Gelcompar 2 software

After digestion with Xba I and Apa I endonucleases, the DNA was separated using a CHEF-DR II system (BioRad, Birmingham, UK) [42 (link),43 (link)]. The Salmonella serotype Braenderup strain (H9812) was included in each PFGE gel as an internal control. Computer analysis of PFGE profiles was done using the Gel Compar II software, v.6.6.11 (Applied Maths, Inc.; Sint-Martens-Latem, Belgium) after visual inspection using the criteria of Tenover [44 (link)]. The Dice coefficients were calculated and were then transformed into an agglomerative cluster by the unweighted pair group method with arithmetic average (UPGMA).

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)].

Total community DNA was used as the template for amplification of the partial 16S rRNA gene fragment using Taq DNA polymerase (Bioline, Lückenwalde, Germany) with primer F968 with a GC clamp attached to the 5′ end and universal bacterial primer R1401.1b. For ITS1 amplification, primers EF4/ITS4 were used; this PCR was followed by a second amplification with primers ITS1f-GCITS2. Primer sequences, the reactions mixtures, and cycling conditions have been described (Pereira e Silva et al., 2012 (link)). The DGGE was performed in 6 % (w/v) polyacrylamide gels with 45–65 % and 20–50 % denaturant gradients for bacterial and fungal communities, respectively (100 % denaturant is defined as 7.0 M urea with 40 % deionized formamide). Electrophoresis was carried out at 100 V and 75 mA, for 16 h at 60 °C. The gels were subsequently stained for 40 min in 0.5 % TAE buffer with SYBR gold (final concentration 0.5 μg/L) (Invitrogen, Breda, the Netherlands) (Fig. S1 in the Supplementary Material). Gel images were digitized using Imagemaster VDS (Amersham Biosciences, Buckinghamshire, UK). The DGGE patterns were then transformed to a band-matching table using GelCompar II software (Applied Maths, Sint Martens Latem, Belgium).