Plasmid profile analysis and conjugation assays.

Plasmid DNA was prepared according to the rapid alkaline lysis method and separated by horizontal electrophoresis in 0.7% agarose gels in Tris-borate-EDTA (TBE) buffer at room temperature at 100 V (50 mA) for 4 h (19). The molecular weights of unknown plasmids were determined by comparing their band patterns with those obtained from plasmids used as standards. Plasmids present in E. coli PDK-9 (140, 105, 2.7, and 2.1 MDa), R1 (62 MDa), RP4 (36 MDa), Sa (23 MDa), and V-517 (35.8, 4.8, 3.7, 3.4, 3.1, 2.0, 1.8, and 1.4 MDa) strains were used as molecular weight standards (20). Conjugation was carried out by both broth mating and filter mating assays at 30°C using NDM-1-producing isolates as a donor and sodium azide-resistant E. coli J53 as a recipient. Transconjugant selection was performed on MacConkey agar plates containing meropenem (0.5 mg/liter) and sodium azide (100 mg/ml). Transconjugants were tested for the NDM-1 gene, as described above, by PCR. The MICs of antibiotics against all transconjugants were determined as described above. Plasmid analysis of the transconjugants was carried out as described previously (18).