Apai restriction enzyme

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).

Molecular subtyping of the 50 ACB complex isolates was carried out using pulsed field gel electrophoresis (PFGE), as previously described [16 (link)]. Briefly, chromosomal DNA of these ACB complex isolates was prepared in agarose gel blocks, followed by digestion using ApaI restriction enzyme (Promega, Madison, WI, USA). Then, restricted DNA fragments were separated using CHEF Mapper (Bio-Rad, Hercules, CA, USA). Gels were run at 14°C in 0.5x TBE buffer for 24 hours, with pulse time of 2–40 seconds. The XbaI-digested Salmonella enterica ser. Braenderup H9812 was used as the molecular marker in these runs. Cluster analysis of the PFGE profiles was based on the Dice coefficient of similarity analyzed by BioNumerics 6.0 software (Applied Maths, Kortrijk, Belgium) using the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) algorithm at 1.5% band position tolerance. Only DNA bands within the molecular marker (20.5 kb–1135 kb) were scored.

Genetic relatedness of the isolates was revealed using PFGE. Briefly, genomic DNA of CRAB contained in the plug was digested using ApaI restriction enzyme (Promega, Madison, WI, USA). The fragmented DNA was separated using PFGE CHEF-MAPPER system (Bio-Rad, Hercules, CA, USA) under the condition of 6 Vcm⁻¹ for 20 h, with initial and final switch time of 2 s and 40 s, respectively [53 (link)]. Salmonella enterica, serotype Braenderup H9812, was used as the reference standard marker. Bionumerics software, version 7.6.2 (AppliedMaths, Sint–Martens–Latem, Belgium), was used for the analysis of banding patterns. A dendrogram was generated based on dice correlation coefficients and clustered by unweighted pair group method using arithmetic averages (UPGMA) with band position tolerance and optimization of 1.5% and 2.0%, respectively. Isolates grouped together based on > 85% similarity in dendrogram were considered to represent the same PFGE cluster.