Lambda 48.5 kb concatemers

Genomic relatedness among A. baumannii isolates was investigated by Pulsed-Field Gel Electrophoresis (PFGE). The genomic DNA of the isolates was digested with ApaI restriction enzyme (35 U/sample; Promega Corporation, Madison, WI, USA) and fragments were separated on a CHEF-DR II system (Bio-Rad) at 14 °C for 25 h at 6 V/cm with an initial pulse time of 0.5 s and a final pulse time of 30 s. Lambda 48.5 kb concatemers (New England BioLabs, Beverly, MA, USA) were used as molecular size markers. DNA restriction patterns and the dendrogram of strains relatedness were analyzed and obtained with Fingerprinting II version 3.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the Unweighted Pair Group Method with Arithmetic Averages (UPGMA). The Dice correlation coefficient was used with a 1.2% position tolerance. Only bands larger than 40 kb were considered for the analysis. Strains were considered clonally related in the case of >85% similarity [46 (link)]. A. baumannii RUH875 and RUH134 were used as reference strains representative of the International Clonal Lineages I (ICL I) and II (ICL II).

PFGE of A. baumannii was performed after ApaI digestion using a method described previously (Bou et al., 2000 (link)). Genomic DNA was included in agarose plugs, and DNA restriction was carried out at 30°C for 16 h. PFGE was performed in a CHEF DRII system (Bio-Rad, Hercules, CA, United States), with pulses ranging from 0.5 to 15 s at a voltage of 6 V/cm at 14°C for 20 h. Lambda 48.5-kb concatemers (New England BioLabs, Beverly, MA, United States) were used as molecular size markers. Isolates showing three or fewer band differences were regarded as a single PFGE type, according to the criteria described previously by Tenover et al. (1995) (link).