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Solar disinfection (SODIS) and subsequent darkstorage of Salmonella typhimurium and Shigellaflexneri monitored by flow cytometry Franziska Bosshard,1,2 Michael Berney,13 Michael Scheifele,1Hans-Ulrich Weilenmann1 and Thomas Egli1,2 1Eawag, Swiss Federal Institute of Aquatic Science and Technology, PO Box 611, CH-8600 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zu¨rich, 8092 Zu¨rich, Switzerland Pathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality indeveloping countries. Solar disinfection (SODIS) is an effective means to fight this problem. In thepresent study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonellatyphimurium, was investigated with a variety of viability indicators including cellular ATP levels,efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmicmembrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlightand UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state ofthe bacterial cells continued to deteriorate even in the absence of irradiation: apparently thecells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh.
flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage ofbottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (.700 W m”2) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (,400 W m”2).
is crucial to understand the way in which SODIS damagesbacteria – and whether repair can occur.
The availability of safe drinking water is a key health issuein developing countries. The United Nations have declared The effectiveness of SODIS has been proven by cultivation- it a millennium development goal to reduce the number of based techniques with Escherichia coli and some pathogenic people without sustainable access to safe drinking water by organisms (Acra et al., 1980; Berney et al., 2006b; half by 2015. Solar disinfection (SODIS) is one of the McGuigan et al., 1998; Wegelin et al., 1994), and recently means to reach this goal. Its success is based on easily we have applied cultivation-independent methods to available and low-cost tools: one day of exposure to the sun characterize the inactivation of E. coli by sunlight (Berney of hygienically unsafe drinking water in PET bottles leads et al., 2006a). In that study we used flow cytometry to a significant increase in microbiological water quality. A combined with viability staining to characterize the loss of positive impact on health has been documented in several essential cellular functions in irradiated bacterial cells. The epidemiological field studies, e.g. in India, where a total of recorded cellular functions include membrane integrity, 40 % of diarrhoeal diseases and 50 % of severe diarrhoea membrane potential, efflux pump activity and glucose episodes were prevented by the use of SODIS (Rose et al., uptake activity. We showed that a reproducible sequence of 2006). SODIS water treatment is already used by 2 million membrane-function breakdown takes place when E. coli is people and the number is increasing. But despite the fact irradiated with sunlight or UVA light (Berney et al., 2006a).
that the method works, the exact mechanism of inactiva- However, it is important to know whether these results tion of microbial pathogens is not yet known. Therefore, it translate to enteric pathogens like Salmonella or Shigella,the inactivation of which is the primary goal of SODIS. So 3Present address: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9016, New Zealand.
Reliability of the SODIS method depends not only on the Abbreviations: DiBAC4(3), bis-(1,3-dibutylbarbituricacid)trimethine oxo- light dose leading to damage in target cells, but also on nol; EB, ethidium bromide; 2-NBDG, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose; PET, poly(ethylene terephthalate); PI, possible recovery processes in injured cells after irradiation.
So far, no regrowth or recovery of membrane functions in Solar disinfection of S. typhimurium and Sh. flexneri injured E. coli cells has been found (Berney et al., 2006a; Staining procedures. Five fluorescent dyes were used alone or in Joyce et al., 1996; Oates et al., 2003; Reed, 1997; Wegelin different combinations: Syto 9 (Invitrogen Molecular Probes), propidium iodide (PI; Invitrogen), bis-(1,3-dibutylbarbituricacid)-trimethine oxonol (DiBAC4(3); Invitrogen), ethidium bromide (EB; The present work extends our knowledge about the Fluka) and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy- inactivation mechanism of solar light from the indicator D-glucose (2-NBDG; Invitrogen). Samples taken from irradiation bacterium E. coli to two important enteric pathogens, experiments (sunlight and artificial UVA) were divided into five Salmonella typhimurium and Shigella flexneri. In addition, subsamples and immediately stained with two mixtures of fluorescentdyes (Syto 9/PI and Syto 9/EB) and three single fluorescent dyes we have investigated the ability of these enteric pathogens [DiBAC4(3), Syto 9 and 2-NBDG]. Samples were incubated in the to survive and repair damage after solar irradiation.
