Confirmation of mcr Gene Location

To confirm mcr genes location biologically, conjugation was performed with an azide-resistant E. coli J53 strain. Selection of transconjuguants was done on MacConkey agar (Beckton Dickinson, Le Pont de Claix, France) supplemented with 120 mg/L sodium azide and 2 mg/L colistin. In case of unsuccessful conjugation, transformation of pure plasmid by electroporation method with Top10 electrocompetents E. coli (Thermo Fisher Scientific, Waltham, MA, United States) was performed and transformants were selected on LB agar (Beckton Dickinson, France) with 2 mg/L colistin. Plasmid curing was performed at high temperature on K. pneumoniae LH94 strain (Letchumanan et al., 2015 (link)). Then, the presence of mcr genes and plasmid types were controlled by standard PCR (Carattoli et al., 2005 (link); García-Fernández et al., 2009 (link); Rebelo et al., 2018 (link); Wang et al., 2018 (link)).