Verification of mcr-2 Gene Location

The genomic location of the mcr-2 gene was verified by S1 nuclease digestion of genomic DNA followed by electrophoretic separation and Southern hybridization. A digoxigenin-labeled DNA probe (“DIG luminescent detection Kit”, Boehringer Mannheim GmbH, Mannheim) targeting a 567-bp PCR fragment specific for the mcr-2 gene using the aforementioned primers (Xavier et al., 2016 (link)) was used. In addition, we performed an in silico search of whole genome sequences with mlplasmids v. 1.0.0 (Arredondo-Alonso et al., 2018 (link)). To test whether the colistin resistance determinant was transferable, conjugation was performed by the broth filter mating method at 37°C using plasmid-free sodium azide resistant E. coli K12-J53 (J53 AziR) as recipient. Prior to the conjugation assays, all mcr-2-positive isolates were tested for their susceptibility to sodium azide. Transconjugants were selected on Endo agar plates containing 100 mg/L sodium azide and 2 mg/L colistin sulfate or containing 100 mg/L sodium azide and 4 mg/L colistin sulfate (Sigma-Aldrich, Germany, Karlsruhe, Germany). To confirm successful plasmid transfer, antimicrobial susceptibility testing of transconjugants, a PCR targeting the mcr-2 gene and plasmid profiling was performed as described above.