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Swiss Federal Institute of Aquatic Science and Technology (EAWAG), PO 611, CH-8600 Dübendorf, Switzerland
Correspondence
Thomas Egli
egli{at}eawag.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Every day, diarrhoeal diseases such as cholera (Fewtrell et al., 2005) claim the lives of 5000 young children throughout the world and most of the cases could be easily prevented (Hinrichsen et al., 1997). The World Health Organization (WHO) and the United Nations Children's Fund (UNICEF) have recently claimed that improvement of drinking water quality and basic sanitation can cut this toll, and simple, low-cost, household water treatment has the potential to save further lives because it cuts the primary transmission route for diarrhoeal diseases (World Health Organization/United Nations Children's Fund, 2005).
Solar disinfection (SODIS) is such a water treatment method. Through exposure of drinking water in poly(ethylene terephthalate) (PET) bottles to sunlight (
6 h), enteric bacteria in the water are inactivated (Acra et al., 1984; Wegelin et al., 1994). Both UVA light and mild heat have been shown to have inimical potential and, if the water temperature rises above 50 °C, a synergistic effect is observed (Wegelin et al., 1994). Field trials in different geographic regions, carried out by the Swiss Federal Institute of Aquatic Science (EAWAG) Department of Water and Sanitation in Developing Countries (SANDEC), have shown that temperatures >50 °C are rarely reached (R. Meierhofer, personal communication). Acra et al. (1984) have proposed that solar UVA irradiation accounts for >70 % of the negative effects of sunlight. Today, SODIS is one of the recommended methods for household drinking water disinfection (World Health Organization/United Nations Children's Fund, 2005). Nevertheless, the mechanism(s) of disinfection are not yet known precisely. The two primary sources for bacterial inactivation in this method are believed to be mild heat and UVA light (Wegelin et al., 1994). The first report on the inimical effect of sunlight on bacteria was published by Downes (1886) and, 100 years later, Acra et al. (1984) proposed the use of sunlight to disinfect oral rehydration solutions. Subsequently, it was demonstrated to be effective against bacteria and higher organisms (Joyce et al., 1996; Lonnen et al., 2005; Wegelin et al., 1994), and its applicability has been shown in a recent health impact study (Hobbins, 2004). The effectiveness of SODIS has been shown using classical cultivation of micro-organisms on solid agar substrates, which represents the reference procedure for estimating the number of viable bacteria. This method, however, only enumerates culturable bacteria that are able to initiate cell division and replication on agar plates. By using conventional culturability assays, there is a risk of overestimation of effectiveness of disinfection processes. For example, bacteria that are injured can fail to proliferate on plates and may appear dead (Aldsworth et al., 1999). It has been shown that injured bacteria do not grow due to the stress of high nutrient concentrations, which causes a free radical burst (Dodd et al., 1997). This behaviour has been referred to as a suicide response (Aldsworth et al., 1999). Postgate had already observed this behaviour much earlier and called it substrate-accelerated death (Postgate & Hunter, 1964), but did not link it to oxidative stress. Furthermore, in view of the discussion about the viable but non-culturable (VBNC) state, a careful analysis of bacterial viability after SODIS treatment is of great importance (Barer & Harwood, 1999; Colwell, 2000; Nystrom, 2001). It is still an ongoing debate if such a state exists at all and how this can be proven.
Multi-parameter flow cytometry has enjoyed increasing popularity in microbiology, particularly in biotechnological processing, food preservation and chemical disinfection (Hewitt et al., 1999; Nebe-von-Caron et al., 2000; Porter et al., 1997). Hewitt & Nebe-Von-Caron (2004) have shown that multi-parameter flow cytometry allows a functional classification of the physiological state of single-celled micro-organisms beyond that of culturability alone.
In the work presented here, the SODIS method was assessed with flow cytometry and viability stains. A set of essential bacterial functions of E. coli, namely, membrane integrity, membrane potential, efflux pump activity, glucose uptake activity and culturability were measured.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Growth media and cultivation conditions.
