Fingerprinting 2 version 3

The P. mirabilis isolates were grown on MacConkey (Scharlab) agar at 37 °C for 18 h and then in Luria Bertani Broth (Liofilchem Diagnostic Ltd, Roseto degli Abruzzi TE, Italy,). The restriction digestion was performed using SfiII (35 U/sample; Promega Corporation, Madison, Wisconsin, USA) at 37 °C for 20 h. Fragments were separated in a 1% (w/v) Pulsed Field Certified Agarose gel (Bio-Rad Laboratories, Inc. Hercules, California, USA) in a 0.5x Tris-Borate-EDTA (TBE )buffer on a CHEF MAPPER (Bio-Rad Laboratories, Inc. Hercules, California, USA) apparatus at 14 °C at 6 V/cm for 25 h with an initial pulse time of 9.06 s and a final pulse time of 1m34s. Lambda 48.5 kb ladder (New England BioLabs, Ipswich, Massachusetts, USA) were used as molecular size markers. The gels were stained with ethidium bromide (Sigma Aldrich,,St. Louis, Missouri USA ), digitally photographed with Gel Doc 2000 (Bio-Rad Laboratories, Inc.) and normalized as TIFF images. Dendrograms of strain relatedness were created with Fingerprinting II version 3.0 software (Bio-Rad Laboratories, Inc.) using UPGMA. The Dice correlation coefficient was used with a 1.2% position tolerance in order to analyse the similarities of the banding patterns; the strains were considered clonally related in the case of >85% similarity.

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).

The pulsed-field gel electrophoresis (PFGE) was performed using the XbaI restriction enzyme, and the obtained genomic fragments were separated on a CHEF-DR II apparatus (Bio-Rad, Milan, Italy) for 22 h at 14°C. Bacteriophage λ concatenamers were used as DNA size markers. DNA restriction patterns of scanned gel pictures were interpreted following cluster analysis using the Fingerprinting II version 3.0 software (Bio-Rad) using the unweighted pair-group method with arithmetic averages (UPGMA). Only bands larger than 48 kb were considered for the analysis. The Dice correlation coefficient was used with a 1.0% position tolerance to analyse the similarities of the banding patterns, and a similarity threshold of 90% to define clusters. The restriction patterns of the genomic DNA from the isolates were analysed and interpreted according to the criteria of Tenover et al. (1995) (link).