Rapid assessment of susceptibility of bacteria and erythrocytes to antimicrobial peptides by single-cell impedance cytometry

Abstract

Antimicrobial peptides (AMPs) are natural molecules, produced by all organisms as a defense against infections. They usually kill pathogens by perturbing the permeability of their cell membranes. Due to this mechanism, AMPs exhibit a low propensity to induce bacterial resistance and are therefore promising compounds to fight antibiotic-resistant infections. In addition, they are often selective, killing bacteria at concentrations lower than those at which they are toxic to the host. However, clinical applications of AMPs are hindered by a limited understanding of their interactions with bacteria and human cells. Standard susceptibility testing methods are based on the analysis of the growth of a bacterial population and therefore require several hours. Moreover, different assays are required to assess the toxicity to host cells. In this work, we propose the use of microfluidic impedance cytometry to explore the action of AMPs on both bacteria and host cells, in a rapid manner and with single-cell resolution. By using a simple coplanar-electrode impedance cytometer, we show that the electrical signatures of Bacillus megaterium (B. 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-PMAP23 bactericidal activity and toxicity to RBCs. The impedance-based characterization is validated by comparison with standard antibacterial activity assays and absorbance-based hemolytic activity assays. Furthermore, we demonstrate the applicability of the technique to a mixed sample of B. megaterium cells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in presence of both cell types.