dark at 37 uC for 5 min (2-NBDG) or at room temperature for10 min [DiBAC4(3)], 15 min (Syto 9/EB), 20 min (Syto 9/PI) and25 min (Syto 9), respectively, before analysis. Prior to flow-cytometric analysis, samples (~1–56107 cells ml21) were diluted with sterile-filtered bottled water (Evian) to 1 % (v/v) of the initial cell Bacterial strains. Salmonella enterica serovar Typhimurium ATCC concentration (~1–56105 cells ml21 final concentration). Stock 14028 (referred to in this paper as Salmonella typhimurium) and solutions of the dyes were prepared as follows: PI and Syto 9 were Shigella flexneri ATCC 12022 were used in this study.
used from the LIVE/DEAD BacLight kit (Invitrogen), EB wasprepared in distilled and filtered water at 25 mM, DiBAC4(3) was Growth media and cultivation conditions. Cells were grown as prepared in DMSO at 10 mM, and 2-NBDG was dissolved in distilled described by Berney et al. (2006a) with modifications. In drinking and filtered water at 5 mM. All stock solutions were stored at 220 uC.
water, cells grow very slowly or not at all; therefore, we used The working concentrations of Syto 9, PI, EB, DiBAC4(3) and 2- stationary-phase cells, which were shown to be more resistant to NBDG were 5, 30, 30, 10 and 5 mM, respectively. 2-NBDG was added SODIS than cells in the exponential growth phase (Berney et al., in combination with 2,4-dinitrophenol (final concentration 2 mM) 2006b; Reed, 1997). Luria–Bertani (LB) broth, which was filter- (Natarajan & Srienc, 2000). At the beginning of each experiment a sterilized with membrane filters (0.22 mm, Millipore) and diluted to sample was incubated at 90 uC for 3 min (in a 2 ml Eppendorf tube) 33 % (v/v) of its original strength with ultrapure water, was used for as a control measurement for inactive bacteria. By comparing the batch cultivation. Precultures were prepared for each individual batch staining pattern of heat-inactivated with untreated samples, electronic experiment from the same cryo-vial, streaking the stock solution onto gates were set to differentiate negatively and positively stained Hektoen agar plates (Oxoid) selective for Shigella and Salmonella species. After 15–18 h of incubation at 37 uC, one colony was picked,loop-inoculated into a 125 ml Erlenmeyer flask containing 20 ml LB Flow-cytometric measurements. The methods used here have broth, and incubated at 37 uC on a rotary shaker at 200 r.p.m. At an been described recently (Berney et al., 2006a, 2007). Flow-cytometric OD546 between 0.1 and 0.2, an aliquot of the culture appropriate to measurements were made using a Partec Cyflow space flow cytometer obtain an initial OD546 of 0.002 was transferred into a 500 ml with 488 nm excitation from an argon ion laser running at 50 mW or Erlenmeyer flask containing 50 ml prewarmed LB broth. With this (for the fluorescent glucose analogue 2-NBDG) 200 mW. Green procedure, no lag phase was observed. These flasks were then shaken fluorescence was collected in the FL1 channel (520±20 nm), and red at 200 r.p.m. on a rotary shaker at 37 uC for approximately 18 h until fluorescence in the FL3 channel (.590 nm); all data were processed stationary phase was reached. Stationary phase was confirmed from with the Flowmax software (Partec), and electronic gating with the five consecutive OD546 measurements within 1 h.
software was used to separate positive signals from noise. The specificinstrumental gain settings for these measurements were as follows: Sample preparation and plating. Cells were harvested from batchculture by centrifugation (16 000 g, 3min), washed three times with FL15490, FL35600, speed 3 (implying an event rate never exceeding filter-sterilized commercially available bottled water (Evian) and 1000 events s21). All samples were collected as logarithmic (3 decades) signals and were triggered on the green fluorescence channel 546 of approximately 0.01 (corresponding to 1– 56107 cells ml21). To allow the cells to adapt to the mineral water, (FL1). A routine check of the flow cytometer was performed every day light exposure of bacterial suspensions was not started until 1 h after for correct alignment with an FITC bead standard. This ensured dilution. During exposure, aliquots were withdrawn at different time accuracy in counting (volumetric counting device) and measured points and diluted in decimal steps (1021 to 1026) with sterile-filtered fluorescence intensities. Microscopic observation was performed on bottled mineral water (Evian). Volumes of 1 ml of appropriate an Olympus BX50 microscope equipped with filters HQ-F41-007 for dilutions were withdrawn and mixed with 7 ml liquid tryptic soy agar PI and EB and HQ-F41-001 for Syto 9, DiBAC4(3) and 2-NBDG (all (TSA) (Biolife) at 45 uC (pour-plate method). After 20 min, the solidified agar was covered with another 4 ml liquid TSA (40 uC).
