Xba 1 or s1 nuclease

The Salmonella enterica serovar Braenderup H9812 (as size marker) and E. coli cells harboring the mcr-1 gene were fixed by SeaKem Gold Agarose (Lonza Group AG, United States) and subsequently lysed. The embedded DNAs were digested using Xba I or S1 nuclease (Takara Bio, China) in a 37°C water bath for 3 h. The restricted DNA fragments were separated in 0.5 × Tris-Borate-EDTA buffer (Sangon Biotech, Shanghai, China) at 14°C for 18 h using a pulsed-field electrophoresis system (CHEF Mapper, Bio-Rad, United States) with pulse times of 2.2 to 63.8 s. The gel block was stained and observed with a gel imager (Tang et al., 2019a (link); Liang et al., 2021b (link)). The mcr-1-specific probe was labeled using a DIG High Prime DNA Labeling and Detection Starter Kit (Roche, Sant Cugat del Vallés, Spain) following the manufacturer’s instructions. E. coli J53 was used as the recipient strain, and E. coli strains harboring mcr-1 were used as the donor strain in the conjugation transfer assay. Donor and recipients bacteria were grown together overnight and then inoculated onto LB plates with colistin and sodium azide.

Pulsed-field gel electrophoresis (PFGE) was performed to determine the genotypic relatedness of E. fergusonii isolates, and S1-PFGE was performed to demonstrate the approximate size of plasmids harbored by E. fergusonii. The restriction endonucleases Xba I and S1 nuclease were used in PFGE analysis and S1-PFGE analysis, respectively. Briefly, after E. fergusonii cells were fixed by SeaKem Gold Agarose (Lonza Group AG, USA) and subsequently lysed, the embedded DNAs were digested using Xba I or S1 nuclease (Takara Bio, China) in a 37°C water bath for 3 h. The restricted DNA fragments were separated in 0.5× Tris-Borate-EDTA buffer (Sangon Biotech, Shanghai, China) at 14°C for 18 h using a CHEF Mapper electrophoresis system (Bio-Rad, USA) with pulse times of 2.2 to 63.8 s. PFGE images were processed by cluster analysis and band-matching using BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) to determine the E. fergusonii genotypes and kinships. The band matching was performed with optimization and tolerance settings of 1.0% and 1.5%, respectively. Homology cutoff values of 70% and 85% were used to group the related isolates within the same PFGE-type. Salmonella Braenderup H9812 was used as a standard DNA ladder strain.

Pulsed-field gel electrophoresis (PFGE) was performed to determine the genotypic relatedness of E. fergusonii isolates, and S1-PFGE was performed to demonstrate the approximate size of plasmids harbored by E. fergusonii. The restriction endonucleases Xba I and S1 nuclease were used in PFGE analysis and S1-PFGE analysis, respectively. Briefly, after E. fergusonii cells were fixed by SeaKem Gold Agarose (Lonza Group AG, USA) and subsequently lysed, the embedded DNAs were digested using Xba I or S1 nuclease (Takara Bio, China) in a 37°C water bath for 3 h. The restricted DNA fragments were separated in 0.5× Tris-Borate-EDTA buffer (Sangon Biotech, Shanghai, China) at 14°C for 18 h using a CHEF Mapper electrophoresis system (Bio-Rad, USA) with pulse times of 2.2 to 63.8 s. PFGE images were processed by cluster analysis and band-matching using BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) to determine the E. fergusonii genotypes and kinships. The band matching was performed with optimization and tolerance settings of 1.0% and 1.5%, respectively. Homology cutoff values of 70% and 85% were used to group the related isolates within the same PFGE-type. Salmonella Braenderup H9812 was used as a standard DNA ladder strain.