Inoculum effect of antimicrobial peptides

The activity of many antibiotics depends on the initial density of cells used in bacteria growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, since bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules to fight drug-resistant bacteria, since they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here we report the first systematic characterization of the inoculum effect for this class of antibacterial compounds. Thirteen peptides (including all-D enantiomers) and peptidomimetics were analyzed by measuring minimum inhibitory concentration values, covering more than 7 orders of magnitude in inoculated cell density. In all cases, we observed a significant inoculum effect for cell densities above 5 x 104 cells/mL, while the active concentrations remained constant (within the micromolar range) for lower densities. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide-cell association and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause a killing effect. The observed effects question the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the inoculum dependence of the activity of antimicrobial peptides and peptidomimetics should be considered. Significance statementBacterial drug resistance is a crucial threat to global health and antimicrobials with novel mechanisms of action are severely needed. Antimicrobial peptides are natural molecules that kill bacteria mostly by perturbing their membranes and represent promising compounds to fight resistant microbes. Their activity is normally tested under standardized conditions of bacterial density. However, the bacterial load in clinically relevant infections varies by many orders of magnitude. Here we showed that the minimum peptide concentration needed for bacterial killing can vary by more than 100 times with an increase in the density of cells in the initial inoculum of the assay (inoculum effect) These findings question utility of the presently used activity screening assays and our current understanding of antimicrobial peptides.