Chef mapper pfge system

PFGE molecular typing was performed according to the PFGE protocol for C. jejuni [42 (link),43 ]. Briefly, restriction digestion was conducted by using 40 U SmaI (Takara, Dalian, China), and run on a CHEF Mapper PFGE system (Bio-Rad Laboratories, Hercules, Canada) for SeaKem gold agarose (Lonza, Rockland, MD, USA) in 0.5×Tris-borate-EDTA. Bionumerics v6.6 software was used for the clustering analysis. Similarity greater than 95% was considered as the same genetic group. The similarity between chromosomal fingerprints was scored using the Dice coefficient. The unweighted pair group method, with arithmetic means (UPGMA) at the cut-off of 1.5% tolerance and 1.00% optimization, was used to obtain the dendrogram in the PFGE profile.

All 21 S. Choleraesuis isolates included in this study were analyzed by PFGE according to the standard methods outlined by PulseNet [23 (link)]. The DNA was digested by XbaI restriction enzyme (Takara, Dalian, Japan) at 37°C for 3 h. Then, electrophoresis of the digested DNA was performed using a CHEF Mapper PFGE system (Bio-Rad, Hercules, CA, USA) using 1% SeaKem agarose in 0.5× Tris-borate-EDTA for 19 h. Macrorestriction patterns were compared using BioNumerics Fingerprinting software version 6.01 (Applied Math, Austin TX, USA). The dendrogram was based on unweighted pair-group method by use of average linkages and pairwise Dice coefficients.

Plasmid DNA was extracted from donor, recipients, and transconjugants by Kado and Liu’s (1981) (link) method and analyzed by gel electrophoresis using 0.8% agarose. Presence of bla_NDM-1 in the purified plasmid of the donor and transconjugants was determined by PCR. For size determination, plasmid analysis by S1 nuclease-pulsed-field gel electrophoresis (PFGE) (Barton et al., 1995 (link)) was performed with the donor and CT-E. coli J53. Total bacterial DNA prepared in agarose plugs was digested with S1 nuclease (Fermentas, Waltham, MA, USA) and separated using a CHEF-Mapper PFGE system (Bio-Rad, Hercules CA, USA), as reported previously (Kumarasamy et al., 2010 (link)). The PFGE conditions were run time 18 h gradient 6 V/cm, temperature 14°C, included angle 120° and initial and final pulses conducted for 2.16 s and 54.17 s, respectively. DNA of S. enterica serovar Braenderup H9812 digested with Xba1 (Roche) was included as control size marker. Incompatibility group of plasmid was determined for the Salmonella isolate and E. coli J53 transconjugant (as E. coli J53 Az^R was devoid of plasmids) using the PCR-based replicon typing (PBRT) method as described by Carattoli et al. (2005) (link).

PFGE of 28 isolates was performed following the PulseNet standardized protocol (www.cdc.gov/pulsenet). Genomic DNA fragments were prepared from each isolate with the restriction enzyme XbaI (NEB), incubated at 37°C for 2 h. Macrorestriction fragments were run on a 1% agarose gel in 0.5 × TBE buffer (containing 50 μM thiourea to prevent DNA degradation) in a CHEF Mapper PFGE system (BioRad). Gels were stained with ethidium bromide and photographed with UV transillumination. Salmonella Braenderup H9812 XbaI-digested DNA was used as the molecular weight standard. The settings used were: 6 V/cm, an angle of 120°, initial switch time of 2.16 s, final switch time of 63.8 s, and a run time of 19.5 h.
Macrorestriction fragments were compared using the fingerprint analysis module of the BioNumerics software (Version 7.6, Applied Math). The isolates were considered dissimilar if one or more DNA bands appeared to have different molecular weights. The similarity index was calculated using the Dice similarity coefficient, and a similarity dendrogram was constructed using the unweighted pair group method using average linkages (UPGMA) with 1% optimization and 1% band position tolerance.

The conjugation experiment was carried out to investigate the transferability of mcr gene. Procedures were performed according to the method described previously (Hadjadj et al., 2019 (link)). Sodium azide-resistant E. coli J53 (MIC of 200 mg/L) was used as recipient strain. The transconjugants were selected on the medium containing azide at 200 mg/L and PMB, at 1 mg/L was consistent with the MIC of the donor. Resultant colonies were screened for the presence of mcr by PCR.
S1-PFGE analysis of mcr-positive isolates was used to estimate the sizes of mcr-positive plasmids. XbaI-restricted DNA of Salmonella enterica serovar Braenderup H9812 was used as a DNA marker. Shortly, agarose plugs were made using the colonies in the medium of a single colony inoculated and digested with S1 nuclease (TaKaRa, Dalian, China). The DNA was separated using the CHEF-MAPPER PFGE system (Bio-Rad) under the following conditions: 14°C, 6 V/cm, and a 120° pulse angle for 16 h, with the initial and final pulses conducted for 2.16 s and 63.8 s, respectively. Then the dyed gel was visualized with the imaging system.

To estimate the sizes of mcr-positive plasmids, S1-PFGE and Southern hybridization were performed. Briefly, bacterial whole-cell DNA of mcr-positive isolates and their transconjugants was prepared in agarose plugs and digested with S1 nuclease (TaKaRa, Dalian, China). The DNA was separated using the CHEF-Mapper PFGE system (Bio-Rad) under the following conditions: 14°C, 6 V/cm, and a 120° pulse angle for 16 h, with the initial and final pulses conducted for 2.16 and 63.8 s, respectively. The separated DNA fragments were transferred to nylon membranes, hybridized with digoxigenin-labeled mcr-10- or mcr-8-specific probes, and detected using the nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP) color detection kit (Roche, catalog no. 11745832910).