Dinuclear polypyridylruthenium(II) complexes: flow cytometry studies of their accumulation in bacteria and the effect on the bacterial membrane

Objectives: To determine the energy dependency of and the contribution of the membrane potential to the cellular accumulation of the dinuclear complexes [{Ru(phen)₂}₂{μ-bb(n)}]⁴⁺ (Rubb(n)) and the mononuclear complexes [Ru(Me₄phen)₃]²⁺ and [Ru(phen)₂(bb₇)]²⁺ in Staphylococcus aureus and Escherichia coli, and to examine their effect on the bacterial membrane. Methods: The accumulation of the ruthenium complexes in bacteria was determined using flow cytometry at a range of temperatures. The cellular accumulation of the ruthenium complexes was also determined in cells that had been incubated with the metal complexes in the presence or absence of metabolic stimulators or inhibitors and/or commercial dyes to determine the membrane potential or membrane permeability. Results: The accumulation of ruthenium complexes in the two bacterial strains was shown to increase with increasing incubation temperature, with the relative increase in accumulation greater with E. coli, particularly for Rubb₁₂ and Rubb₁₆. No decrease in accumulation was observed for Rubb₁₂ in ATP-inhibited cells. While carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did depolarize the cell membrane, no reduction in the accumulation of Rubb₁₂ was observed; however, all ruthenium complexes, when incubated with S. aureus at concentrations twice their MIC, depolarized the membrane to a similar extent to CCCP. Except for the mononuclear complex [Ru(Me₄phen)₃]²⁺, incubation of any of the other ruthenium complexes allowed a greater quantity of the membrane-impermeable dye TO-PRO-3 to be taken up by S. aureus. Conclusions: The results indicate that the potential new antimicrobial Rubb(n) complexes enter the cell in an energy-independent manner, depolarize the cell membrane and significantly permeabilize the cellular membrane