Fingerprinting 2 informatix software

Pulsed-field gel electrophoresis (PFGE) was used to reveal the clonal relationship among the K. pneumoniae isolates. Strains were grown overnight (10 h) on LB agar at 37 °C. The ODs were adjusted to 1.3 to 1.4 (~10⁸ CFU/mL) at 540 nm. Genomic DNA was prepared in low-gelling point agarose (BioRad, USA) by a procedure developed at the Centre for Disease Control (CDC) [79 (link)]. DNAs were in cube digested with XbaI (New England Biolabs, San Diego, CA, USA) restriction endonuclease for 12 h, with 10 units/mL. Separation of the fragments was performed by using the CHEF-DR II system (BioRad, Hercules, CA, USA). DNA was electrophoresed for 24 h at 14 °C in a 1.2% agarose gel (Sigma-Aldrich, St. Louis, MO, USA) at 6 V/cm with a linear gradient pulse time of 54 s. Interpretation of PFGE patterns was based on the criteria of Tenover et al. [80 (link)]. After photographing, gels were analysed and interpreted with Fingerprinting II Informatix Software (BioRad, USA). Levels of similarities were calculated with the Dice coefficient, and unweighted pair group method with arithmetic averages (UPGMA) was used for the cluster analysis of the PFGE patterns. Pulsotypes (PTs) were defined at 85% similarity between macrorestriction patterns [20 (link)].

Available clinical isolates underwent phylogenetic group determination and pulsed-field gel electrophoresis (PFGE). Clinical isolates were screened using a specific 16S rRNA gene polymerase chain reaction (PCR) assay, and sequenced to confirm taxonomic identities. PCR was performed using primers SM1f (5′-GTTGGGAAAGAAATCCAGC-3′) and SM4 (5′-TTAAGCTTGCCACGAACAG-3′) as described previously [16 (link),17 (link)]. Sequence analysis of PCR products was conducted with MEGA version 3.1 using the maximum likelihood method. AB695350 (S. maltophilia strain 4APB) was used as a control [18 (link)]. S. maltohpilia clinical isolates were typed using PFGE with Xba I digestion as described previously [19 (link)]. PFGE was performed with a CHEF-DR III apparatus (Bio-Rad Korea, Seoul, Korea) using 5 to 35 s of linear ramping at 6 V/cm for 20 h at 14°C. Digital images were analyzed with Fingerprinting II Informatix software (Bio-Rad, Hercules, CA, USA) using the Dice coefficient and UPGMA with a 1% tolerance and 0.5% optimizing setting value. The results were interpreted using the criteria of Tenover et al. [20 (link)].