Chef dr 3 pfge system

For amplicons used in plasmid constructions, PCR was performed using the Q5 Polymerase (NEB #M0491S) following the manufacturer’s instructions. For transformant screenings (yeast and mycoplasma), PCR was performed with the Advantage2 polymerase mix (Takara #639201) following the manufacturer’s instructions. Multiplex PCR amplification was performed with the QIAGEN Multiplex PCR kit (QIAGEN #206143). The primer pairs used during simplex and multiplex PCR are all listed in Table S1.
Mccp agarose plugs and yeast agarose plugs were subjected to PFGE in a 1 % Bio-Rad Pulsed Field Certified Agarose (Bio-Rad #162–0137) gels in 0.5× TBE buffer (Bio-Rad #161–0733), with a CHEF-DR III PFGE system (Bio-Rad). Running conditions were set up as follows: switch angle 120 °, voltage gradient 6 V cm⁻¹, ramped switch time from 50 (initial time) to 90 s (final time), duration 22 h and temperature 14 °C. After electrophoresis, the gel was stained with SYBR Gold and visualization was performed using a Vilbert Lourmat E-BOX VX2 Complete Imaging system. The Bio-Rad Marker 0.225–2.2 Mb S. cerevisiae chromosomal DNA (Bio-Rad #170–3605) was used for DNA size estimations.

PFGE was performed according to the international PulseNet CDC guidelines [25 ] and using the XbaI restriction enzyme. PFGE was performed using a CHEF DR–III PFGE system (Bio–Rad Laboratories, Inc., Hercules, CA, USA), and the following parameters were applied: separation on a 1% agarose gel (Pulsed Field Certified Agarose, Bio–Rad) in 0.5 M Tris–Borate–EDTA (TBE) buffer at 14 °C for 20 h (pulse times of 2.2–63.8 s). The gels were stained with 0.5 μg/mL of ethidium bromide for 15 min and photographed under UV transillumination using a QuantityOne (BioRad, Madrid, Spain) software and GelDoc 2000 (BioRad, Madrid, Spain) system. The banding patterns were analyzed with bionumerics Gel Compar II 6.5 software (Applied Maths, Sint–Martens–Latem, Belgium) using the Dice coefficient and the UPGMA (Unweighted Pair-Group Method with Arithmetic mean) algorithm. A position tolerance of 1% was adopted for the generation of a dendrogram. Salmonella strains with more than 95% similarity were clustered together as identical.

Genomic relationships were analyzed by XbaI restriction digestion followed by pulsed-field gel electrophoresis (PFGE) using the CHEF DR III PFGE System (BioRad, Hercules, CA, USA). Electrophoresis conditions consisted of an initial time of 2.2 s, a final time of 54.2 s at a gradient of 6 V cm^− 1 and an included angle of 120°. The gels were electrophoresed for 18 h. The results were evaluated with BioNumerics (version 7.6; Applied Maths, Austin, TX, USA) using the cut-off value of 80% similarity to distinguish PFGE types.

All the phenotypically efflux pump positive (carbapenemase non-producing) isolates were typed by pulsed-field gel electrophoresis (PFGE). Agarose blocks were made to prepare DNA and were subsequently digested with XbaI (Promega, Madison, WI). The digested DNA fragments were further separated by using CHEF-DR III PFGE system (Bio-Rad, Hercules, CA) for 24 h at 4 V/cm with pulses at 120° angle in a 10 s to 40 s pulse time.

DNA concentration was always tested by fluorometric and spectrophotometric methods. Quality was checked with a spectrophotometer and acceptable values are: ~1.8 for A260/280 and ~2.2 for A260/230, according to the official recommendations of ONT. Pulsed field gel electrophoresis was used to assess the length of isolated DNA, sheared DNA and prepared library. DNA fragments were separated by PFGE on a 1% agarose gel run in 0.5× TBE buffer at 6 V/cm, 14 °C, included angle of 120°, switch time 1–10 s for 14 h using a Bio-Rad CHEF-DR III PFGE system. The gel was then stained in 1µg/mL ethidium bromide solution at RT on a shaker for 30 min.

The 23 O. anthropi isolates were grown overnight and suspended in SE buffer (75 mM NaCl, 25 mM EDTA, pH 7.5). The cell suspensions (4 McFarland units) were mixed with an equal volume of 2% low-melting point agarose, moulded into plugs at 4°C, and lysed with lysis buffer (1% N-lauryl sarcosine, 0.5 M EDTA, pH 8) supplemented with Proteinase K (500 μg/mL). The DNA contained in each plug was digested by 20 U of SpeI restriction enzyme in accordance with the manufacturer's instructions. Macrorestriction fragments were separated using the CHEF-DR III PFGE system (Bio-Rad) at 10°C for 20 h, at a field strength of 6 V/cm, with an initial switch time of 5 s and a final switch time of 35 s. A lambda ladder of phage DNA concatemers was used as a size marker. O. anthropi ATCC 49188 T and O. intermedium LMG 3301 T were also genotyped by PFGE, and fragment patterns were compared according to the criteria described by Tenover [14 (link)].

The ampicillin-sulbactam resistance phenotype, IntI1 integrase positive genotype, and strains isolated within season of high infection rate were chosen as the selection criteria for listing candidates MDRAB strains. Standard protocol for PFGE analysis was employed for the A. baumannii isolates. Briefly A. baumannii were plated on blood agar and incubated in a 5% CO₂ atmosphere at 35°C for 16–24 h. Plug slices were digested with 20 U of SgrAI. The DNA fragments were then separated in 1% Seakem Gold agarose gels (FMC BioProducts) at 14°C using a Bio-Rad CHEF DRIII PFGE system (Bio-Rad Laboratories, Hercules, CA, USA). Gels were run in 0.5× Tris-borate-EDTA (TBE; pH 8) at a 120° fixed angle and a fixed voltage (6 V/cm), with pulse intervals from 4–40 s for 20 h. Following staining and imaging, the chromosomal DNA restriction patterns produced by PFGE were interpreted using Tenover’s categorization [16 (link)].