Inoculum effect of antimicrobial peptides

Abstract

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 10⁵ cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. 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 bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.

Keywords: antimicrobial activity; antimicrobial peptides; inoculum effect.

Fig. 1.

IE for the MIC values of different AMPs and peptidomimetics against E. coli ATCC 25922 (or S. epidermidis ATCC 12228, where indicated). Each measurement was repeated in triplicate, and all three data points (which often overlap) are reported (in different shades of blue). Error bars indicate the interval between the lowest concentration inhibiting bacterial growth and the highest concentration not causing inhibition. For membrane-active peptides and peptidomimetics, the best fit to Eq. 2 is reported as a red line. When the MIC was higher than the highest concentration tested (i.e., 64 µM), the data point was reported arbitrarily as 96 ± 32 µM, as a crossed square, but it was not included in the fit.

Fig. 2.

Comparison of the IE for the MIC values of enantiomeric peptides against E. coli ATCC 25922. Data for the all-L and all-D enantiomers are reported in shades of blue and red, respectively. For the definition of symbols and error bars, please refer to Fig. 1.

Fig. 3.

IE of LL-37 against E. coli ATCC 25922 after 20 h at 37 °C in 100, 50, and 10% MHB. Results of three independent measurements are reported in different shades of blue, green, and red, for 100, 50, and 10% broth, respectively. For the definition of symbols and error bars, please refer to Fig. 1.

Fig. 4.

Peptide adsorption to the microtiter plate surface as determined by the fluorescence in the center of the well volume. Peptide concentrations of 2, 4, and 8 µM are reported in blue, green, and red, respectively. The dashed lines in the indolicidin plot correspond to a 40 µM tryptophan solution, used as a control for possible photobleaching effects. Top: 20 h; bottom: 60 min.

Fig. 5.

Bacterial growth curves for different inocula of E. coli ATCC 25922 (5 × 10¹, 5 × 10², 5 × 10³, 5 × 10⁴, 5 × 10⁵, 5 × 10⁶, 5 × 10⁷, and 1 × 10⁸ CFU/mL, shown in violet, purple, blue, light blue, green, orange, red, and dark red, respectively), untreated (Left), or in the presence of 1/2 × MIC of indolicidin (Right). A representative growth curve (5 × 10⁵ CFU/mL), in the presence of an indolicidin concentration corresponding to the MIC, is also reported as a black dashed line (Right). The OD of the bacterial culture was measured at 590 nm during incubation at 37 °C. Results are the mean of duplicate samples from a single experiment, representative of two different experiments.

Fig. 6.

Model predictions for peptide-cell binding and the IE. The fraction of cell-bound peptide molecules (f_b, Left) and MIC relative to the plateau value at low cell densities (Right) are both shown as a function of the inoculum cell density relative to the apparent partition constant. The blue and orange zones correspond to inocula in which the peptides are predominantly free in solution or predominantly bound to cells, respectively.