Recent Advances on Multi-Parameter Flow Cytometry to Characterize Antimicrobial Treatments

Abstract

The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non-culturable (VBNC). Recent advances on flow cytometry (FCM) and especially on multi-parameter flow cytometry (MP-FCM) provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling) in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments.

Current Perspectives on Viable but Non-culturable State in Foodborne Pathogens

“…Fluorescence techniques combined with direct optical detection methods for the rapid assessment of bacterial viability have been increasingly followed for several years. Among these techniques, flow cytometry (FCM) has been shown to be a powerful tool for rapidly analyzing populations on a cell-by-cell basis and can be applied in many areas of food safety or medical microbiology (Léonard et al, 2016). The main principle is that particles in suspension are pumped into a narrow flow stream intersected by one or more laser beams.…”

“…Recently, Mathur et al (2016) reviewed the advancement of FCM and the introduction of novel fluorochromes allow to study the viability of cells, the membrane structure and its integrity, and the membrane potential at a single-cell level. The ability to use FCM to visualize, enumerate and analyze a population of cells into subpopulations of varying physiological status is a valuable aid to understanding this intricate area for the microbiologists (Léonard et al, 2016). Indeed, the use of personalized probes and dyes for the detection of changes in specific targets and intracellular activities permits the targeted use of FCM to ascertain the structural and functional characteristics of a population of VBNC cells.…”

Plant antimicrobial polyphenols as potential natural food preservatives

“…Recent advances in multiparameter flow cytometry offer the opportunity to obtain high‐speed information on the mechanism of action of antimicrobials at real time at the single‐cell level . Flow cytometric analysis revealed that resveratrol‐ trans ‐dihydrodimer disturbs the membrane potential and hinders DNA synthesis of microorganisms .…”

Membrane Active Peptides and Their Biophysical Characterization

“…Its principle is based on light scattering and fluorescence emission of the particle, with fluorescent features derived from antibodies or dyes, upon illumination with a series of laser beams. The detected signals are then processed for analysis [250][251][252][253]. FC gives a statistically relevant and quantitative signal of fluorescence very quickly and allows sensitive detection.…”