Conjugation and plasmid curing with

Conjugation and plasmid curing with an antibiotic resistance cassette.The E. coli S17-1 λpir strains containing each constructed plasmid were used as conjugal donors for A. baumannii strains. The transconjugants were conjugated and isolated as follows. Donor and recipient strains were grown in LB broth until the late-log phase (optical density at 600 nm [OD600], 0.8). The bacterial cells were then mixed at an equal ratio and were spotted onto an LB plate. The bacterial cells were incubated for 12 h at 30°C. The bacteria were resuspended in LB broth and were then plated onto LB agar plates containing ampicillin and kanamycin or erythromycin in order to eliminate the donor strains and select the merodiploid strains. Plasmids with the antibiotic resistance cassette were excised from the bacterial chromosome on solid LB medium with 10% sucrose and without NaCl (13). Each mutant was confirmed by PCR analysis.