Luria–Bertani (LB) broth (10 g tryptone l–1, 5 g yeast extract l–1, 10 g NaCl l–1) that was filter-sterilized with membrane filters (Millex GP, 0.22 µm pore size; Millipore), and diluted to 33 % (v/v) of its original strength (unless indicated otherwise) with ultrapure water (deionized and activated carbon-treated), was used for batch cultivation (Miller, 1972). Precultures were prepared for each individual batch experiment from the same cryo-vial stored at –80 °C by streaking the stock solution onto LB agar plates. After 15–18 h of incubation at 37 °C, one colony was picked, loop-inoculated into a 125 ml Erlenmeyer flask containing 20 ml diluted LB broth, and incubated at 37 °C on a rotary shaker at 200 r.p.m. At an OD546 between 0.1 and 0.2 (measured spectrophotometrically at 546 nm in glass cuvettes with a 1 cm light path using a Jasco V550 UV/VIS spectrophotometer), an aliquot of the culture was transferred into 500 ml Erlenmeyer flasks containing 50 ml prewarmed LB broth to obtain an OD546 of 0.002. With this procedure, no lag phase was observed. These flasks were then shaken at 200 r.p.m. in a temperature-controlled water bath (SBK 25D; Salvis AG) at 37 °C for 18 h until stationary phase (specific growth rate, µ=0 h–1) was reached. The specific growth rate µ was calculated from five consecutive OD546 measurements. In drinking water, cells grow very slowly or not at all; therefore, we used stationary-phase cells, which were shown to be more resistant to SODIS than cells in the growth phase (Berney et al., 2006; Reed, 1997).
Sample preparation and plating.
Cells were harvested by centrifugation from batch culture (at 13 000 g, Biofuge Fresco; Kendro), washed three times with filter-sterilized (Nuclepore Track-Etch Membrane, 0.22 µm pore size; Sterico) commercially available bottled water (Evian) and diluted to an OD546 of 0.01 (corresponding to 1–5x107 cells ml–1). During exposure, aliquots were withdrawn at different time points and diluted in decimal steps (10–1–10–5) with sterile-filtered (0.22 µm), bottled mineral water (Evian). Volumes of 1 ml of appropriate dilutions were withdrawn and mixed with 7 ml liquid tryptic soy agar (TSA) (Biolife) at 40 °C (pour-plate method). After 20 min, the solidified agar was covered with another 4 ml liquid TSA (40 °C). Plates were incubated for 48 h at 37 °C until further analysis. For cultivation under reduced oxidative stress conditions, a 1 ml aliquot of the appropriate test solution was mixed with 7 ml TSA supplemented with 0.05 % sodium pyruvate, covered after 5 min with the same agar, and then placed in an anaerobic jar (Oxoid; Khaengraeng & Reed, 2005). Just before closure of the jars, an AnaeroGen sachet (Oxoid) was placed in the container to generate an anaerobic gas phase. The jars were incubated for up to 72 h at 37 °C. Plate counts were determined with an automatic plate reader (Acolyte; Synbiosys).
Sunlight exposure.
Samples of 10 ml bacterial suspension (see above) were exposed to solar light in 30 ml quartz tubes, which were placed into a temperature-controlled, acrylic glass container with a quartz front glass, holding 25 tubes in total. A water bath was used to control the temperature of the sample tubes in the container. The container was adjusted regularly so that sunlight met the tubes at an angle of 90±2°. At each time point, one tube was withdrawn and its content immediately processed as described above. Irradiation intensity data were obtained from a weather station, which was located 300 m away from the exposure site (BUWAL/NABEL, EMPA Dübendorf, Switzerland). The fluence rates for sunlight irradiation given in this work refer to the wavelength range 350–450 nm, which reflects that of the UVA lamp used (see below). Conversion factors and calculations were used as described by Wegelin et al. (1994). The light spectra were recorded with a calibrated LI-1800 portable spectroradiometer (LI-COR), 8 nm bandwidth, fitted with a model 1800-10 detector head.
UVA exposure.
