Pulsed field gel electrophoresis (PFGE)

PFGE was performed according to the “One-Day (24–28 hr) Standardized Laboratory Protocol for Molecular Subtyping of Non-typhoidal Salmonella by PFGE” [21]. A single colony of each isolate was streaked on tryptic soy agar (TSA) and incubated overnight at 37 °C. Using a cotton swab, a portion of the growth on agar plate was transferred to 2 ml of Cell Suspension Buffer (100 mM Tris: 100 mM EDTA, pH 8.0) and the concentration of cell suspensions adjusted to 14–15% in a bioMerieux Vitek colorimeter. Immediately, 400 μl of adjusted cell suspension was transferred to 1.5 ml micro-centrifuge tubes with 20 μl of proteinase K (20 mg/ml stock), subsequently mixed with 400 μl of melted 1% SeaKem Gold (Cambrex, East Rutherford, NJ): 1% SDS agarose was prepared with TE Buffer (10 mM Tris: 1 mM EDTA, pH 8.0), and pipetted into disposable plug moulds. Three plugs were transferred to 50 ml polypropylene screwtubes with 5 ml of Cell Lysis Buffer (50 mM Tris: 50 mM EDTA, pH 8.0 with sarcosyl) and 25 μl of proteinase K (20 mg/ml stock) and incubated at 54 °C in a shaker water bath for 2 h with agitation. Thereafter, the plugs were washed twice with 15 ml of sterile water and three more times with TE Buffer at 50 °C for 15 min. Chromosomal DNA was digested with 50 U of XbaI (Promega, Madison, WI, USA.), and PFGE was performed on a CHEF Mapper XA System (Bio-Rad Lab., Richmond, CA, USA) in 0.5X Tris-Borate-EDTA buffer (Bio-Rad Lab.) with water circulation at 14 °C. Pulse times were ramped from 2.2 to 63.8 s during an 18 h run at 6.0 V/cm. After electrophoresis, the gels were stained within 2 μg of aqueous ethidium bromide (Sigma-Aldrich. St. Louis, MO, USA) per ml for 15 min and were photographed using 300 nm UV light. Similarities in PFGE patterns were calculated by using a computer-based similarity and clustering program (BioNumerics 3.0, Applied Maths, Biosistematica, Devon, UK). The dice coefficient was used to express similarities, and a similarity matrix was shown graphically by using the unweighted pair group method with arithmetic mean (UPGMA). The relatedness of the PFGE profiles of Salmonella isolates was estimated based on the presence or absence of shared bands. A PFGE type was defined as a group of isolates with similarities ≥85% [12].