Conjugation Experiment: Bacterial Resistance Transfer

The conjugation experiment of biparental mating on the sterile nitrocellulose filter was performed using the multiple resistant P. aeruginosa as the donor cell and the rifampin-resistant Escherichia coli C600 (EC600) as the recipient (He et al., 2021 (link)). The transconjugants were selected on Mueller–Hinton agar plates containing 600 μg/ml rifampin and 32 μg/ml ceftazidime or 8 μg/ml meropenem. The target resistance gene in the transformant was verified by PCR with primers of the corresponding β-lactamase resistance genes (Table 1). The PCR product was sequenced and the sequence was compared with those in the public database by the BLASTN program (see footnote 15). The plasmid of the transformant was extracted with an AxyPrep Plasmid Miniprep Kit (Axygen Scientific, Union City, CA, United States), and the target resistance gene on the plasmid was also verified by PCR and PCR product sequencing as mentioned above.