed-field gel electrophoresis (PFGE). Bacterial DNA extracted with a technique described by Husičková et al. [19 (link)] was digested by the XbaI restriction endonuclease (New England Biolabs, Ipswitch, MA, USA) for 24 h at 37 °C in Klebsiella pneumoniae isolates and by the SmaI restriction endonuclease (New England Biolabs, Ipswitch, MA, USA) for 24 h at 25 °C in Staphylococcus aureus strains. The obtained DNA fragments were separated by PFGE on 1.2% agarose gel for 24 h at 6 V/cm and pulse times of 2–35 s for both Klebsiella pneumoniae and Staphylococcus aureus strains. Subsequently, the gel was stained with ethidium bromide. The resulting restriction profiles were analyzed with the GelCompar II software (Applied Maths, Kortrijk, Belgium) using the Dice coefficient (1.2%) for comparing similarity and unweighted pair group method with arithmetic means for cluster analysis. The results were interpreted according to criteria described by Tenover et al. [20 (link)].

The similarity of the VRE isolates was performed with pulsed-field gel electrophoresis (PFGE). Bacterial DNA was isolated using a technique described by Husickova et al. and digested by the SmaI restriction endonuclease (New England Biolabs, Ipswich, MA, USA) for 24 h at 25 °C [33 (link)]. The obtained DNA fragments were separated by PFGE on 1.2% agarose gel for 24 h at 6 V/cm and pulse times of 2–35 s. Subsequently, the gel was stained with ethidium bromide. The resulting restriction profiles were analyzed with GelCompar II software (Applied Maths, Kortrijk, Belgium). The coefficient of similarity (CS) was calculated using the Dice algorithm. Individual clusters were analyzed with the unweighted pair group method with arithmetic mean algorithm, and the results were interpreted using the criteria defined by Tenover et al. [34 (link)]. Optimization and band matching tolerance was set at 1.2%.