Plates were incubated for 48 h at 37 uC until further analysis. The Total ATP. For the determination of total ATP, the BacTiter-Glo standard error of pour plating was always ,10 %.
system (Promega) was used. The BacTiter-Glo buffer was mixed withthe lyophilized BacTiter-Glo substrate and equilibrated at room Sunlight and UVA exposure. Samples of 10 ml bacterial suspension temperature. The mixture was stored overnight at room temperature (see above) were exposed to sunlight or UVA light as described earlier to ensure that all ATP was hydrolysed (‘burned off’) and the background signal had decreased. A cell suspension of 100 ml wasmixed in a 2 ml Eppendorf tube with an equal volume of the Dark storage. Samples of 10 ml bacterial suspension were exposed to previously prepared BacTiter-Glo reagent (stored on ice). The sample UVA light (see above). Cellular damages were assessed immediately was then briefly mixed by once pipetting up and down and put into a after irradiation and at different time points during dark storage, water bath at 37 uC for 30 s. The luminescence of the sample was which was performed at 37 uC for 48 h, holding the cells in the same measured in a luminometer (model TD-20/20; Turner BioSystems) medium (sterile-filtered bottled water) as during irradiation. To immediately after incubation. A calibration curve with dilutions of exclude the possibility of regrowth, nalidixic acid was included in all pure rATP (Promega, P1132) was measured for each batch of the samples at a concentration of 100 mg ml21.
BacTiter-Glo buffer. ATP concentration per cell was then calculated using this calibration curve and the total count measurements (Syto threefold, the reciprocity law started to fail not only for the culturability of Sh. flexneri (Fig. 1) but also for all othermeasured viability indicators (data not shown). This clearly Reproducibility. All field experiments were conducted in three demonstrates that for these enteric pathogens high biological replicates on three different days. Irradiation intensity datawere obtained from a weather station located 300 m from the irradiation intensities in laboratory experiments result in exposure site (BAFU/NABEL, EMPA Du¨bendorf, Switzerland).
an overestimation of the effect in comparison with the Sunlight intensity varied with the weather conditions, so the light same light dose under natural sunlight conditions and that dose at the sampling points was never exactly the same. Therefore, we such data are hence of limited use. Therefore, the doses listed in Table 1 were obtained exclusively from experi-ments performed at light intensities that were in the rangeof natural conditions.
The difference in the fluence needed to achieve a three-logreduction when exposing the cells to different light intensities was a result of two effects. Firstly, the shape of The susceptibility of different properties of S. typhimurium the inactivation curve followed the ‘log-linear with a and Sh. flexneri to artificial UVA light is shown in Table 1.
shoulder’ model for samples irradiated with natural sunlight Both organisms were less susceptible to UVA light than the or artificial UVA of a corresponding intensity. However, the indicator bacterium E. coli (Berney et al., 2006a). For shoulder disappeared when cells were irradiated with very example, when assessed with PI staining, the light dose high intensities (919 W m22 and 1315 W m22 in Fig. 1).
needed for membrane permeabilization of S. typhimurium Secondly, the inactivation was three times faster with very was approximately three times, and for Sh. flexneri high intensities as compared to natural conditions (slope of approximately two times, higher than for E. coli.
20.0031 for intensities around 360 W m22 vs 20.0011 with However, for all three enteric bacteria, the same sequential inactivation pattern was observed with the measured In general, the reciprocity law was valid for Sh. flexneri as long as intensities not exceeding 400 W m22 were applied.
Because of this higher resistance, some of the laboratory Experiments conducted in this irradiation intensity range experiments were conducted with much higher irradiation compared well with natural conditions. Reciprocity for S.
intensities than those normally achieved with natural typhimurium held over a much wider range (from 50 to sunlight. Typically, the maximum natural sunlight intens- 700 W m22). However, when very high irradiation ity at noon on mid-European longitude is about 130 W intensities (1000 W m22) were applied, the shoulder of m22 (integrated for the wavelength spectrum 350– the inactivation curve was shortened (data not shown).
