Plasmid Analysis of NDM-1-Producing Bacteria

Plasmid DNA was extracted from donor, recipients, and transconjugants by Kado and Liu’s (1981) (link) method and analyzed by gel electrophoresis using 0.8% agarose. Presence of bla_NDM-1 in the purified plasmid of the donor and transconjugants was determined by PCR. For size determination, plasmid analysis by S1 nuclease-pulsed-field gel electrophoresis (PFGE) (Barton et al., 1995 (link)) was performed with the donor and CT-E. coli J53. Total bacterial DNA prepared in agarose plugs was digested with S1 nuclease (Fermentas, Waltham, MA, USA) and separated using a CHEF-Mapper PFGE system (Bio-Rad, Hercules CA, USA), as reported previously (Kumarasamy et al., 2010 (link)). The PFGE conditions were run time 18 h gradient 6 V/cm, temperature 14°C, included angle 120° and initial and final pulses conducted for 2.16 s and 54.17 s, respectively. DNA of S. enterica serovar Braenderup H9812 digested with Xba1 (Roche) was included as control size marker. Incompatibility group of plasmid was determined for the Salmonella isolate and E. coli J53 transconjugant (as E. coli J53 Az^R was devoid of plasmids) using the PCR-based replicon typing (PBRT) method as described by Carattoli et al. (2005) (link).