Chef mapper

Agarose-embedded (Lonza, USA) SA DNA was digested with SmaI (Fermentas, Lithuania) followed by gel electrophoresis in the CHEF MAPPER (Bio-Rad) system. Electrophoresis conditions were 14 °C, 0.5 × Tris-borate-EDTA buffer (Sigma, Switzerland), initial pulse 5 s, final pulse 50 s, 6 V, 18 h. Salmonella Braenderup H9812 restricted with XbaI (Fermentas, Lithuania) was used as a marker. PFGE restriction patterns were analyzed by the BioNumerics software (Applied Maths). The pulsotypes were compared using the band-based DICE similarity coefficient with 1% optimization and tolerance. The un-weighted pair group method with arithmetic mean (UPGMA) algorithm was used for cluster analysis [37 (link)].

Agarose-embedded (Lonza, USA) DNA was digested with XbaI (Fermentas, Lithuania) followed by gel electrophoresis in the CHEF MAPPER (Bio-rad) system. Electrophoresis conditions were 14°C, 0.5×Tris-borate-EDTA buffer (Sigma, Switzerland), initial pulse 2.2 s, final pulse 54.2 s, 6V, 18 h. Salmonella Braenderup H9812 restricted with XbaI (Fermentas, Lithuania) was used as a marker. PFGE restriction patterns were analyzed by the BioNumerics 7.6 software (Applied Maths). The pulsotypes were compared using the band-based DICE similarity coefficient with 1% optimization and tolerance. The un-weighted pair group method with arithmetic mean (UPGMA) algorithm was used for cluster analysis

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 DNA was prepared in agarose plugs (0.5 × 10⁶ cells per plug) and restriction digested with EcoRV in the buffer recommended by the manufacturer. The digested DNA was separated by CHEF (contour-clamped homogeneous electric field) gel electrophoresis (CHEF Mapper, Bio-Rad) (autoprogram, 50–1100 kb range, 16 h run or 250–2500 kb range, 48 h run), transferred to a nylon membrane (Amersham Hybond-N+), and blot-hybridized with a 421 bp probe specific for the human rDNA spacer sequence (IGS probe). The DNA sequence for the probe was amplified by PCR using the primers FOR-F/REV-R (Table S1). The blot was incubated for 2 h at 65 °C in pre-hybridization Church’s buffer (0.5 M Na-phosphate buffer containing 7% SDS and 100 µg/ml of salmon sperm DNA). The labeled probe was heat denatured in boiling water for 5 min and snap-cooled on ice. The probe was added to the hybridization buffer and allowed to hybridize overnight at 65 °C. The blots were then washed twice in 2 × SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0), 0.05% sodium dodecyl sulfate (SDS) for 10 min at room temperature, then twice in 2 × SSC, 0.05% SDS for 5 min at 60 °C, twice in 0.5 × SSC, 0.05% SDS for 5 min at 60 °C and twice in 0.25 × SSC, 0.05% SDS for 5 min at 60 °C. The blots were exposed to X-ray film for 24–72 h at − 80 °C to visualize labeled DNA band(s).

Cronobacter isolates were analyzed as described in the PulseNet standardized PFGE Cronobacter protocol using XbaI (TaKaRa, Japan) as the restriction enzyme (Brengi et al., 2012 (link); Yan and Fanning, 2015 ). DNA fragments were separated by electrophoresis (CHEF Mapper, Bio-Rad Laboratories, Hercules, California, US) through a 1% (w/v) agarose gel (Seakem Gold, Rockland, Maine, US) in 0.56 TBE buffer at 6 V/cm with an initial switch time of 1.8 s and a final switch time of 25 s. Gels were stained in deionized water containing GelRed Nucleic Acid Gel Stain (Biotium, CA, US), and visualized under UV light using a GelDoc XR⁺ system (Bio-Rad laboratories, Hercules, California, US). XbaI-digested Salmonella Braenderup H 9812 was used as the molecular weight standard. Dendrograms were constructed using Bionumerics software (Version 5.1, Applied-Maths, Belgium), and the cluster analysis was conducted using the DICE coefficient and unweighted pair group method (UPGMA) with arithmetic means, with a 1.2% band position tolerance. When comparing the DNA fingerprint patterns, a cutoff value of 100% similarity was applied.

Southern-blot hybridization was performed with a ³²P-labelled DNA probe [27 (link)]. The genomic DNA from 5 × 10⁵ cells was digested by SpeI in an agarose plug. The digested CHEF DNA (CHEF Mapper, Bio-Rad Laboratories, Hercules, CA, USA) was gel-separated (5–250 kb range, 16 h run), transferred onto membrane (Amersham Hybond-N⁺), and hybridized with a 201-bp YAC/BAC DNA probe specific for alphoid^tetO-HAC. The DNA probe was PCR-amplified from the genomic DNA in the presence of ³²P-labeled dNTPs, using the 5′-GGGCAATTTGTCACAGGG-3′ and 5′-ATCCACTTATCCACGGGGAT-3′ primers. The blot was pre-hybridized for two hours at 65 °C in Church’s buffer containing 7% SDS and 0.5 M Na-phosphate buffer supplemented with 100 µg/mL salmon sperm DNA, then hybridized overnight at 65 °C with heat denatured 201-bp YAC/BAC DNA probe. The blot was washed twice in 0.05% SDS, 2× SSC for 10 min at RT, then twice in 0.05% SDS, 2× SSC for five minutes at 60 °C, twice in 0.05% SDS, 0.5× SSC for 5 min at 60 °C and twice in 0.05% SDS, 0.25× SSC for 5 min at 60 °C, developed for 24–72 h at −80°C.