Chef dr 2

DNA preparation, restriction enzyme digestion, gel electrophoresis, Southern blotting, hybridisation, and autoradiography were essentially as described [90 ]. To prepare S. pombe spheroplasts, logarithmically-growing cells were treated with zymolase (Zymo Research, Irvine, CA, USA) [84 (link),91 (link)]. High molecular weight (HMW) DNA from yeast, insect and chicken cells was prepared in agarose blocks using standard methods and resolved by pulsed-field gel electrophoresis (PFGE) using either a Chef DRII (BioRad, ‎Hercules, CA, USA) or Biometra Rotaphor Type V (Analytik, Jena, Germany) apparatus [26 (link)]. DNA size markers used were: lambda DNA ladder, S. cerevisiae chromosomes, H. wingei chromosomes (BioRad). DNA was transferred to nylon membrane (Hybond N+, GE Healthcare Life Sciences, Little Chalfont, UK). Probe DNA was radioactively labelled by random-priming [92 (link)]. Southern blot hybridisations were carried out in a Hybaid oven and membranes washed at high stringency (0.2 × SSC/0.1% SDS at 65 °C).

DNA fragments were separated using a clamped homogenous electric field device (CHEF-DR II, BioRad) with pulse times of 5–15 sec over 10 h, and 15–60 sec over 13 h at 6 V, and run through a 1.2% gel of Agarose NA (GE Healthcare, Uppsala, Sweden) in 0.5 × Tris-borat EDTA-buffer (0.9 M Tris-borat, 20 mM EDTA; National Veterinary Institute, Uppsala, Sweden). Then, gels were stained with GelRed (Biotium, CA, USA).
For each bacterial species, one isolate with an easily interpreted pattern was selected among our collected isolates, as an internal reference and was run every 5^th to 6^th lane on all gels. In addition, a Lambda Ladder PFG Marker (New England Biolabs, MA, USA) was used in the first and last lane on each gel.
The protocol was repeated for untypeable isolates.

DNA for PFGE was extracted in low-melting point agarose from cells grown to saturation as described previously^{86 (link)}. The chromosomes were separated in a BioRad CHEF-DR II. The gel was then stained using SYBR Gold (Invitrogen # S11494) for 1 h. The chromosomes from the gel were transferred to nylon membranes (Hybond N+) and hybridized to radioactively labeled PCM1 to identify Chr V fragments.

DNA plugs for PFGE were prepared as described [71 (link)]. Electrophoresis was performed using a Bio-Rad CHEF-DRII apparatus at 6 V/cm, with a 60 to 120 s switch time for 25 h. The gels were stained with ethidium bromide and imaged. The DNA in the gel was transferred to Hybond-XL membranes by neutral capillary blotting. The DNA was crosslinked to the membrane by UV irradiation in a Stratalinker (Stratagene) apparatus at maximum output for 60 seconds. The MCM3 probe was generated by amplifying MCM3 from genomic DNA using the primers 5’-CTGTGCAAGAAATGCCCGAAATG-3’ and 5’-GCCCCGGAGTTGGAATGCTC-3’ followed by random primer labeling of the PCR product with the Biotin DecaLabel DNA Labeling Kit (Thermo Scientific). Probe hybridization was performed at 50°C for 1 hr. Biotin signal was detected using Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific).

Typing MRSE and MRSH was performed using the PFGE method as previously described by Talebi et al. [3 (link)]. Briefly, the enzyme digestion of the plugs was carried out with the restriction enzyme SmaI (New England Biolabs). A Salmonella serotype Braenderup strain (H9812) was used as a molecular size marker (kindly provided by the Research Center of Health Reference Laboratories, Tehran, Iran). In addition, DNA separation was carried out by programming two states under the following conditions in a pulsed-field electrophoresis system (CHEF DR-II; Bio-Rad Laboratories, Hercules, CA, USA) at the temperature of 14°C; voltage 6 V/cm; switch angle, 120°C; switch ramp 1-30 second for 20 hours. Ethidium bromide (SinaClon, Iran) 0.5 mg/mL was used for staining gels. For PFGE pattern analysis, BioNumerics software version 7.5 (Applied Maths, St-Martens-Latem, Belgium) was applied. The unweighted pair group method by using mathematical averaging (UPGMA), dice correlation coefficient with 1.5% optimization, and a 1.5% tolerance setting was used for the calculation of dendrograms. Isolates were considered genetically indistinguishable, closely related, possibly related, and different when there were 0, 1, 2, and >3 banding differences, respectively [25 (link)].

All S. aureus isolates were genotyped by pulsed-field gel electrophoresis (PFGE) using the SmaI digestion enzyme (Sigma, Poole, Dorset, UK) as described before [29 (link)]. PFGE of the H. influenzae isolates was performed similarly, with some supplementation taken from the protocol by Saito et al [30 (link)]. Briefly, after solidification, the plugs were incubated first in 100 mM EDTA containing lysozyme for three hours at 37°C, then in 100 mM EDTA containing proteinase K overnight at 54°C. The purified chromosomal DNA was digested by SmaI enzyme. DNA fragments were separated in 1% pulse-field certified agarose gel (Bio-Rad) in 0.5x TBE (Tris-boric acid-EDTA) buffer in a CHEF-DR® II apparatus, at 14°C, 6 V/cm, for 10 h with pulse times of 5 s to 15 s, and for 10.5 h with pulse times of 15 s to 60 s. S. pneumoniae isolates were also compared based on their SmaI restriction patterns as described before [31 (link)]. PFGE profiles were analysed with the BioNumerics software version 2.5 (Applied Maths, Sint-Martens-Latem, Belgium), applying unweighted pair group method using arithmetic averages (UPGMA) and the different bands similarity coefficient, with a band position tolerance of 2.0%. During interpretation, the Tenover’s criteria [32 (link)] and the suggested designation by van Belkum et al. [33 ] were applied.