450 nm), whereas in this study, intensities between163 W m22 and 1315 W m22 were used for artificial UVA exposure to achieve high enough fluences within areasonable time period. We observed that, as soon as A decrease in culturability of more than 99 % was observed natural irradiation conditions were exceeded two- to (Figs 2a and 3a) for S. typhimurium and Sh. flexneri during Table 1. Comparision of the susceptibility of S. typhimurium and Sh. flexneri to artificial UVA light and sunlight Figures indicate the approximate light doses (±20 %) in kJ m22 at which .90 % of the cells exhibited the properties indicated. Cells were exposedcontinuously to artificial UVA light with an intensity of 620 W m22 for S. typhimurium and 360 W m22 for Sh. flexneri. In the case of sunlight, cellswere exposed during two consecutive days with a night break of approximately 12 h. Sunlight exposure on the first day reached 2300 kJ m22 for S.
typhimurium and 2500 kJ m22 for Sh. flexneri, respectively. An asterisk (*) indicates that this parameter changed during the night break. Forcomparison, corresponding data for E. coli are shown (Berney et al., 2006a).
Loss of culturability (0.1 % survival) at .90 % of cells with inactivated efflux pumps at .90 % of cells unable to take up glucose at .90 % of cells with depolarized membranes at .90 % of cells with permeabilized membranes at Solar disinfection of S. typhimurium and Sh. flexneri lowest level on the first day of irradiation (Fig. 2b, e).
Interestingly, a more than twofold increase in ATP concentration was observed initially before it rapidly decreased at approximately 1000 kJ m22. A similar increase in cellular activity with increasing exposure in the initial phase was observed also for the uptake of the fluorescent For Sh. flexneri a similar general pattern was observed, but with some distinct differences in the magnitude of the effects. Culturability dropped over more than 3 additional log-units during the night break (Fig. 3a). After the night break, 80 % of the cells had lost their membrane potential and 20 % were even permeabilized (Fig. 3c, d). About 40 % of the cells were unable to take up glucose (Fig. 3f). As in S.
typhimurium, ATP concentration and efflux pump activity in Sh. flexneri had already reached final levels on the first day. Comparable to S. typhimurium, a slight increase inglucose uptake activity was observed during irradiation onthe first day (Fig. 3d).
Fig. 1. Deviations from light dose reciprocity appearing withdifferent intensities of artificial UVA light irradiation in comparison The overall fluence rate resulting in membrane permeabi- to long, low-intensity irradiation with sunlight, shown for the lization in .90 % of the cells (8000 vs 4500 kJ m22 in S.
culturablity of Sh. flexneri. Bacterial cells were harvested from the typhimurium, and 6000 vs 4500 kJ m22 in Sh. flexneri) and stationary phase of an LB batch culture, washed three times and loss of membrane potential in .90 % of the cells (6000 vs diluted in bottled mineral water. C.f.u. were measured by pour 2300 kJ m22 in S. typhimurium, and 4500 vs 2500 kJ m22 plating and sensitivity was recorded as c.f.u./(c.f.u. at time zero).
in Sh. flexneri) was clearly reduced with discontinuous Horizontal dashed lines indicate the detection limits. Lines represent modelled inactivation curves with the program GInaFIT(Geeraerd et al., 2005). Empty diamonds (e) represent averagedcontrols. (a) Artificial UVA light was applied at the following Dark storage of S. typhimurium after irradiation intensities: ¾, 163 W m”2; $, 332 W m”2; #, 370 W m”2; &, For a better understanding of the overnight processes in 710 W m”2; +, 732 W m”2; h, 786 W m”2; -, 802 W m”2; m, the outdoor experiments, irradiation and subsequent 919 W m”2 and g, 1315 W m”2. (b) Sunlight irradiation on three storage in the dark of S. typhimurium was investigated in different days in biologically independent triplicates: ¾, $ and # the laboratory (Fig. 4). This was done because of the higher on 23 August 2006; &, + and h on 31 August 2006; -, m and g resistance of S. typhimurium resulting in only 2-log reduction during one full day of exposure. Bacterial cellsuspensions were irradiated with a dose of 1500 kJ m22, one day of solar irradiation (8 h; 2300 and 2500 kJ m22, corresponding to half a day of sunlight or one full day of respectively). Efflux pump activity and ATP concentration exposure under overcast conditions. Culturability, efflux had reached their lowest level by the end of the day (Figs pump activity, membrane potential, membrane permeab- 2b, e and 3b, e). In contrast, inactivation of glucose uptake ility and ATP content per cell were measured immediately ability, loss of membrane potential and loss of membrane after exposure and for a period of up to 48 h at eight integrity were not observed with S. typhimurium and Sh.
