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.

    • "Similar results were achieved with lactic acid bacteria (Moreno et al., 2006) using fluorescent flow cytometric measurements (Boulos, Prevost, Barbeau, Coallier, J., &amp; Desjardins, 1999). According toJoux and Lebaron (2000), bacterial cells cannot be necessarily considered active if they show intact membranes, but it would seem to be more accurate to assume that membranecompromised cells are dead (Berney, Weilenmann, &amp; Egli, 2006). The EOs antimicrobial activity is due to their hydrophobic nature affecting the lipid bilayer of microbial cells, as confirmed by the evidences of this assays, since the kit used enables differentiation only between bacteria with intact and damaged cytoplasmic membranes, differentiating between active and dead cells (Sachidanandham, Yew-Hoong Gin, & Laa Poh, 2005). "
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    ABSTRACT: The antimicrobial activity of eight essential oils (EOs) extracted from the fruit peel of Citrus genotypes (orange, mandarin and lemon) was evaluated against 76 strains of Listeria monocytogenes, previously isolated from different food matrices. EOs showing the most (EO L2 and EO L8) and least (EO O3 and EO M7) effective inhibition activities were chemically characterized by gas chromatography coupled with mass spectrometry (GC/MS) to compare their composition. EO L2 and EO L8 were chosen to determine the MIC and to evaluate the cell viability of the most sensitive strains (L. monocytogenes LM35 and LM69) after 1, 2, 4 and 6 h of exposure. The effectiveness of chitosan (CH) and methylcellulose (MC) edible films, alone and in combination with EO L2 and EO L8, was determined against LM35 and LM69 at 37 °C for 0, 8 and 24 h and at 8 °C for 0, 1, 3 and 7 days. In addition, the analysis of the microstructure of the films were performed by scanning electron microscope (SEM) to evidence the interactions between the polymers and EOs. Thirty-five and twenty-nine strains were clearly inhibited by EO L2 and EO L8, respectively, while the other Citrus EOs showed poor (EO M1, O4, O5, O6) or minimal (EO O3 and M7) antimicrobial activity. A total of 36 chemical volatile substances was identified by GC/MS to detect the compounds that might play an important role in the characterization of the EOs. The chemical characterization points to oxygenated monoterpenes as relevant compounds in inhibiting Listeria strains, since they have been detected in lemon EOs in concentrations four/five folds higher than orange EOs. Generally, CH- and MC-based films containing EO L2 and EO L8 showed antilisterial activities, even though, the best performances were observed in case of CH-films at 8 °C, with a major reduction up to 3 log (CFU/cm2) in case of EO L2 incorporation. The microstructures observed by SEM suggested a better incorporation of the EOs in CH matrix, where a higher amount of oil droplets was distinguished. Therefore, lemon EOs incorporated into chitosan films could be an efficient tool to control Listeria monocytogenes, especially in refrigerated applied conditions.
    No preview · Article · Jan 2016 · Food Control
    • "FCM combined with advanced fluorescent dye technology can express detailed metabolic states of bacterium in aquatic environment and during water treatment process [19]. Until now, a range of fluorescent dyes can be obtained which target the cell components such as membrane integrity and potential, enzyme activity, and DNA [20] [21]. With the FCM analysis, a better insight of the metabolic dynamic changes can be obtained during the cell injuries or disinfection process [22]. "
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    ABSTRACT: Up to now, the effects of ultraviolet (UV) and chlorine disinfection on microbes have been evaluated by heterotrophic plate counting (HPC). Recently, with the application of flow cytometry (FCM) in aquatic environment, it is used to analyze the membrane integrity, DNA damage and enzymatic activity of bacteria. The present work investigated the disinfection efficacy of UV-C and chlorine on E. coli (Gram negative) and Bacillus subtilis (Gram positive) and compared the detection methods for disinfection efficacy with HPC, Adenosine triphosphate (ATP), and FCM. The results show that there are considerable discrepancies among HPC, ATP, and FCM detection for E. coli and B. subtilis when disinfected by UV and chlorine. Specifically, the bacterium was sharply inactivated when evaluated by HPC, whereas the more gentle inactivation trend was observed when detected by ATP and FCM during both UV and chlorine disinfections for E. coli. In addition, for B. subtilis disinfection, the results between ATP and HPC detection were of little difference, especially with chlorine disinfection; thus, ATP measurement could be a replacement of HPC as it was much more fast and convenient. The results of FCM demonstrated that most of the bacteria undergo viable but non-culturable (VBNC) states which could not be detected by HPC but had the infection ability. Considering the risk of VBNC cells and the limitation of HPC, the following was suggested: when applied UV-C disinfection, HPC should combine ATP or FCM to evaluate the microbial viability, whereas FCM was a powerful tool to distinguish viable, but non-culturable cells (VBNC) applied for chlorine disinfection.
    No preview · Article · Sep 2015 · Desalination and water treatment
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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.
    Full-text · Article · Jun 2015 · Solar Energy
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