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

Abstract

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight antibiotic-resistant infections. In most cases, they kill bacteria by making their membrane permeable and therefore exhibit low propensity to induce bacterial resistance. 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. Impedance measurements are particularly wellsuited to detect the effects of AMPs on bacteria, due to the fact that the mechanism of action involves perturbation of the permeability of cell membranes. 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-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 the presence of both cell types.

Addressing variability in cell electrorotation through holographic imaging and correction factors

“…Subsequently, the polarization and EROT torque were calculated using ( 1) and (6). Finally, the simulated electric field strength and EROT torque distribution within the device were time-averaged, extracted and used as input for (8). Both electrostatic simulations were done simultaneously but independently with a time-dependent study over 1 frequency cycle (20 steps), then the polarization and EROT torque calculation were done afterwards.…”

“…Among electrical characterization techniques, electrorotation (EROT) stands out for probing the cytoplasm and membrane properties without cell contact or displacement [7]. In contrast to impedance spectrometry, EROT does not require prior calibration and is less affected by background noise [8,9]. Consequently, EROT was used to measure the electrical properties of cells [4], cell infection [10], viability assays [11], and cell differentiation [12].…”

Convolutional Neural Network-Driven Impedance Flow Cytometry for Accurate Bacterial Differentiation

“…Although IFC technology has identified unprecedented advancements in cell-typing research, it is not yet systematic enough for bacteria differentiation, which is of vital importance in clinical diagnosis, water quality detection, food safety, and ambient air monitoring . In order to obtain a higher sensitivity and resolution for bacterial analysis, the geometric dimension and the electrode layout were further optimized to achieved various applications for bacterial study, such as bacteria quantification, , the antimicrobial susceptibility test (AST), − and activity analysis . Although IFC has been applied to the measurement and classification of bacteria and submicrometer particles, direct differentiation of various species of bacteria remains a challenge.…”

On the compatibility of single-cell microcarriers (nanovials) with microfluidic impedance cytometry

“…[1][2][3] By means of multifrequency electrical impedance measurements, MIC measures the electrical phenotype of individual biological cells, which is related to cell type and status. MIC has been extensively used for the label-free characterization of mammalian cells, human pathogens, and plant cells, for applications including viability assessment, [4][5][6] subpopulation distinction, [7][8][9][10] antimicrobial susceptibility testing [11][12][13][14] and deformability assessment. 15,16 MIC has also been reported as a tool for the non-invasive monitoring of 3D cell cultures under continuous flow, 17,18 and impedance-based microfluidic sorters have been proposed for sorting cells upon their electrical phenotype.…”