subsequent time points during storage in the dark. This flexneri during one day of sunlight irradiation (Figs 2f, c, d aspect was investigated in more detail because it is possible and 3f, c, d). Therefore, we decided to continue exposure that a small part of the cells might survive the irradiation and that these survivors might start growing on either lysedcells or assimilable organic carbon in the water. For Interestingly, S. typhimurium and Sh. flexneri lost cellular example, it has been shown that some pathogenic bacteria activity during dark storage at 37 uC overnight. This is are able to grow on natural substrates in bulk water (Vital clearly shown for S. typhimurium, where an additional one- et al., 2007, 2008). To exclude this possibility and to make log reduction in c.f.u. was observed after the night break sure that only the cells originally exposed to light were (Fig. 2a). Accordingly, 60 % of the cells lost their analysed, we added nalidixic acid to inhibit cell division.
membrane potential during the night break and a slight The concentration of nalidixic acid required to keep S.
reduction in membrane integrity was also observed typhimurium from dividing was determined in a minimal (Fig. 2b). Furthermore, the percentage of cells able to take inhibitory concentration experiment.
up glucose increased by 40 % on the first day and wasfollowed by a loss of 80 % during the night break (Fig. 2f).
After the bacteria had received a ‘half-day’ UVA dose, c.f.u.
ATP concentration and efflux pump activity reached their had decreased by approximately 1 log-unit (Fig. 4a).
Fig. 2. Viability parameters of stationary-phase cells of S. typhimurium exposed tosunlight. (a) Culturability [log(c.f.u. ml”1)] ($),with the dashed line indicating the detectionlimit, and log(total cell concentration ml”1) (.).
(b) EB-positive cells, (c) DiBAC4(3)-positive cells, (d) PI-positive cells. Values were calcu- lated as percentage of total cell concentration.
(e) Average ATP concentration per cell. (f) 2- NBDG-positive cells (able to take up glucose)calculated as percentage of total cell concen-tration. Light dose on day 1, 2300 kJ m”2; day2, 1200 kJ m”2 (overcast conditions). Thenight break is indicated by a dotted line in eachgraph. In all graphs, unirradiated control samples are displayed as empty symbols.
Error bars represent standard deviations from three biologically independent experiments.
During subsequent dark storage, c.f.u. decreased by 5 log- unirradiated control cells were also hampered at the units over 24 h. Efflux pump activity was lost completely beginning of the experiment, when about 50 % of the just after irradiation and was not regained (Fig. 4b). The population showed no efflux pump activity, probably as an Fig. 3. Viability parameters of stationary-phase cells of Sh. flexneri exposed to sunlight.
(a) Culturability [log(c.f.u. ml”1)] ($), with thedashed line indicating the detection limit, andlog(total cell concentration ml”1) (.). (b) EB-positive cells, (c) DiBAC4(3)-positive cells, (d) PI-positive cells. Values were calculated as percentage of total cell concentration. (e) Average ATP concentration per cell. (f) 2- NBDG-positive cells (able to take up glucose) calculated as percentage of total cell concen-tration. Light dose on day 1, 2500 kJ m”2; day2, 1300 kJ m”2 (overcast conditions). Thenight break is indicated by a dotted line in eachgraph. In all graphs, non-irradiated control samples are displayed as empty symbols.
Error bars represent standard deviations from three biologically independent experiments.
