Monitoring of active but non-culturable bacterial cells by flow cytometry

Sachidanandham, Ramaiah; Gin, Karina Yew‐Hoong; Poh, Chit Laa

doi:10.1002/bit.20304

Cited by 43 publications

(48 citation statements)

References 60 publications

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“…Under various stress conditions, many bacteria are able to enter a state where they are no longer culturable on standard media but still main- tain metabolic activity (3,5,7,18,24). As previously shown for other bacteria (23), the present results demonstrate for the first time that FCA is a valuable tool for characterizing VBNC cells of the Legionella genus. The 12 Legionella strains used in this study exhibited different heat inactivation curves as evaluated by FCA (Fig.…”

supporting

confidence: 85%

Use of Flow Cytometry To Monitor Legionella Viability

Allegra

Berger

Berthelot

et al. 2008

Appl Environ Microbiol

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Legionella viability was monitored during heat shock treatment at 70°C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.Legionellae are widespread in natural and manmade aquatic habitats. Sources of contamination are aerosols from showerheads, air-cooling towers, and other systems distributing water. To prevent outbreaks, surveillance of Legionella environmental contamination by use of culture methods has been set up for hot sanitary water systems in collective settings such as hospitals, hotels, and thermal spas (4). However, the findings of environmental surveillance do not always correlate with the occurrence of Legionnaires' disease, since the concentration of Legionella bacteria in environmental samples is largely underestimated when culture on GVPC medium (8,11,12), the reference method required by current norms (1, 16), is used. The existence of viable but nonculturable (VBNC) bacteria (13,21,24), which reduce the sensitivity of culture-based assays, has been demonstrated. Analysis of membrane integrity in order to distinguish between viable and dead cells in various bacterial species has been proposed (2, 14, 17, 19); some of these assays are based on double staining combining Syto 9 and propidium iodide (PI) (6, 9, 26). To date, only three studies dealing with the detection of Legionella cells by flow cytometry have been reported (15,27,29), and none of them analyzed the presence of VBNC cells, which was undertaken in this study.The flow cytometric assay (FCA) was performed on a BD FACSCanto II flow cytometer (Becton Dickinson Biosciences, Le Pont-de-Claix, France). A threshold was applied on the FL1 channel to eliminate background noise, and analyses were performed at a low flow rate setting. The concentrations of the two dyes were adjusted for the optimal discrimination of green-and red-fluorescing bacteria. One microliter of 2.3 mM Syto 9 and 5 l of 1-mg/ml PI (Invitrogen SARL, Cergy Pontoise, France) were used for cell staining. A cytogram was generated for each different Legionella strain; unstained cells and double staining of sterilized water were used to define the background noise (data not shown).During a heat shock treatment from 0 to 60 min at 70°C, the concentration of dead cells increased proportionally with the duration of heat exposure. As shown for Legionella pneumophila serogroup 1 (sg 1) in Fig. 1, the bacteria could be segregated within three regions: dead cells stained with PI were located in the P1 region (Fig. 1, dot plot at 60 min); bacteria with intact membranes that were stained with Syto 9 were used to delineate the P2 region (Fig. 1, dot plot at 0 min); and a third population, located in the P3 region and appearing as early as 1 min after the beginning of the heat shock, represented an intermediate physiological state that could correspond to cells that were st...

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supporting

confidence: 85%

Use of Flow Cytometry To Monitor Legionella Viability

Allegra

Berger

Berthelot

et al. 2008

Appl Environ Microbiol

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“…Several studies have successfully used LIVE/DEAD BacLight kit for the measurement of bacterial viability in environmental isolates (e.g., Haznedaroglua et al 2008;Portaels et al 2008). These observations, together with our earlier work conducted on osmotically stressed cultures of E. coli and coliform environmental isolate (H03N1) (Sachidanandham et al 2005), forecasted that no abnormalities in the staining of LIVE/DEAD BacLight kit in the viability measurements were observed (also see Fig. 5, live and dead cell count of Enterobacter sp.…”

