Standardized PFGE Typing for Klebsiella pneumoniae

We used the 1-day, standardized PFGE protocol for K. pneumoniae [35 (link)]. Cell suspensions were placed in polystyrene tubes (Falcon; 12 × 75 mm), and their optical densities were adjusted to 3.8–4.0 using a Densimat photometer (BioMérieux, Marcy l’Etoile, France). Slices of K. pneumoniae agarose plugs were digested using 50 U of XbaI (Takara) per slice for 4 h at 37°C, and electrophoresis was performed using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was conducted with a switch time of 6 s to 36 s for 18.5 h, and images were captured using a Gel Doc 2000 system (Bio-Rad) and converted to TIFF files. The TIFF files were analyzed using BioNumerics version 5.1 software (Applied Maths, Kortrijk, Belgium). A similarity analysis of the PFGE patterns was performed by calculating the Dice coefficients (SD) [36 (link)] and clustering was performed using the unweighted-pair group method with average linkages (UPGMA).