Plasmid Transferability via Conjugation

To investigate the transferability of tet(X4)-bearing plasmid, conjugation assays were performed using streptomycin-resistance E. coli C600 as the recipient strain. Briefly, overnight cultures of donor and the recipient strains were 1:1 mixed and incubated at 37°C for 16 to 20 h. After incubation, 10-fold serial dilutions were mixed in sterile saline, and 100-μL samples were spread onto LB agar plates containing 4 mg/L tigecycline and 1,500 mg/L streptomycin. The tet(X4)-positive transconjugants were confirmed by PCR and ERIC-PCR (Versalovic et al., 1991 (link); Sun et al., 2019b (link)). Susceptibility of transconjugants was detected as mentioned previously. Plasmid analysis was performed using whole-genome sequence as described previously. Plasmid DNA was extracted using a Qiagen Prep Plasmid Midi Kit (Hilden, Germany).