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 well-suited 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.

Keywords: Bacillus megaterium; antimicrobial peptides (AMPs); antimicrobial susceptibility testing (AST); electrical sensing; erythrocyte; microfluidic impedance cytometry; single-cell analysis.

Figure 1

Overall experimental protocol. (a) Sample preparation: B. megaterium cells and human RBCs were incubated at 37 °C for 30 min with the DNS-PMAP23 peptide at six different concentrations in the range of 0.025–0.80 μM. As negative control, samples without peptide were prepared. As positive control, samples with peptide concentration over 1 μM (for the bacteria) or samples treated with osmotic shock (for the RBCs) were prepared. (b) Standard bactericidal assay: colony-forming unit (CFU) count after overnight culture. (c) Standard RBC hemolysis assay: absorbance measurements at 414 nm of supernatant after centrifugation (N_R, number of replicates). (d) Microfluidic impedance cytometry: bacterial suspensions, RBC suspensions, or mixed samples were measured with an impedance cytometer at 0.5, 11, and 20 MHz stimulation frequency. Thousands of single cells were acquired for each experimental condition (N_E, number of events).

Figure 2

Results of the B. megaterium analysis. (a) Impedance-based characterization of B. megaterium cells at 0.5 MHz (first row), 11 MHz (second row), and 20 MHz (third row) stimulation frequencies. The density plot of the phase against the electrical diameter is shown for the negative control sample (0 μM, first column), the sample at 0.10 μM (second column), and the sample at 2 μM (third column). For each stimulation frequency (i.e., in each row), the red contour line in the first column denotes the region enclosing 95% of the datapoints of the negative control sample (0 μM). This contour line is plotted as a reference also in the density plot of the samples at 0.10 μM (second column) and 2 μM (third column). (b) Empirical probability density function of the phase at 0.5 MHz (first row), 11 MHz (second row), and 20 MHz (third row) for the samples at 0 μM (in red), 0.10 μM (in green), and 2 μM (in blue). (c) Median values of the phase at 0.5 MHz (first row), 11 MHz (second row), and 20 MHz (third row) as a function of the peptide concentration. Interquartile ranges are also shown. In each panel, the horizontal line indicates the median value of the phase of the negative control sample (0 μM). (d) Density plot of the phase at 20 MHz against the electrical diameter at 0.5 MHz (sample at 0.10 μM), along with exemplary snapshots of a flowing B. megaterium cell (three consecutive frames, fr.; scale bar is 20 μm). (e) Comparison of the killing curve based on microfluidic impedance cytometry (at 20 MHz), in blue, with the killing curve based on CFU counts, in red. Markers denote experimental datapoints (circles and triangles refer to different experiment repetitions), and continuous lines denote fits of the results.

Figure 3

Results of the RBC analysis. (a) Impedance-based characterization of RBCs at 0.5 MHz (first row), 11 MHz (second row), and 20 MHz (third row). For each peptide concentration (column-by-column), the density plot of the phase against the electrical diameter is shown (last column refers to the osmotic sample). Two RBC populations are found at 11 and 20 MHz, denoted by H (high phase) and L (low phase). (b) Density plot of the phase at 20 MHz against the electrical diameter at 0.5 MHz (sample at 0.20 μM). RBCs of subpopulation H are clearly detectable in the high-speed video (three consecutive frames, fr.; scale bar is 20 μm). RBCs of subpopulation L, despite having comparable electrical size, resulted invisible with the present optical setup. (c) Comparison of the RBC toxicity assay based on microfluidic impedance cytometry (at 20 MHz), in blue, with the standard toxicity assay based on hemoglobin release, in red. Markers denote experimental datapoints (circles and triangles refer to RBCs from two different donors), and continuous lines denote fits of the results.

Figure 4

Results of impedance-based analysis of mixed samples (i.e., containing both B. megaterium cells and RBCs): (a–d) untreated sample (0 μM) and (e–h) treated sample (0.35 μM). (a, e) Density plot of the electrical diameter at 0.5 MHz against the electrical diameter at 20 MHz, with highlight of relevant subpopulations. The gating line is also shown (electrical diameter at 0.5 MHz equal to 3.8 μm). (b, f) [resp. (c, g)] Density plot of the phase at 20 MHz against the electrical diameter at 20 MHz for the bacterial cells [resp. for the RBCs]. (d, h) Empirical probability density function of the phase at 20 MHz, for each subpopulation.