Revisiting the mode of action of daptomycin considering the inoculum effect and a novel liposome-MIC-competition assay.

Daptomycin (DAP) is a cyclic lipopeptide drug for intravenous application against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). In spite of numerous studies, its mode of action remains controversial. This is partially due to the fact that no attention has been paid to the strong dependency of DAP activity on the cell count - referred to as the inoculum effect when minimal inhibitory concentrations (MICs) are concerned. The first objective is to quantify the inoculum effect of DAP acting against MRSA and, on this basis, review comparisons of data recorded at different cell counts. The data imply that the aqueous DAP concentration in equilibrium with minimally growth-inhibited MRSA is 0.24 μg/mL and the corresponding inhibitory drug load bound per bacterium is Rebac∗ = 1.0E+7. To directly compare phenomena observed at different cell counts, the samples must agree in Rebac∗; not in DAP concentration. The second objective has been to render data on liposomal model systems quantitatively comparable to in vivo data. This is achieved by the liposome-MIC-competition assay combining bacteria and liposomes in a single MIC experiment. It suggests an equivalent phosphatidylglycerol concentration of 3.5E-8 μM/(CFU/mL), giving rise to a re-assignment of model studies to comparable in vivo systems. Finally, the data obtained here are combined with common estimates regarding the lipid numbers per bacterium, suggesting that the majority of the bacteria-bound DAP is not localized in the membrane but most likely in the cell wall. Off-target binding is a crucial issue for membrane-active antibiotics.