Chef mapper 2

Pulsed-field gel electrophoresis was conducted according to the instructions from the 2009 USA PulseNet protocol for Campylobacter (CDC, 2009 ). The isolates previously identified by PCR were grown on Columbia agar (48 h at 42°C) in a microaerophilic atmosphere and embedded in agarose blocks (Seakem Gold agarose, Lonza, Rockland, MD, USA). Following DNA purification, 1 mm of agar plugs slices was digested 18 h with SmaI restriction enzyme (Promega, Milan, Italy) and the DNA fragments were separated by PFGE (Chef Mapper II, Biorad Laboratories, Hercules, CA, USA) in 1% agarose gel (Seakem Gold agarose, Lonza).
Salmonella Braenderup H9812, digested with XbaI enzyme (Promega, Milan, Italy), was used as the standard molecular weight marker. The gel was stained with SYBR Safe DNA gel stain (Invitrogen, Cergy Pontoise, France) and photographed on a UV transilluminator (Alpha Innotech Corporation, San Leandro, CA, USA). The image analysis was performed using Bionumerics program v. 6.6 (Applied Maths NV, Sint-Martens-Latem, Belgium). The similarity analysis was carried out using the Dice coefficient (position tolerance, 1%). The unweighted pair group mathematical average was used to cluster patterns. Isolates with <90% similarity were clustered as separate pulsotypes.

PFGE was performed according to the instructions of the 2009 U.S. PulseNet protocol for Campylobacter. Bacteria, previously identified by PCR, were subcultured onto Columbia agar and embedded in agarose blocks (Seakem Gold agarose, Lonza, Rockland, ME, USA). The blocks were then lysed, washed, digested with SmaI restriction enzyme (Promega, Milan, Italy) and subjected to pulsed-field electrophoresis in 1% agarose gel (Seakem Gold agarose, Lonza) for 18 h (Chef Mapper II, Biorad Laboratories, Hercules, CA, USA). Salmonella serovar Branderup H9812 was used as standard molecular weight size. After electrophoresis run, the gel was stained with Sybr Safe DNA gel stain (Invitrogen) and photographed at transilluminator (Alpha Innotech). The image analysis was performed using the program Bionumerics v. 6.6 (Applied Maths NV, Sint-Martens-Latem, Belgium). Pair comparisons and cluster analyses were carried out using the Dice correlation coefficient and the unweighted pair group mathematical average (UPGMA) clustering algorithm. The optimization tolerance was set at 2.5% and the position tolerance for band analysis was set at 1%.

MLST was performed as described by Curran et al. [3 (link)]. Pulsed-field gel electrophoresis (PFGE) of SpeI-digested DNA fragments from each strain was performed on a CHEF-MAPPER II apparatus (Bio Rad Laboratories, Rockland, Maine, USA) at 6 V/cm, for 19.7 h with pulse times running from 5.3 to 34.9 s in 0.5 × TBE buffer at 14°C. PFGE patterns in the dendrogram were analyzed with Molecular Analyst Software Fingerprinting II (Bio-Rad Laboratories). Only restriction fragments larger than 50 kb were used for analysis. PFGE genotypes were distinguished by the unweighted pair-group method using average linkages (UPGMA) and Dice coefficient, and isolates with ≥80% similarity were assigned into the same pulsotype.