Molecular typing

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]. 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.

At least 50% of the CRAB isolates from each PFGE cluster and at least one isolate from each participating hospital, endowed with different bla_OXA gene content were selected for MLST analysis. Overall, 37 isolates were typed by MLST under the Pasteur MLST scheme which involved PCR amplification, purification and sequencing of seven housekeeping genes (fusA, gltA, pyrG, recA, cpn60, rpoB, and rplB), as indicated previously [29]. Sequence types (STs) were assigned using the same scheme from the MLST website (http://pubmlst.org/abaumannii/).