Samples of 10 ml bacterial suspension (see above) were exposed to UVA light in 30 ml quartz tubes placed in a carousel reactor (holding 10 tubes) (adapted from Wegelin et al., 1994) equipped with a medium-pressure mercury lamp (Hanau TQ718 Z4), which was operated at 500 W (for wavelength spectrum, see Berney et al., 2006). The lamp was placed in a cooling jacket (Duran 50 borosilicate glass) in the centre of the carousel reactor. The light emitted from the lamp passed through the glass jacket and through 35 mm of filter solution before reaching the cells in the quartz tubes. The temperature of the filter solution was maintained at 37 °C, and the solution consisted of 12.75 g sodium nitrate l–1 with a cut-off at 320 nm and a half maximum at 340 nm. The transmission property of the filter solution was measured before each experiment. Chemical actinometry with p-nitroanisole/pyridine was used to determine the fluence rate at the tube position (Wegelin et al., 1994). Bacterial solutions were mixed intermittently on a magnetic stirrer. At each time point, one tube was withdrawn and its contents immediately processed as described above. UVA and light spectra were recorded as outlined above.
Flow-cytometric measurements.
Flow-cytometric measurements were made using a Partec PAS III flow cytometer with 488 nm excitation from an argon ion laser at 20 mW. Five fluorescent dyes were used alone or in different combinations: Syto 9 (Molecular Probes), propidium iodide (PI; Molecular Probes), bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3); Molecular Probes], ethidium bromide (EB; Fluka Chemie) and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG; Invitrogen). Samples taken from irradiation experiments (sunlight and artificial UVA) were divided into five subsamples and immediately stained with two mixtures of fluorescent dyes (Syto 9/PI and Syto 9/EB) and three single fluorescent dyes [DiBAC4(3), Syto 9 and 2-NBDG]. Samples were incubated in the dark at 37 °C for 5 min (2-NBDG) or at 20 °C for 10 [DiBAC4(3)], 15 (Syto 9/EB), 20 (Syto 9/PI) and 25 min (Syto 9), respectively, before analysis. Prior to flow-cytometric analysis, cell samples were diluted with sterile-filtered bottled water (Evian) to 1 % (v/v) of the initial concentration (1x105 cells ml–1 final concentration). Stock solutions of the dyes were prepared as follows: PI and Syto 9 were used from the LIVE/DEAD BacLight kit (Invitrogen), EB was prepared at 25 mM in distilled and filtered water, DiBAC4(3) was prepared in dimethylsulfoxide at 10 mM, and 2-NBDG was dissolved in distilled and filtered water at 5 mM. All stock solutions were stored at –20 °C. The working concentrations of Syto 9, PI, EB, DiBAC4(3) and 2-NBDG were 5, 30, 30, 10 and 5 µM, respectively. 2-NBDG was added in combination with 2,4-dinitrophenol (final concentration 2 mM) (Natarajan & Srienc, 2000). In the flow cytometer, optical filters were set up such that PI and EB were measured above 590 nm and Syto 9, DiBAC4(3) and 2-NBDG at 520 nm. The trigger was set for the green fluorescence (520 nm) channel FL1.
The 2-NBDG uptake kinetics of E. coli K-12 MG1655 were measured prior to the irradiation experiment by incubating a bacterial sample with a mixture of 2-NBDG and 2,4-dinitrophenol at 37 °C for 40 min (Natarajan & Srienc, 1999). Every 5 min, a sample was withdrawn, diluted with sterile-filtered mineral water (Evian) to 1 % (v/v) of the initial concentration and analysed on the flow cytometer. The peaks of the bacterial population were gated (range-gate RN1) and the geometric mean of green fluorescence intensity was used for data analysis.
Fluorescence stains and their function.