Solar disinfection of S. typhimurium and Sh. flexneri Fig. 4. Dark storage of S. typhimurium afterexposure to a dose of 1500 kJ m”2 artificial UVA light, applied with an irradiation intensityof 205 W m”2 for 120 min. To exclude the possibility of regrowth, nalidixic acid (100 mgml”1) Culturability [log(c.f.u. ml”1)] ($), with thedashed line indicating the detection limit, andlog (total cell concentration ml”1) (.). (b) EB-positive cells, (c) DiBAC4(3)-positive cells, (d)PI-positive cells. Values were calculated as percentage of total cell concentration. (e) Average ATP concentration per cell. (f) 2- NBDG-positive cells (able to take up glucose), calculated as percentage of total cell concen- tration ($, numbers on left side of graph), andgeometric mean of green fluorescence of the2-NBDG-positive cell population (., numberson right side of graph and dashed line for detection limit). In all graphs, non-irradiated effect of nalidixic acid. Efflux pump activity was later functions during continuous artificial UVA exposure was regained in the control sample. Membrane potential was similar in both tested organisms and corroborates our lost in only a very small part of the cell population during results for E. coli (Berney et al., 2006a). The similarity in irradiation, but a relevant fraction of the cells (about 60 %) inactivation pattern suggests that the molecular mechan- lost their membrane potential in the following 24 h of dark isms involved in the inactivation and killing of bacterial storage (Fig. 4c). Integrity of the cell membranes did not pathogens due to solar irradiation are similar or even change significantly during dark storage: only a slight identical in the three enteric bacteria. Therefore, E. coli can increase in the percentage of permeabilized cells was be considered a good model organism for such investi- observed (Fig. 4d). ATP content of the cells was reduced to gations. However, as suggested in an earlier study (Berney 10 % compared to the control just after the treatment and et al., 2006b), both Sh. flexneri and S. typhimurium were levelled off to ,5 % during the first 10 h after the found to be more resistant than E. coli.
treatment. Also the fraction of cells able to take up glucosedecreased from about 90 % just after the treatment to zero during the first 24 h. The population that was still able totake up glucose during dark storage became increasingly In experiments with Sh. flexneri and S. typhimurium, we less fluorescent, which indicates that the uptake rate of observed two modes of exposure that caused deviation fluorescently labelled glucose decreased with time.
from the reciprocity law: firstly, exposure to either veryhigh or very low irradiation intensity, and secondly, split We never observed a regain of culturability in irradiated S.
exposure by a pause in irradiation of up to 14 h.
typhimurium or Sh. flexneri cells after 24 h or 48 h dark Interestingly, light dose reciprocity was valid for E. coli storage after irradiation with different light doses in (Berney et al., 2006b) and S. typhimurium (this study) laboratory or field experiments (data not shown).
within a much broader intensity range (50–700 W m22)than for Sh. flexneri, which already showed distinctdeviation when light intensities were two- to threefold higher than those occurring under natural conditions.
Susceptibility of S. typhimurium and Sh. flexnerito solar light High irradiation intensity reduces the light doserequired for inactivation For the first time, cellular functions in S. typhimurium andSh. flexneri during solar and UVA exposure were followed Light dose reciprocity has been observed in a majority of not only by plating but also with viability staining and ATP biological and medical applications, while so called measurements. The pattern of sequential loss of cellular ‘reciprocity law failures’ have been mostly observed in experiments conducted at either very low or very high radiant fluxes (Martin et al., 2003). In the case of Sh.
The data presented in this study strongly suggest that flexneri irradiated with UVA light we observed that the SODIS inactivates S. typhimurium and Sh. flexneri by shoulder of the inactivation curve became less pronounced inhibiting the respiratory chain. The ATP content per cell or was even eliminated with high irradiation intensities.
decreased rapidly in E. coli (Berney et al., 2006a) and Sh.
