Apai enzyme

As previously mentioned, for fosmid DNA isolation, clones were grown in 100-ml flasks containing LB-Cm broth medium supplemented with 2 μl/ml of CopyControl Fosmid Autoinduction solution (Epicentre) for higher DNA yields, followed by incubation at 37°C for 12 to 16 h and with continuous stirring at 150 rpm. Fosmid DNA was extracted using the FosmidMax DNA purification kit (Epicentre) according to the manufacturer’s instructions and was then subjected to restriction analysis using ApaI enzyme (Thermo Scientific) to determine if different DNA insert sequences were present. Subsequently, fosmids were sequenced on the Illumina NextSeq 500, with a NextSeq 500/550 High Output reagent kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols. The KneadData pipeline (v. 0.6.1) (http://huttenhower.sph.harvard.edu/kneaddata) was used to perform quality trimming using Trimmomatic (v. 0.38) (42 (link)), and sequences mapping to the cloning host and vector sequences were removed using bmtagger (v. 3.101) (ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger/). The remaining reads were assembled using Unicycler (v. 0.4.7) (43 (link)), annotated with Prokka (v. 1.13) (44 (link)), and antibiotic resistance was profiled using the Resistance Gene Identifier web portal (45 (link)).

The preparation of samples was performed as previously described [15 (link)]. DNA restriction was done with ApaI enzyme (Thermo Scientific, Lithuania) at 37^°C for 3 hours. PFGE was performed with a 2015 Pulsafor unit (LKB Instruments, Broma, Sweden) equipped with a hexagonal electrode array for 16h at 300V at 9^°C. The gels were stained with ethidium bromide and photographed under UV illumination. A dendrogram was derived from the Ward linkage of correlation coefficients between PFGE patterns of different genotypes by using SPSS cluster analysis software (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.).

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.