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.
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.
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