Rapid assessment of susceptibility of bacteria and erythrocytes to antimicrobial peptides by single-cell impedance cytometry
Author:
Troiano Cassandra, De Ninno AdeleORCID, Casciaro BrunoORCID, Riccitelli Francesco, Park Yoonkyung, Massoud RenatoORCID, Mangoni Maria LuisaORCID, Bisegna PaoloORCID, Stella LorenzoORCID, Caselli FedericaORCID
Abstract
AbstractAntimicrobial 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. We show that the electrical signatures ofBacillus 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 ofB. megateriumcells and RBCs, which paves the way to study AMP selectivity for bacterial versus eukaryotic cells in presence of both cell types.
Publisher
Cold Spring Harbor Laboratory
Reference58 articles.
1. Bamford, R.A. , Smith, A. , Metz, J. , Glover, G. , Titball, R.W. , Pagliara, S. , 2017. Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy. BMC Biol. 15. https://doi.org/10.1186/s12915-017-0465-4 2. Bertelsen, C.V. , Franco, J.C. , Skands, G.E. , Dimaki, M. , Svendsen, W.E. , 2020. Investigating the use of impedance flow cytometry for classifying the viability state of E. Coli. Sensors (Switzerland) 20. https://doi.org/10.3390/s20216339 3. Bobone, S. , Stella, L. , 2019. Selectivity of antimicrobial peptides: A complex interplay of multiple equilibria, in: Advances in Experimental Medicine and Biology. https://doi.org/10.1007/978-981-13-3588-4_11 4. Bocchinfuso, G. , Palleschi, A. , Orioni, B. , Grande, G. , Formaggio, F. , Toniolo, C. , Park, Y. , Hahm, K.S. , Stella, L. , 2009. Different mechanisms of action of antimicrobial peptides: Insights from fluorescence spectroscopy experiments and molecular dynamics simulations, in: Journal of Peptide Science. https://doi.org/10.1002/psc.1144 5. Cama, J. , Pagliara, S. , 2021. Microfluidic single-cell phenotyping of the activity of peptide-based antimicrobials, in: Methods in Molecular Biology. https://doi.org/10.1007/978-1-0716-0928-6_16
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