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 Trametinib in vitro using the Student’s t-test; a P-value of ≤ 0.05 was considered significant. Transformation experiments were performed as described previously by electroporation CH5424802 order 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). Vasopressin Receptor 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.