The cellular properties and physiological functions indicated by the fluorescent stains used here are visualized in Fig. 1. Syto 9, a green fluorescent nucleic acid stain, has been shown to stain living and dead Gram-positive and Gram-negative bacteria (Haugland, 2002). Therefore, it can be used for total count measurements. EB binds to nucleic acid, and can cross the intact cytoplasmic membrane, but is actively pumped out of the cell via a non-specific, proton antiport transport system in active cells (Midgley, 1987). It has been shown that the ethidium efflux system is dependent on a transmembrane proton electrochemical gradient, but is ATP independent. PI is a red fluorescent dye that intercalates with dsDNA and only enters permeabilized cytoplasmic membranes. If EB or PI is combined with Syto 9, a quenching effect on the green fluorescence intensity is observed as soon as the red fluorescent dye enters the cells. It is important to mention that only the cation (ethidium or propidium) enters the cells; nevertheless, the stains are always referred to together with their anionic constituent (bromide or iodide). DiBAC4(3) is a lipophilic and anionic bis-oxonol. The uptake of this membrane potential-sensitive dye is restricted to depolarized cells or cells with disrupted cytoplasmic membranes. The fluorescent dye accumulates inside the cells by binding to intracellular proteins and membranes. For further information about the dyes described above, refer to Hewitt & Nebe-Von-Caron (2004). 2-NBDG is a fluorescent glucose analogue, which is taken up by the glucose-specific phosphoenolpyruvate-phosphotransferase system (PEP-PTS) (Natarajan & Srienc, 1999; Yoshioka et al., 1996). The system consists of five proteins and uses PEP as an energy source.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Culturability and total counts
Three independent experiments were conducted to measure all cellular functions during artificial UVA irradiation. Culturability on TSA was measured in all three experiments to ensure comparability of results. The inactivation based on this parameter was reproducible (Fig. 2). Exposure to sunlight was repeated twice (on two different days) while, in the experiment presented here, triplicate measurements (three independent cultures were grown, diluted and exposed to the same sunlight) of culturability were made (Fig. 3). To prevent oxidative bursts in the cells during aerobic incubation, samples were also incubated on pyruvate-supplemented agar in anaerobic jars, which resulted in higher c.f.u. In all experiments, total counts remained constant over the whole irradiation period (Fig. 4a). UVA light appeared to be slightly more effective in inactivating E. coli cells than sunlight when assessed with plating (Fig. 4b).
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5 % of the initial value (Fig. 4c
). The uptake of EB (non-pumping cells) closely followed the decline in ATP concentration per cell (Fig. 4d). EB-positive cells (non-pumping cells) showed a decrease in green fluorescence intensity (quenching effect) and an increase in red fluorescence intensity on the flow cytometer (Fig. 5a–c). The loss of culturability of bacterial cells appeared to be time-delayed when compared to the decrease in ATP.
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). In the flow cytometer, E. coli cells with polarized cytoplasmic membranes did not appear, because the signal trigger was set on the green fluorescence channel (FL1, 520 nm; Fig. 5d), therefore, only depolarized (green fluorescent) cells were counted (Fig. 5e, f). The percentage of depolarized cells was calculated using the total count measurements. In our experiment, depolarization of the bacterial cells occurred later than the cessation of efflux pump activity but before membrane integrity was lost (Fig. 4f). PI, a dye which only enters cells with permeabilized cytoplasmic membranes, was only detected after a dose of 2000–2500 kJ m–2 had been applied. The permeabilized cell population showed a typical increase in red fluorescence and decrease in green fluorescence intensity because of quenching (Fig. 5a–i). Interestingly, green fluorescence intensity increased by a factor of 4.3 before PI was able to enter the cells significantly. At the end of the sunlight experiment, after a fluence of 2088 kJ m–2, 50 % of the cell population had lost membrane integrity. From the UVA light experiment, we learned that, after 2500 kJ m–2 of UVA light, most of the E. coli cell population (>95 %) had lost membrane integrity. Artificial UVA light seemed to be slightly more effective in permeabilizing E. coli cells than sunlight.
Glucose uptake activity
The 2-NBDG uptake kinetics of healthy E. coli cells followed a typical pattern (Fig. 6a). The mean green fluorescence intensity of the cells increased linearly with time during the first 10 min of incubation with 2-NBDG. Therefore, irradiated cells were always incubated for 5 min to ensure that the mean fluorescence intensity correlated with the uptake rate. The mean 2-NBDG uptake rate of the cells remained constant over the first 2 h of sunlight exposure (800 kJ m–2), thereafter, it dropped along with the number of fluorescent cells, indicating that the PEP-PTS system was compromised (Fig. 6b). Bacterial cell size did not change significantly during exposure, as was indicated by the forward angle light scatter (data not shown). This loss of the ability to take up glucose correlated well with the loss of membrane potential.
