Xbai 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)].

Intact chromosomal DNA of clinical ETEC strains was prepared and digested with the XbaI restriction endonuclease (New England Biolabs) according to the PulseNet program descried elsewhere [37 (link)]. DNA fragments were separated by pulsed-field gel electrophoresis on a CHEF-MAPPER apparatus (Bio-Rad) under the following conditions: switching time from 2.2 to 54.2 s at 6 V cm⁻¹ for 20 h at 14°C. Analysis of the TIFF image was carried out by the BioNumerics software version 4.5 (Applied Maths, Belgium) with average linkages to generate dendrogram at 1.5% tolerance level using the dice coefficient and unweighted-pair group method.

A single plug for each sample was transferred to a 1.5 ml tube and equilibrated for 20 min at room temperature in 1× New England Biolabs (NEB) buffer-2 supplemented with 1× bovine serum albumen (BSA) buffer (New England Biolabs, MA, USA). The buffer was removed and replaced with 200 μl of 1× NEB buffer-2/1× BSA containing 400 units of XbaI restriction endonuclease (New England Biolabs, Ipswich, MA, USA). Digests were performed overnight at 37°C. A 1.0% agarose gel was prepared in 0.5× Tris-borate-EDTA (TBE) buffer (50 mM Tris-Cl pH8.3, 50 mM boric acid, 1 mM EDTA) using PFGE-certified agarose (Bio-Rad Laboratories, Hercules, CA, USA). Samples were separated in 0.5× TBE at 14°C using a Bio-Rad CHEF Mapper XA System (Bio-Rad Laboratories, Hercules, CA, USA) set to resolve DNA fragments of 100–400 kb. Upon run completion, the gel was transferred to a solution of 1 μg/ml ethidium bromide in ultrapure water for 30 min at room temperature, before destaining with two 15-min washes with ultrapure water. Images were captured and the migration distance of the molecular weight markers was measured in millimeters. Molecular weight markers include MidRange PFG Marker I and II (New England Biolabs, MA, USA), and Lambda HindIII marker (Life Technologies, Grand Island, NY, USA).