Section: Flow Cytometric Analysis and Fluorescence-activated Cell Sorsupporting

confidence: 67%

A dormancy state in nonspore-forming bacteria

Sachidanandham

Gin

2009

Appl Microbiol Biotechnol

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While cultivation is a convenient way of proliferating and understanding bacteria, studies have shown the formation of nonculturable cells in nonspore-forming bacteria in response to environmental stress and thus in turn have generated immense interest. Whether these cells are in a state of dormancy or in a stage preceding cell death has been considered of paramount importance for the past couple of decades. In this study, osmotic-stress-induced dormant bacterial cells were separated by cell sorting and revived by osmotic down-shift in the absence of nutrients, source(s) that potentially could supply nutrients, and/or the external addition of resuscitation factor(s). Reversal of dormancy followed a definite pattern akin to population asynchrony of dormant cells, and the phenomenon was observed across three species, namely, Enterobacter sp. strain mcp11b, Klebsiella pneumonia strain mcp11d and Escherichia coli. In addition, our study precisely forecasted the presence of multiple subpopulations in dormant cells, which is explained by an emerging theory of survival mechanisms in stressful environments. These observations reveal that the state of dormancy induced by environmental stress in these nonspore-forming bacteria is "reversible" and also implies that it is an orderly and spontaneous adaptation to circumvent adverse conditions.

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“…Notably, it has been established that when starter cultures are subjected to various forms of sublethal stress, some cells may be injured and fail to grow on a medium adequate for the growth of unstressed cells, but they are capable of growth when they are given a suitable environment (6). Evidence of recovery of growth ability by damaged or injured, stressed cells by repairing or replacing damaged molecules under suitable conditions has been reported (3,29).…”

Section: Resultsmentioning

confidence: 99%

“…In this way, different cellular functions, such as reproductive growth, metabolic activity, and membrane integrity, can be detected, allowing a definition of the term viable-but-not-culturable cells (VBNC) (24) (other authors have called them active but nonculturable). Previous studies have reported that there are two major adaptations that cells undergo during the formation of VBNC states: cell wall toughening and DNA condensation (29). The detection of metabolic activity provides presumptive evidence of reproductive growth by the demonstration of enzyme activity, such as esterases, along with membrane integrity.…”

mentioning

confidence: 99%

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

Herrero

Quirós

Garcı́a

et al. 2006

Appl Environ Microbiol

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Analysis of microbiological samples from fermentation processes in the beverage industry (beer, wine, and cider) by traditional indirect, culture-based standard methods is timeconsuming, and the methods do not produce direct information about the physiological state of the microorganisms. Moreover, the plate-counting method detects only cells able to form colonies under the conditions of the medium that is used, ignoring the presence of cells that do not form colonies but are nevertheless metabolically active (6). As is known, standard laboratory culture media rarely resemble natural environmental conditions (32). The application of flow cytometry (FC) with fluorescent dyes is faster and more direct and has made it possible to distinguish stages beyond the classical definition of viability generally demonstrated by culturing (24). FC can be used to quantitatively measure the optical characteristics of cells as they pass, in single file, into a focused beam of light (for a review, see reference 33). As particles pass through the beam, three parameters are measured: forward light scatter, side angle light scatter, and fluorescence at selected wavelengths. Light scatter is related to cell mass, structure, surface properties, and the optical density of the internal medium. A variety of fluorescent dyes may be used to detect structural or functional cellular properties. In this way, different cellular functions, such as reproductive growth, metabolic activity, and membrane integrity, can be detected, allowing a definition of the term viable-but-not-culturable cells (VBNC) (24) (other authors have called them active but nonculturable). Previous studies have reported that there are two major adaptations that cells undergo during the formation of VBNC states: cell wall toughening and DNA condensation (29). The detection of metabolic activity provides presumptive evidence of reproductive growth by the demonstration of enzyme activity, such as esterases, along with membrane integrity. In the absence of metabolic activity, it is still possible to determine membrane integrity by either dye retention or dye exclusion, the latter using propidium iodide (PI). Cells without an intact membrane cannot maintain or generate the electrochemical gradient that generates the membrane potential and can be considered dead cells (24). The use of FC techniques was reported for the "at-line" study of Escherichia coli fermentations (14), which detected a considerable drop in cell viability (about 20%) during the latter stages of small-scale (5-liter), well-mixed fedbatch fermentations.Flow-cytometric applications using different dyes have been compared to standard methods to assess yeast cultures in baking, wine making, cider making, and brewing (1,5,7,8,10,11,17,19). The technique has also been used to study the membrane integrity of ethanol-stressed Oenococcus oeni cells (9). Most studies of FC applications have focused mainly on microorganims as pure cultures, and only a small number have analyzed mixed populations over time (30).The purp...

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Monitoring of active but non-culturable bacterial cells by flow cytometry

Cited by 43 publications

References 60 publications

Use of Flow Cytometry To Monitor Legionella Viability

Use of Flow Cytometry To Monitor Legionella Viability

A dormancy state in nonspore-forming bacteria

Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

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