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Syto 9 staining characteristics
The analysis of membrane integrity with the Syto 9/PI mixture during UVA irradiation revealed a unique fluorescence pattern for the bacterial cell population (Fig. 5g–i). On a two-dimensional dot plot, the whole population moved in a circle from low green and red fluorescence intensity (Fig. 5g) to an 4.3-fold increased green fluorescence intensity (Fig. 5h), and then to a state of increased red and decreased green fluorescence intensity (Fig. 5i). The increase of green fluorescence intensity coincided with the loss of culturability of >97 % of the cell population and, only after prolonged irradiation, was PI able to enter the cells and quench Syto 9 fluorescence. Hence, membrane integrity was lost long after the culturability of the population dropped below the detection limit. An increase in green fluorescence intensity was also observed when E. coli was exposed to sunlight. In these experiments, the geometrical mean of green fluorescence intensity of stained cells increased 3.7-fold, while culturability was lost by >98 % of the population. This observation is probably related to an increased uptake of Syto 9. This was tested by using the Syto 9/PI mixture and Syto 9 alone in the same irradiation experiment (Fig. 7). Using Syto 9 alone also led to a significant 3.7-fold increase in green fluorescence intensity. Furthermore, staining with DiBAC4(3) revealed that the loss of membrane potential coincided with the increase in green fluorescence intensity in samples that were stained with the Syto 9/PI mixture (Fig. 5e, h). This implies that Syto 9 is also subject to active dye exclusion and accumulates intracellularly when efflux pump activity and membrane potential are lost.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Inactivation pattern of vital cellular functions
The inactivation of cellular functions in E. coli measured during exposure to light followed a typical inactivation pattern. Shortly after the start of exposure, the total ATP concentration decreased rapidly, indicating the cessation of ATP synthesis by ATPases. At the same time, efflux pump activity stopped as well. Both functions are dependent on the proton motive force. Gradually, the membrane potential was also lost and the glucose uptake rate diminished accordingly. Finally, the cytoplasmic membrane of bacterial cells became permeable. The loss of culturability was dependent on the incubation method and the growth medium. Anaerobic incubation on sodium pyruvate-supplemented agar, a procedure known to also recover injured cells (Bromberg et al., 1998; Czechowicz et al., 1996), showed a decrease in culturability that closely correlated with the loss of membrane potential (r2=0.96).
The rapid decrease of total ATP during irradiation implies a direct or indirect inhibition of ATP synthesis and the utilization of the remaining ATP by ATP-dependent functions. Nevertheless, a certain level of ATP was still measured after prolonged irradiation. This implies that cells could either maintain alternative ways of ATP synthesis, such as glycolysis, or that the remaining ATP could not be used due to damage to systems which require ATP. It has been shown that the activity of ATPase from Saccharomyces cerevisiae solubilized from the plasma membrane and exposed to UVA light remains constant irrespective of dosage, indicating that the ATPase molecule itself is not damaged by UVA irradiation (Arami et al., 1997a, b). The authors proposed that the reduction of ATPase activity in the membrane by UVA irradiation is attributable to conformational changes resulting from an alteration in the higher-order structure of the membrane due to photochemical decomposition of ergosterol. For E. coli K-12 growing on succinate, it has been shown that ubiquinone Q-8 is a chromophore for inhibition of ATP synthesis (Lakchaura et al., 1976). Nevertheless, the same authors concluded that the oxidative phosphorylation system is not a primary factor in the induction of growth inhibition in E. coli by UVA light, because the doses required for inhibition of growth are only one-sixth of those required for inhibition of ATP synthesis. This contradicts our findings that the inhibition of ATP synthesis and efflux pump activity occurs earlier than the loss of culturability. However, it has to be mentioned that, in their experiments, stressed cells were plated on unsupplemented complex media. Such media can enhance an oxidative burst in bacterial cells, which eventually leads to cell death (Aldsworth et al., 1999; Dodd et al., 1997; George et al., 1998). Furthermore, the strain used in their study (AB2480) was recA-deficient and, therefore, was probably more susceptible to UVA light. These circumstances can lead to an overestimation of the negative effects of UVA light on the culturability of E. coli.
