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.

Lactoferricins access the cytosol of Escherichia coli within few seconds

“…It is currently not clear whether the present findings can also be extended to other AMPs. Yet, similar conclusions were drawn for the peptide LL-37 by Zhu et al ( 2019 ), and an emergent number of AMPs has been reported to show comparable partitioning behavior in bacteria ( Loffredo et al, 2021 ). We thus propose, that the combination of membrane translocation speed and efficient shut down of bacterial metabolism are generic factors that should be considered in designing future AMPs to combat infectious diseases.…”

The Attenuated Protective Effect of Outer Membrane Vesicles Produced by a mcr-1 Positive Strain on Colistin Sensitive Escherichia coli

“…We noticed that the protective effects of 2 mg/ml E. coli ATCC25922 OMVs and 20 mg/ml E. coli 08-85 OMVs in the growth curve assay were not exactly the same as in the killing protection assay. This could be due to the inoculum effect in which the initial density of cells affects the activity of many antibiotics including membrane-active peptides (Loffredo et al, 2021). The growth curve assay was started with a 1:1,000 dilution of the overnight culture (starting inoculum of 10 5 CFU/ml), while the log-phase culture was used as the starting inoculum which was 10 7 CFU/ml in the killing protection assay.…”

How to Combat Gram-Negative Bacteria Using Antimicrobial Peptides: A Challenge or an Unattainable Goal?

“…AMPs which have been approved or are in clinical development have had 1.7 to 16 µM MIC values against Gram-negative bacteria ( Table 1 ). It is worth noting that although such values can act as reference regarding activity, determining MIC is influenced by many experimental factors such as the ion concentration in the medium used, initial bacterial inoculum and pH [ 37 , 38 , 39 ]. AMPs in clinical development (colistin, pexiganan and LL-37) have haemolytic activity at their MIC so their administration route has been limited to just topical use as their toxicity prohibits systemic application.…”

“…Engineered cationic antimicrobial peptide (eCAP) WLBU2 (PLG0206) is in phase I clinical trials (ACTRN12618001920280); it has been projected for the treatment of prosthetic joint infections due to its anti-Gram-positive bacteria activity [ 34 ]. However, this broad-spectrum peptide could be useful for treating Gram-negative bacteria infections such as P. aeruginosa and other multi-resistant bacteria [ 39 ]. WLBU2 has proven to be superior to natural peptides such as colistin and LL-37 due to it having less potential for inducing resistance and greater stability in in vivo conditions [ 36 , 37 ].…”

“…WLBU2 has proven to be superior to natural peptides such as colistin and LL-37 due to it having less potential for inducing resistance and greater stability in in vivo conditions [ 36 , 37 ]. In spite of this, WLBU2′s poor selectivity continues to be a limiting factor [ 39 ].…”