The transferability of the carbapenem resistance phenotype was determined by conjugation experiments (Lorenzo-Diaz and Espinosa, 2009 (link)) using the streptomycin-resistant E. coli C600 strain. Transconjugants were chosen on MacConkey agar plates (Huankai Co. Ltd., Guangzhou, China) supplemented with cefotaxime (1 mg/L) and streptomycin (1,500 mg/L).
The electrotransformation experiments (Choi et al., 2006 (link)) were further performed for isolates that failed conjugation experiments. In brief, plasmid DNA was extracted using a QIAPrep Plasmid Midi Kit (QIAGEN, Hilden, Germany) and transformed into electro-competent E. coli DH5α (TaKaRa Biotechnology, Dalian, China). electrotransformants were selected on LB agar plates (Huankai Co. Ltd., Guangzhou, China) supplemented with cefotaxime (1 mg/L).
We further confirmed the transconjugants or electrotransformants by PCR for the blaKPC-2 gene and tested for antimicrobial susceptibility as described above.
The electrotransformation experiments (Choi et al., 2006 (link)) were further performed for isolates that failed conjugation experiments. In brief, plasmid DNA was extracted using a QIAPrep Plasmid Midi Kit (QIAGEN, Hilden, Germany) and transformed into electro-competent E. coli DH5α (TaKaRa Biotechnology, Dalian, China). electrotransformants were selected on LB agar plates (Huankai Co. Ltd., Guangzhou, China) supplemented with cefotaxime (1 mg/L).
We further confirmed the transconjugants or electrotransformants by PCR for the blaKPC-2 gene and tested for antimicrobial susceptibility as described above.