It has been hypothesized that when a cell is stressed energetically, the active transport systems will cease first, then the cytoplasmic membrane will depolarize, and finally permeabilization will occur, indicating cell death (Nebe-von-Caron et al., 2000). These authors observed that Salmonella typhimurium, which was stored for 25 days on nutrient agar at 4 °C and resuspended in growth medium, could be differentially stained with a combination of EB, DiBAC4(3) and PI. In their case, 35 % of the depolarized cells could still form colonies when sorted onto nutrient agar plates, while all permeabilized cells were unable to grow. In our experiments, depolarization of bacterial cells correlated well with the loss of culturability. In contrast to their measurements, we did not use EDTA to facilitate permeabilization of the outer membrane. Still, the uptake of DiBAC4(3) seemed to be specific without the addition of EDTA, because permeabilization of the cytoplasmic membrane was observed at a much higher fluence. Without disrupting the outer membrane with EDTA, one probably overestimates the fluence needed to depolarize and permeabilize the cytoplasmic membrane of E. coli. In the case of SODIS this is rather favourable, because an underestimation could have severe consequences for the user of SODIS.
The specific glucose uptake rate by the PEP-PTS decreased simultaneously with the loss of membrane potential, although the cause of inhibition remains unclear. Changes in uptake rates due to cell size can be ruled out, because bacterial cell size remained constant during exposure (data not shown). Also, the loss of membrane potential cannot be a direct cause, because 2-NBDG uptake is measured in combination with an uncoupler (2,4-dinitrophenol) which itself causes a collapse of the bacterial membrane potential (by preventing ATP synthesis, hence the degradation of 2-NBDG) (Natarajan & Srienc, 1999; White, 1995). One can speculate that enzymes of the PEP-PTS are inactivated by UVA light, and that glucose is taken up only until all PEP is used up. It has been proposed that components of the electron transport chain, possibly menaquinone or dehydrogenases, are damaged by UVA light and that this inhibits permeases such as the lactose permease (Jagger, 1981). Therefore, the high-affinity glucose permease could be an indirect target of UVA. Recently, it has been revealed by two-dimensional PAGE analysis that pyruvate kinase (PykF) and elongation factor G (EF-G) (FusA) of S. typhimurium specifically increased in cells just after heat treatment and recovery in tryptic soy broth (TSB) (Kobayashi et al., 2005). PykF catalyses the synthesis of pyruvate from PEP in the sugar phosphotransferase system. Kobayashi and coworkers suggest that the increase of PykF in cells just after heating and recovery of cells might facilitate the production of ATP in the electron transport system, because pyruvic acid is a key substrate for the citric acid cycle and subsequent electron transport system (Kobayashi et al., 2005). The same authors state that ATP seems to be particularly important, because many reactions involved in recovery require ATP as an energy source. If PykF were a direct or indirect target of UVA light, it would prevent recovery by the process described above. This is in line with our finding that cells which are inhibited in ATP synthesis can still be recovered on sodium pyruvate-supplemented agar. Furthermore, inhibition of PykF would not directly inhibit glucose uptake by the PEP-PTS, because PEP is not converted to pyruvate and is still available for the uptake of glucose molecules, as discussed above.
All viability indicators were applied again to the irradiated cells 5 days after irradiation. No regrowth or recovery of injured bacterial cells was observed (data not shown). Moreover, a significant decrease in all measured cellular functions, especially in cells which received >1500 kJ m–2 solar UVA (corresponding to 530 W m–2 global sunlight intensity for 6 h), was observed. This indicates that the damage in E. coli caused by UVA light is irreversible. It has to be mentioned, however, that resuscitation attempts with sterile supernatant, as carried out in previous studies (Kaprelyants & Kell, 1993; Mukamolova et al., 1998), were not performed in this study. However, to our knowledge, the resuscitation of permeabilized cells has never been shown and seems highly unlikely.
