Rapid Assessment of Susceptibility of Bacteria and Erythrocytes to Antimicrobial Peptides by Single-Cell Impedance Cytometry

Antimicrobial peptides (AMPs) represent a promising classof compoundsto fight antibiotic-resistant infections. In most cases, they killbacteria by making their membrane permeable and therefore exhibitlow propensity to induce bacterial resistance. In addition, they areoften selective, killing bacteria at concentrations lower than thoseat which they are toxic to the host. However, clinical applicationsof AMPs are hindered by a limited understanding of their interactionswith bacteria and human cells. Standard susceptibility testing methodsare based on the analysis of the growth of a bacterial populationand therefore require several hours. Moreover, different assays arerequired to assess the toxicity to host cells. In this work, we proposethe use of microfluidic impedance cytometry to explore the actionof AMPs on both bacteria and host cells in a rapid manner and withsingle-cell resolution. Impedance measurements are particularly well-suitedto detect the effects of AMPs on bacteria, due to the fact that themechanism of action involves perturbation of the permeability of cellmembranes. We show that the electrical signatures of Bacillus megaterium cells and human red blood cells(RBCs) reflect the action of a representative antimicrobial peptide,DNS-PMAP23. In particular, the impedance phase at high frequency (e.g.,11 or 20 MHz) is a reliable label-free metric for monitoring DNS-PMAP23bactericidal activity and toxicity to RBCs. The impedance-based characterizationis validated by comparison with standard antibacterial activity assaysand absorbance-based hemolytic activity assays. Furthermore, we demonstratethe applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the wayto study AMP selectivity for bacterial versus eukaryotic cells inthe presence of both cell types.