Lambda ladder 48.5 727 5 kb pfg marker

Genotyping and the clonal relationship between A. baumannii strains were determined by pulsed-field gel electrophoresis (PFGE) using ApaI restriction enzyme (Thermo Fisher Scientific, USA) for digestion. The Lambda Ladder 48.5–727.5 kb PFG Marker (New England Biolabs, US) was used as DNA size marker. DNA fragments were separated in 1% ultra-pure agarose gels (Invitrogen) using the CHEF Mapper apparatus (Bio-Rad, Munich, Germany) under the following conditions: voltage 6 V/cm; temperature at 14 °C; with switch times ranging from 5 s to 30s; at an angle of 120°, for 19 h. After that, the gels were stained by ethidium bromide, and the DNA bands were visualized and photographed under UV transilluminator. DNA banding pattern images were analyzed using Bionumeric 7.6.3 software (Applied Maths NV, St-Martens-Latem Belgium) and the dendrogram was constructed by UPGMA method (Unweighted Pair Group Method with Arithmetic Mean), based on Dice’s similarity coefficient at a 1.5% tolerance [6 (link)]. The similarity of 80% or higher was defined as the same PFGE genotype, whereas the similarity of < 80% indicated various PFGE genotypes.

PFGE included 60 CRAB isolates randomly selected from all participating hospitals, with respect to the specimen types and gene content. PFGE was performed with a 2015 Pulsafor unit (LKB Instruments, Broma, Sweden), as previously described [28 (link)]. DNA restriction was done with ApaI enzyme (Thermo Scientific, Lithuania). The Lambda Ladder 48.5–727.5 kb PFG Marker (New England Biolabs, US) was used as DNA size marker. The stained gels were scanned using the Diversity Database software image-capturing system (Bio-Rad Laboratories Ltd., UK). The Dice coefficient was used to calculate similarities of the banding patterns with a tolerance of 1.5%, and the unweighted-pair group method using average linkages (UPGMA) was used for cluster analysis with BioNumerics software, version 4.0 (Applied Maths, St-Martens-Latem, Belgium). The isolates with more than 80% similarity in their DNA patterns were defined as genetically related and part of the same cluster.