The existence of a shoulder was interpreted earlier by either flexneri (this study) upon irradiation with sunlight, while the presence of repair mechanisms that are able to slow in S. typhimurium an initial increase was observed before a down the light effect and/or damage to more than one sharp sustained decline. We propose that after an initial target within the cell (Harm, 1980; Sommer et al., 2001). If activation of energy metabolism, which is reflected in a shoulder is eliminated, as seen in our experiments for increased glucose uptake and ATP level, respiration stops high-intensity irradiation of Sh. flexneri and also S.
and the remaining ATP is either consumed by various typhimurium, repair mechanisms might be too slow or recovery processes (Kobayashi et al., 2005), or, more likely, damaged themselves. Elimination of the shoulder results ina reduction of the light dose required for inactivation.
through the maintenance of the membrane potential viathe F1F0-ATPase. Consistent with this proposal is theobserved increase in glucose uptake activity, which Split exposure reduces the light dose required for provides ATP via substrate-level phosphorylation to fuel the proton-pumping ATPase to maintain the membrane We found that S. typhimurium and Sh. flexneri are more potential at a critical level even in the absence of a susceptible to the same light dose when exposed to sunlight functioning electron-transport chain. Since the medium over two days (with a break overnight) than with continuous used in this study (bottled mineral water) contains only artificial UVA light. This finding was reproduced in the low levels of assimilable organic carbon, the cells will laboratory with discontinuous irradiation on two consec- eventually die from ATP exhaustion and loss of the utive days (data not shown). This is most likely because the membrane potential. In fact, it has been proposed earlier bacterial cells are irreversibly damaged and consequently they that components of the respiratory chain like menaqui- continue to lose viability even when irradiation is stopped.
nones and dehydrogenases could be inactivated by UVAlight (Jagger, 1981).
This is in line with the observation that exposure of cells froma human carcinoma cell line to short intervals of UVA lightwas more cytotoxic than continuous UVA irradiation (Merwald et al., 2005). However, if the time between In our experiments, injured bacterial cells irradiated with exposures exceeded 2 h, the cells were able to recover and, sunlight or UVA light were never observed to be able to therefore, were less susceptible than with one single dose of regrow. This corroborates the work of other authors (Joyce UVA. Similar results were reported for Saccharomyces et al., 1996; Oates et al., 2003; Reed, 1997; Wegelin et al., 1994).
cervisiae and E. coli irradiated with UVC light (Dzidic etal., 1986; Harm, 1968; Salaj-Smic et al., 1985; Sommer et al., The lack of regrowth in cells irradiated with polychromatic 1996) and human dermal fibroblasts irradiated with UVA UV light in water disinfection processes seems to be the light (tanning bed radiation) (Hoerter et al., 2008).
main advantage compared to monochromatic UVC light Therefore, our data suggest either that the repair mechanisms and therefore is of great interest to the water disinfection of S. typhimurium and Sh. flexneri were already inactivated community (Kalisvaart, 2001, 2004; Oguma et al., 2002; after 8 h of continuous irradiation, or that the lack of nutrients in the suspension did not allow the induction of an It has been shown that non-cultivable cells of S.
appropriate repair response. It remains to be determined typhimurium produced by UVA irradiation do not retain whether or not the cells are able to recover when cell damage infectivity for mice (Smith et al., 2000). Our study now is less severe (shorter irradiation period) or when an indicates that this is most likely due to irreversible damage appropriate amount of nutrients is available in the water.
occurring during exposure to sunlight. Bacterial cells that Discontinuous UVC exposure of E. coli was shown to are impaired in glucose uptake and oxidative phosphoryla- induce the SOS response, which increases DNA repair tion may not be able to regrow, because uptake of nutrients activity (Dzidic et al., 1986; Salaj-Smic et al., 1985).
and the maintenance of a membrane potential are regarded However, UVA probably causes more complex damage to as prerequisites for survival and replication.
the cells (several targets may be affected by UVA light, ascompared to UVC, where predominantly DNA damage is observed) (Jagger, 1981). Hence, this is likely to require amore complex repair machinery. In an earlier microarray SODIS and artificial UVA light kill enteric bacteria most study we showed that in E. coli both DNA-repair genes and likely by inactivation of the respiratory chain and genes involved in oxidative stress response are induced subsequent exhaustion of ATP. Our results show that even upon irradiation with sublethal UVA light intensities the resistant strain of S. typhimurium, which appeared to suffer only minor damage after half a day of sunlight, was Solar disinfection of S. typhimurium and Sh. flexneri actually damaged to an extent such that regain of viability was not detected. In fact, our results suggest that it is even Motomatsu, A., Honjoh, K. & Iio, M. (2005). Identification of factors favourable to store the treated water overnight before involved in recovery of heat-injured Salmonella Enteritidis. J FoodProt 68, 932–941.
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