Polycationic antimicrobial peptides are an important component of the innate defence of all species of life. These compounds are active against a broad range of bacterial strains, including antibiotic resistant isolates, and are synergistic with conventional antibiotics. The therapeutic use of a cationic antibiotic polymyxin B (PMB) was abandoned for a long time due to its undesirable side effects. However, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Using different substrates and inhibitors of energy metabolism we obtained information on the mechanism of PMB interaction with bacterial outer membrane (OM) as well as with the plasma membrane (PM). Methods: An electrochemical monitoring of K+, Ca2+, H+, tetraphenylphosphonium (TPP+), and phenyldicarbaundecaborane (PCB-) ion fluxes across envelopes of E. coli and Pseudoalteromonas spp. cells was performed. In parallel, the cell binding of fluorescent compound dansylpolymyxin, OD of bacterial suspensions, ATP content of cells, and bactericidal activity of PMB were studied. Results: Using different conditions of cell incubation, the OM permeabilizing activity of PMB was dissected from the PM depolarizing effects. These two stages were easily distinguishable in the presence of high concentrations of divalent cations and can be separated in time by 1-5 min interval. PMB-induced pores in bacterial envelope were registered, but the pore formation and depolarization of the PM were not obligatory for the dissipation of cell K+ gradient or the bactericidal action of this antibiotic. At conditions of increased ionic strength the dependence of membranotropic activity of PMB on metabolic state of the cells was discovered. Energization of the cells by glucose stimulated the binding of PMB to bacteria and the depolarizing activity of this antibiotic. Membranotropic effects of PMB were considerably stronger when all amount of the drug was added to the cell suspension at one stroke. Conclusions: 1. At low concentrations PMB compromises the barrier of the OM, while at higher concentrations it also depolarizes the PM by forming ion-permeable pores. 2. High ionic strength prevents the self-promoted entry of PMB into bacterial cells, though the ability to bind to the OM surface is not affected. 3. Suppression of energy metabolism of bacteria makes them more resistant to PMB.

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