baumannii BM4547 and P. aeruginosa PU21

as recipients and the five NDM-1-positive E. coli J53 transconjugants as donors. Mixes of donor and recipients cells were incubated for 18 h at 37 °C for S. typhimurium LT2, A. baumannii BM4547, P. aeruginosa PU21 and P. mirabilis CIP103181 and for 3 h at 37 °C for K. pneumoniae CIP15153. In addition, E. coli J53 transconjugant carrying a c. 70-kb IncF-type blaCTX-M-15-positive plasmid was included for comparison, as IncF-type plasmids conjugate efficiently among Enterobacteriaceae (personal data). Transfer frequencies were calculated by dividing the number of transconjugants by the number of donor cells. Statistical analysis was performed Gamma-secretase inhibitor using the Student’s t-test; a P-value of ≤ 0.05 was considered significant. Transformation experiments were performed as described previously by electroporation CAL-101 mw of a plasmid DNA suspension from the five NDM-1-positive E. coli J53 into

rifampicin-resistant P. aeruginosa and A. baumannii reference strains (Potron et al., 2009). pAT-RTG-4 (shuttle vector) and pInt-Veb plasmids were used as positive control for electroporation in A. baumannii and P. aeruginosa (Aubert et al., 2003; Potron et al., 2009). Selection was performed on agar plates supplemented with ticarcillin (50 μg mL−1). MICs of carbapenems and cefotaxime were determined using the E-test strips (bioMérieux, Marcy l’Etoile, France). The five blaNDM-1-carrying plasmids of Enterobacteriaceae studied here belonged to various incompatibility groups (L/M, FII, A/C and two untypeable plasmids). IncL/M, IncA/C and IncFII plasmid types have been frequently described in Enterobacteriaceae carrying other β-lactam resistance determinants (Carattoli, 2009). IncL/M- and IncA/C-type plasmids

are broad-host range plasmids, whereas IncF-type plasmids are narrow-host range plasmids (Novais et al., 2007). Astemizole The five NDM-1-positive plasmids were self-conjugative using E. coli J53 as recipient at frequencies ranging from 10−4 to 10−8 transconjugants/donor (Table 1). The blaNDM-1 gene was the single carbapenem resistance marker located on those plasmids. Using blaNDM-1-positive E. coli J53 transconjugants as donors, second-step transconjugants were obtained using E. coli, K. pneumoniae, S. typhimurium and P. mirabilis as recipient species. In E. coli JM109, transfer frequencies ranged from 10−4 to 10−8 transconjugants per donor depending on plasmid type (Table 2). The lowest transfer frequencies were obtained with the untypeable plasmid p419 and IncA/C-type plasmid pKp7. No difference of transfer rate was observed using E. coli Tc601 and E. coli Tc271 as donors when different temperatures were used during the mating-out assays (Table 2). Using Tc419 and TcKp7 as donors, the transfer rate was significantly higher at 30 °C compared with that observed at 25 °C and 37 °C (P < 0.05), as reported for other blaNDM-1-positive plasmids (Walsh et al., 2011). For E.