Differences in viability stains
The death of a bacterial cell has long been defined as the inability of a cell to grow to a visible colony on bacteriological media. With culturability methods, one can only observe bacterial death in retrospect (Postgate, 1989). Today, several viability indicators can be assessed without culturing cells, and each method is based on criteria that reflect different levels of cellular integrity or functionality. Consequently, the interpretation of viability is often ambiguous. Hewitt & Nebe-Von-Caron (2004) have shown that multi-parameter flow cytometry allows a functional classification of the physiological state of single-celled micro-organisms. They claim that, with this technique, it is possible to resolve a cell's physiological state beyond culturability and to determine population heterogeneity. Our study confirms their findings and shows that the use of only one viability indicator, as often applied, is not sufficient to describe the physiological status of a bacterial cell under stress. Therefore, these viability indicators or indirect methods do not provide short cuts by which viability may be determined (Postgate, 1967). We propose that only the sum of all these methods, including the detection of culturability, can give us some certainty about the physiological state of a bacterium.
Implications for the SODIS method
Our results show that a UVA fluence of >1700 kJ m–2 is needed to depolarize the cytoplasmic membrane of >95 % of the cell population, while 2500 kJ m–2 is needed for permeabilization. This corresponds to about 600–880 W m–2 of sunlight intensity (global intensity) over a period of 6 h, a threshold which can be achieved in most developing countries (Martin-Dominguez et al., 2005). The same study also confirms that temperature is not a predominant factor in the elimination of bacteria with sunlight, but that it is mainly radiation which determines the efficiency of the method. Furthermore, our culturability data fitted well with those of Khaengraeng & Reed (2005), in which a solar simulator at 410 W m–2 for a period of 6 h was used to irradiate stationary-phase E. coli cells. A one log reduction was achieved with a UVA fluence of 1200 kJ m–2. This corresponds well with the 1400 kJ m–2 measured in our work.
Important for the user of SODIS is the loss of infectivity of pathogenic bacteria in the treated water. It has been shown that S. typhimurium C5Nxr exposed to simulated sunlight (probably 700 W m–2) for 8 h and a temperature regime temporarily reaching 55 °C fail to produce detectable infections in BALB/c mice (Smith et al., 2000). Even culturable cells that have been irradiated for 1.5 h are less infective (virulent) than their non-irradiated counterparts. Although infectivity of target organisms is the ultimate parameter to measure in disinfection processes, it is not a feasible alternative to viability measurements, because appropriate models for certain pathogens are rare and difficult to perform. Our results suggest that cells that (i) have lost culturability under anaerobic conditions in sodium pyruvate-supplemented agar, (ii) are not able to take up glucose, and (iii) have lost membrane potential, will not be able to regain viability in the human intestine. The highest certainty about cell death, however, will be achieved if fluences are applied that lead to membrane permeability. It is likely that these results also apply to other enteric bacteria, but this still has to be investigated. Also, the effect of sunlight on eukaryotes, such as Cryptosporidium or Amoeba, can be assessed with the methods used in this study.
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ACKNOWLEDGEMENTS |
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Received 21 October 2005;
revised 8 February 2006;
accepted 20 February 2006.
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). Bacterial cells were harvested from a stationary-phase LB batch culture, washed and diluted in mineral water (Evian). Initial cell numbers in these experiments were 1.7–1.9x107 cells ml–1 Culturability of bacterial cells was measured with the pour-plate method using TSA.
) incubation] as a percentage of total counts. (c) Total ATP concentration per cell. (d) Non-pumping cells; loss of efflux pump activity measured with Syto 9/EB staining. (e) Depolarized cells; loss of membrane potential measured with DiBAC4(3). (f) Permeabilized cells; loss of membrane integrity measured with Syto 9/PI staining by flow cytometry. All experiments were done at least twice. Data from one representative experiment are displayed.
