DNA Manipulation and Cloning

The bla_KPC–2 and bla_KPC–55 genes were amplified from the total DNA of K. pneumoniae KP1559 and BS407, respectively, using KPC_F (5′-AGGAGGTAAATAATGTCACTGTATCGC CGTCTAGTT-3′) and KPC_R (5′-TTACTGCCCGTTGACGCCCAA-3′) using Phusion^® High-Fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, United States). Each PCR product was purified and cloned into the pCR-Blunt vector (Invitrogen, Thermo Fisher Scientific). The recombinant plasmids were transformed into chemically competent E. coli One Shot^TM TOP10 (Invitrogen, Thermo Fisher Scientific) and selected on MH agar containing kanamycin 50 μg/ml and ampicillin 50 μg/ml. Nucleic acid sequences and the direction of each insert were verified by Sanger sequencing using the universal M13 primers of both directions.