State transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry

Abstract

Background: Vibrio parahaemolyticus, in response to environmental conditions, may be present in a viable but nonculturable state (VBNC), which can still be responsible for cases of infectious diseases in humans.Methods: The characterization of the cellular states of V. parahaemolyticus during entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry (FCM) in combination with SYBR Green I (SG) and propidium iodide allowed us to distinguish between viable, dead, and damagedcells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity.Results: Two groups of bacteria, one with High Nucleic Acid (HNA) and one having Low Nucleic Acid (LNA) content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95% at day 0 to 40% at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure.Conclusions: The methodology used has provided useful data in relation to the state transitions of V. parahaemolyticus regarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state. q

Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces

“…The detection of residual viable cells after phage treatment could also suggest that phages are seemingly immobilized after addition to solid surfaces and therefore could not come into contact with the surviving bacteria through limited diffusion (Guenther et al, 2009). In this study, no intermediate states were observed because microscopic assessment of LIVE/DEAD-stained bacterial cells only allows to distinguish between live (green) from dead (red) cells (Falcioni et al, 2008), due to the human eye cannot dissect the emitted color into separate wavelengths (D'hondt, Höfte, Van Bockstaele, & Leus, 2011). However, flow cytometry (FCM) shows that intermediate states of viability occur during the process of changes in membrane permeability of viable cells, which are characterized by different intracellular concentrations of SYTO 9 and PI.…”

Changes in the Vibrio harveyi Cell Envelope Subproteome During Permanence in Cold Seawater

“…In the case of Vibrio species, some authors proposed that cell reduction together with acquisition of a coccoid-like appearance provide indications of cell entry into the VBNC state [46][47][48]. Consistent with this idea, Sun et al [40] reported that three V. harveyi strains (two clinical strains, SF1 and CW2, and a type strain, VIB295 T ) underwent the above morphological changes once they entered the VBNC state.…”

“…Namely, the presence of OmpW, maltoporin, or agglutination proteins or different ATP synthase subunits did not vary along starvation at low temperature. The continuous presence of ATP synthase subunits, cytochromes, or Na(+)-translocating NADH-quinone reductase is likely indicative of an active electron transport chain, which is consistent with some characteristic of VBNC cells, such as the ability to reduce intracellularly the 5-cyano-2,3-ditoryl tetrazolium chloride (CTC) known to be mediated by the electron transport system (ETS) components or dehydrogenases [46,47,50]. Despite some minor changes in electron transfer systems that were detected in the final stage of the studied process (i.e., when VBNC population predominate [see Fig.…”

Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes

“…Surface antigens/proteins [21,22] and inclusion bodies [23] can be stained through immunofluorescent techniques. This involves the binding of an antibody directed against the target compound or an epitope on a protein that could capture all forms of a specific protein or specific protein modifications like phosphorylation and acetylation.…”