Results were interpreted

as percent sensitive (%S), percent resistant (%R) and percent intermediate (%I) (Pardesi et al., 2007). Determination of the MIC required to inhibit the growth of six strains of A. baumannii using 14 antibiotics from different groups were carried out by an agar dilution method (Deshpande et al., 1993). Antibiotics were checked in the range of 1–1024 μg mL−1 (National Committee for Clinical Laboratory Standards, 2000). Plasmid isolation was done using the O’Sullivan and Klaenhammer method (O’Sullivan & Klaenhammer, 1993). Agarose gel electrophoresis was performed by 0.8% w/v agarose gel prepared in Tris-acetate Selumetinib manufacturer buffer. Plasmid profiles were documented under UV light in gel documentation system (Alpha Innotech Corp.). Molecular weights of plasmids from different A. baumannii isolates were determined using the molecular weight determination parameter in gel documentation system GDC-0449 cell line (Alpha Innotech Corp.). The plasmids from E. coli V517 (MTCC 131) were also included as the positive controls and used for

comparison to test plasmids as well as molecular weight determination (O’Sullivan & Klaenhammer, 1993). Multiple plasmid-containing A. baumannii strains (A1, A2 and A3) with biofilm formation ability were selected for plasmid curing using E. coli MTCC 131 as a standard control. Curing was performed by the use of different curing agents such as ethidium bromide, plumbagin, Rebamipide acriflavin and acridine orange (Shakibaie et al., 1999). The percentage of curing efficiency was expressed as the number of colonies with cured phenotype per 200 tested colonies. The confirmation of cured clones was performed by agarose gel electrophoresis. The MIC of cured colonies was also tested for loss of resistance to antibiotics by an agar dilution method (Shakibaie et al., 1999; Cusumano et al., 2010). Conjugational gene transfer was performed from A. baumannii A3 pUPI 801–807 (Ar, Cur, Cir, Csr, Cpr, Nfr) to E. coli HB 101 (rifampicin-resistant

mutant) by the membrane filter technique (Chopade et al., 1985). The frequency of intergeneric conjugation was determined as the number of transconjugants obtained mL-1 on selective medium divided by total viable count of the recipient (Deshpande & Chopade, 1994). Natural transformation was performed using the plate assay (Ray & Nielsen, 2005). Acinetobacter baylyi 7054 trpE was used as the host for transformation experiments and plasmid DNA from A. baumannii A3 was prepared as the donor strain (O’Sullivan & Klaenhammer, 1993). The experiments were carried out using plasmids: pUPI 801–807 (Ar, Cur, Cir, Csr, Cpr, Nfr) from A. baumannii A3 and competent cells of A. baylyi 7054 trpE as the recipient. They were confirmed for the presence of transferred plasmids according to O’Sullivan & Klaenhammer (1993).