Chef dr 3

Molecular fingerprinting of staphylococci was performed by PFGE using a CHEF-DRIII apparatus (Bio-Rad Laboratories, San Diego, USA). Bacterial cultures were grown overnight on Columbia agar supplemented with 5 % sheep blood (BioMérieux) and a cellular suspension of 5 × 10⁹ CFU/mL incorporated into 1.5 % low melting point agarose (BioRad). Discs were immersed into a lysis solution with lysostaphin (Sigma-Aldrich) (50 μg/ml), lysozyme (Merck) (100 μg/ml) and RNase (Roche) (50 μg/ml) at 37 °C for 3 h. After lysis, discs were incubated with proteinase K (NZYTech, Portugal) (1 mg/ml) for 17 h at 50 °C, followed by overnight digestion with SmaI (Takara) at 25 °C. Digested DNA was submitted to electrophoresis in 1 % agarose gel (Seakem LE) for 23 h at 14 °C and 6 V/cm with pulse times of five to 35 s. Lambda Ladder PFG Marker (BioLabs) 50 μg/ml was used as molecular weight marker. Agarose gels were stained with ethidium bromide and visualized by transillumination under UV (Pharmacia Biotech, Thermal Imaging System FTI- 500). BioNumerics 7.5 software (Applied Maths, Kortrijk, Belgium) was used to register macrorestriction patterns and clustering analysis was performed using DICE similarity coefficient and the unweighted-pair group method with arithmetic mean (UPGMA).

Genotyping of CRP harboring bla_NDM-1 was performed via PFGE. Chromosomal DNA was digested with 50U of SmaI (Takara, Dalian, China) for 3 h at 30 °C. Genomic DNA was separated by CHEF DRIII (Bio-Rad, Hercules, CA, USA) gel electrophoresis in a 1% agarose gel in 0.5 × TBE at 14 °C. The operating conditions are as follows: voltage, 6 V/cm; switch angle, 120°; switch time, 5–20 s for 19 h. XbaI-digested Salmonella Braenderup H9812 was used as the size ladder. The gels were stained with ethidium bromide (Macklin, Shanghai, China), digitally photographed with Gel Doc^TM XR+ (Bio-Rad, Hercules, CA, USA) and normalized as TIFF images. The DNA patterns were analyzed using BioNumerics software v7.6 (Applied Maths, Kortrijk, Belgium) to establish a dendrogram of strain relationships. UPGMA was used as a clustering algorithm and the Dice correlation coefficient was used with a 1.2% position tolerance. The strains with more than 85% similarity were considered to be related.

Isolated BACmid DNA was digested overnight using NheI restriction enzyme (New England Biolabs) at 37°C. Digested DNA fragments were then subjected to 1% agarose pulsed-field gel electrophoresis (PFGE; Bio-Rad) using 0.5% Tris/borate/EDTA (TBE) buffer and the following conditions: 6 V/cm, 120° field angle, and a switch time linearly ramped from 1 to 5 s over 14 to 15 h (CHEF DR III; Bio-Rad). DNA bands were visualized by staining the gel with a solution of 0.5 μg/mL ethidium bromide; excess stain was washed away with deionized water, and the gel was imaged on a ChemiDoc Touch imaging system (Bio-Rad). A DNA molecular weight standard (Bio-Rad; CHEF DNA, size, 8 to 48 kb) was used as reference.