Flow-Cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS)

Swiss Federal Institute of Aquatic Science and Technology (EAWAG), PO 611, CH-8600 Dübendorf, Switzerland.
Microbiology (Impact Factor: 2.56). 06/2006; 152(Pt 6):1719-29. DOI: 10.1099/mic.0.28617-0
Source: PubMed


The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight.

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Available from: Michael Berney, Oct 08, 2015
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    • "Adenosine triphosphate (ATP) synthesis and efflux pump activity in the cell cease shortly after the start of exposure. These are followed by a gradual loss of membrane potential and a reduction in glucose uptake ending in the loss of cultivability (Berney et al., 2006b). "
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    ABSTRACT: The small treated volume (typically ∼2 L) associated with polyethylene terephthalate (PET) bottles that are most frequently used in solar water disinfection (SODIS), is a major obstacle to uptake of this water treatment technology in resource-poor environments. In order to address this problem we have conducted a series of experiments in Spain, Bahrain and India, to assess the efficacy of large volume (19 L) transparent plastic (polycarbonate) water cooler/dispenser containers (WDCs) as SODIS reactors to inactivate Escherichia coli and Enterococcus faecalis, under strong natural sunlight. Reduction values of 6 log10 units (LRV = 6.0) have been observed using WDCs in each location. Additional comparisons between 2-L PET bottles and 19-L indicate that WDCs provide bacterial inactivation similar in both systems. SODIS disinfection experiments in turbid water (100 NTU) in both reactors showed very good inactivation efficiency. LRVs of 6 were obtained for E. coli in both WDC and 2-L PET bottles, and in the case of E. faecalis LRV = 5 and 6 were observed in Spain and Bahrain, respectively. These studies demonstrate that under conditions of strong sunlight and mild temperature, 19 L water dispenser containers can be used to provide adequate volumes of SODIS treated water for households or larger community applications such as schools or clinics in the developing world.
    Solar Energy 06/2015; 116:1-11. DOI:10.1016/j.solener.2015.03.035 · 3.47 Impact Factor
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    • "One ml of each sample was stained with 10 ll SYBR-I (Merck, Darmstadt, Germany), at 1:30,000 final concentration in DSMO and 10 ll of Propidium Iodide (PI) at 1 mg/ml (Invitrogen, Carlsbad, CA, USA) to quantify intact and permeabilized cells (Ziglio et al. 2002). The depolarized cells were measured adding 10 ll of 1 mM DiBAC 4 (3) (Invitrogen, Carlsbad, CA, USA), and only E. coli depolarized cells emit green fluorescence, and the percentage of depolarized cells was calculated using the total count measurements (Berney et al. 2006). Excitation and emission wavelengths were at k ex = 495 nm, k em = 525 nm for SYBR-I; k ex = 536 nm, k em = 617 nm for PI and k ex = 490 nm, k em = 516 nm for DiBAC 4 (3). "
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    ABSTRACT: Supercritical carbon dioxide (SC-CO2) treatment is one of the most promising alternative techniques for pasteurization of both liquid and solid food products. The inhibitory effect of SC-CO2 on bacterial growth has been investigated in different species, but the precise mechanism of action remains unknown. Membrane permeabilization has been proposed to be the first event in SC-CO2-mediated inactivation. Flow cytometry, high performance liquid chromatography-electrospray ionization-mass spectrometry and NMR analyses were performed to investigate the effect of SC-CO2 treatment on membrane lipid profile and membrane permeability in Escherichia coli K12. After 15 min of SC-CO2 treatment at 120 bar and 35 °C, the majority of bacterial cells dissipated their membrane potential (95 %) and lost membrane integrity, as 81 % become partially permeabilized and 18 % fully permeabilized. Membrane permeabilization was associated with a 20 % decrease in bacterial biovolume and to a strong (>50 %) reduction in phosphatidylglycerol (PG) membrane lipids, without altering the fatty acid composition and the degree of unsaturation of acyl chains. PGs are thought to play an important role in membrane stability, by reducing motion of phosphatidylethanolamine (PE) along the membrane bilayer, therefore promoting the formation of inter-lipid hydrogen bonds. In addition, the decrease in intracellular pH induced by SC-CO2 likely alters the chemical properties of phospholipids and the PE/PG ratio. Biophysical effects of SC-CO2 thus cause a strong perturbation of membrane architecture in E. coli, and such alterations are likely associated with its strong inactivation effect.
    Journal of Membrane Biology 03/2014; 247(6). DOI:10.1007/s00232-014-9653-0 · 2.46 Impact Factor
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    • "R. Bigoni et al. / Journal of Cleaner Production xxx (2013) 1e10 6 Please cite this article in press as: Bigoni, R., et al., Solar water disinfection by a Parabolic Trough Concentrator (PTC): flow-cytometric analysis of bacterial inactivation, Journal of Cleaner Production (2013), membrane after pasteurization. PI is a large molecule that only enters cells with a permeabilised cytoplasmic membrane (Berney et al., 2008) and such cells are commonly considered as dead, while an intact membrane provides indication of cell integrity (Hammes et al., 2011); however, there is a long-standing debate on this issue and a cell with an intact membrane is not necessarily alive (Berney et al., 2006). Intact cells showed strong green fluorescence and only weak red fluorescence, whereas permeabilised (damaged) cells typically exhibited enhanced red fluorescence and reduced green fluorescence intensity. "
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    ABSTRACT: An innovative solar water pasteurizer was developed to directly heat the water by solar radiation using a “Parabolic Trough Concentrator” (PTC). The efficiency of drinking water pasteurization by using the PTC was studied with a combination of analytical methods including flow-cytometric determination of total cell concentration and enumeration of cells with damaged membranes before and after treatment. Fluorescent staining of all microbial cells with two nucleic acid stains, SYBR®Green I and Propidium Iodide (live/dead staining), was used. The effectiveness of the pasteurizer to inactivate spiked Escherichia coli cells in contaminated water was also investigated. Flow-cytometric analysis revealed that cellular membranes of all microbial cells were strongly damaged after exposure in all the tested water samples. The pasteurizer reached a maximum daily water production of 66 L on a sunny day and was stable in its E. coli reduction rates. The results of this study suggest that the pasteurization temperature of 87 °C is able to inactivate bacterial cells in drinking water. Despite this, water pasteurized in this way is not sterile and has to be consumed quickly, since treated water samples incubated at 30 °C for 72 h exhibited a potential microbial regrowth.
    Journal of Cleaner Production 03/2014; 67:62–71. DOI:10.1016/j.jclepro.2013.12.014 · 3.84 Impact Factor
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