Get access

Effective antimicrobial activity of Cbf-K16 and Cbf-A7A13 against NDM-1-carrying Escherichia coli by DNA binding after penetrating the cytoplasmic membrane in vitro

Authors


Abstract

New Delhi metallo-beta-lactamase-1(NDM-1)-carrying isolates, which are resistant to most clinical used antibiotics except for tigecycline and colistin, have been found worldwide. Cathelicidin-BF (BF-30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Cbf-K16 and Cbf-A7A13 were obtained by mutating Lys16, Ala7, and Ala13 of BF-30, respectively. To investigate their antimicrobial activities against NDM-1 carrying bacteria, recombinant Escherichia coli BL21 (DE3)-NDM-1 with high NDM-1 activity was constructed by inserting the Klebsiella pneumoniae NDM-1 gene (GenBank accession no. HQ328085) into a pET28a vector and transforming it into E. coli BL21 (DE3). The peptides showed effective antimicrobial activities against NDM-1-carrying E. coli, and the minimum inhibitory concentrations of Cbf-K16 and Cbf-A7A13 were only 4 and 8 µg/ml, whereas those of minimum bactericidal concentrations were 8 and 16 µg/ml, respectively. A time course experiment showed that colony forming unit counts rapidly decreased, and bacteria were thoroughly eliminated within 3 and 6 h by the Cbf-K16 and Cbf-A7A13 treatments, respectively. The peptides penetrated the bacterial cell membrane and enabled β-galactosidase leakage, and caused the cytoplasmic membrane to become permeable, and finally bound to the DNA. The genomic DNA of E. coli was completely unable to migrate on an agarose gel after Cbf-K16 treatment (8 µg/ml). These data demonstrated that Cbf-K16 and Cbf-A7A13 possess effective antimicrobial activity against drug-resistant strains, including NDM-1 carrying E. coli BL21 (DE3)-NDM-1, by binding to DNA after penetrating the cytoplasmic membrane in vitro, which may have potential therapeutic value for the treatment of NDM-1-carrying bacterial infections. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Ancillary