Use of Flow Cytometry for Quantitative Analysis of Metabolism of Viable but Non-culturable (VBNC) <i>Salmonella</i>

Abstract

A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 1-10 mM H 2 O 2 in Luria-Bertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H 2 O 2 -treated SE cells were in the VBNC state and that the extent of H 2 O 2 -induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment.

Benzalkonium Chloride Induces a VBNC State in Listeria monocytogenes

“…The quadrants for the different phenotypic attributes were set by measuring the same cells without BC stress from the exponential growth phase (proliferating living cells with intact cell membrane integrity and metabolic 2-NBDG uptake activity) before and after heat treatment (dead cells with damaged cell membrane and no 2-NBDG uptake), thereby enabling a differentiation between metabolically active and intact cells from other cells and their quantification. Metabolic activity based on 2-NBDG uptake is commonly presented as a proportion in relation to external standards of dead and living cells [16,17]. In our study, controls were directly taken from the exponential growth phase of bacterial cells and therefore the external standard of living cells represented an ideal metabolic state.…”

“…The proportion of cells with intact membranes was determined by calculating the ratio of SYTO9-positive and PI-negative cells [16,17]. Between 80% and 100% of parent and adapted cells were intact after cultivation in BHI without and supplemented with up to 6 µg mL −1 BC (Figure 2, supplementary Figure S1A).…”

“…Recent studies expanded the methodological approaches to determine the metabolic activity and cell membrane integrity of VBNC cells by ATP determination [13], quantitative PCR combined with propidium monoazide treatment [14], the capability to ferment sugars after reuptake [13], and flow cytometric analyses coupled with cell staining [15]. Flow cytometric analysis of SYTO9/PI was also linked to the uptake activity of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to assess both cell membrane integrity and metabolic activity simultaneously [16], thereby quantifying VBNC bacteria at the single cell level [17]. Various stress conditions have been identified to induce the VBNC state in L. monocytogenes, including chlorine-based sanitizing treatments [15] and non-ionic surfactants combined with inorganic salts [13].…”

Structural and proteomic changes in viable but non-culturableVibrio cholerae

“…VBNC cells have previously been reported to exhibit a markedly restrained metabolism [5][6][7]11]. To analyze potential shifts in metabolic pathways, we retrieved the predicted function of metabolic proteins detected in the VBNC proteome data, mapped their respective pathways and expected underlying regulons using KEGG, VchoCyc and RegPrecise 3.0 [25,27,29,30] (Table S5 and (Table S7).…”

“…The facultative human pathogen Vibrio cholerae, like many other bacteria, enters a state of restrained metabolic activity when confronted with cues like low temperatures and or low nutrient availability over extended periods of time [4][5][6][7][8][9][10][11]. Once the cells have entered this state, V. cholerae does not readily start to grow and reproduce when they are returned to more favorable and nutrient-rich media, and their status has, therefore, been termed "viable but non-culturable" (VBNC) [4,12,13].…”