The effect of solar ultraviolet radiation and ambient temperature on the culturability of toxigenic and non-toxigenic Vibrio cholerae in Pretoria, South Africa

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The effect of solar ultraviolet radiation and ambient temperature on the culturability of toxigenic and non-toxigenic Vibrio cholerae in Pretoria, South Africa
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   African Journal of Microbiology Research Vol. x(xx), pp. xxx-xxx, x July, 2012 Available online at http://www.academicjournals.org/AJMRDOI:ISSN 1996-0808 ©2012 Academic Journals Full Length Research Paper  The effect of solar ultraviolet radiation and ambienttemperature on the culturability of toxigenic and non-toxigenic Vibrio cholerae  in Pretoria, South Africa C. C. Ssemakalu 1, 2 , M. Pillay 1,2 and E. Barros 3 * 1 Department of Biosciences, Vaal University of Technology, Vanderbijlpark 1900, South Africa. 2 Department of Life Sciences, College of Agriculture, University of South Africa, Florida Campus, South Africa. 3 Council for Scientific and Industrial Research, Biosciences, P.O. Box 395, Pretoria 0001, South Africa.  Accepted 10 July, 2012   Although solar disinfection (SODIS) is known to be one way of controlling waterborne diseases likecholera, the potential impact that this technology can have in resource poor areas is increasingly beingconsidered as a potential component in water treatment for poor and rural communities and as a meansto alleviate the burden of disease. In this study, comparative growth analysis was conducted on three Vibrio cholerae  strains, two toxigenic and one non-toxigenic, to test the effect of solar ultravioletradiation (SUVR) and ambient temperature. Culturability on solid media was used in addition to flow-cytometry to evaluate the survival and integrity of the cell membrane of these bacteria after exposure toSUVR. The season of the year played an important role in the complete inactivation of the three V.cholerae  strains with autumn and summer being the most significant, requiring only 7 h of exposure torender the bacteria unculturable, due to higher SUVR levels and temperature observed in theseseasons. However, the results also indicated that in winter where the levels of SUVR were comparableto those in spring the extreme variation in the daily recorded ambient temperatures [± 3°C  – ± 30°C] mayhave contributed to the observed disinfection.Key words: Solar disinfection, solar ultraviolet radiation, Vibrio cholerae , cholera.  INTRODUCTION Cholera, a disease well known for its life threateningsecretory diarrhoea characterized by several, capaciouswatery stools, often accompanied by vomiting is awaterborne disease that has infected thousands of people resulting in high mortality rates (Osei and Duker,2008; WHO, 2006, 2011). Vibrio cholerae the causativeagent of cholera is a Gram negative micro-organism thatexists naturally within the aquatic environment (Merrell etal., 2000). To date, two serotypes of  V. cholerae that is(O1 and O139) are known to play an important role in the *Corresponding author. E-mail: EBarros@csir.co.za. Tel: (+27)12 841 3221. Fax: +27 12 841 3651. pathogenesis of cholera. Both these serotypes have beenshown to carry virulence factors (Aoki et al., 2009;Hoshino et al., 1998) expressed by two genetic elements: CTXф which is responsible for the production of cholera toxin (CT) the causative agent of cholera and the VPI pathogenicity island required for the entry of CTXф. The African continent is privileged with the availabilityof freshwater sources such as lakes, rivers, ponds,swamps, dams and boreholes which are essential inmeeting the basic needs of the people such as drinking,cooking and hygiene. However, the inability to protectthese water sources have made them modes of transmission of waterborne diseases such as cholera invarious African communities (WHO, 2006, 2011). One of the millennium development goals is to provide resource   poor communities such as those found in Africa withaccess to clean potable water by the year 2015 (Roselliniand Pimple, 2010). Although a great effort has beengeared towards achieving this goal its realization doesnot appear be within reasonable reach (WHO, 2011) as,more than half a billion people are still lacking access todrinking water and even more are without sanitationfacilities (WHO, 2012). In South Africa as well as in other  African countries sporadic cholera outbreaks due to theconsumption of untreated water have been reported inrural and informal settlements (Mugero and Hoque,2001). The problem is aggravated by the consumption of untreated microbiologically contaminated water or water that is treated and stored inappropriately (Firth et al.,2010; Rufener et al., 2010). As an intervention solar ultraviolet radiation (SUVR), a priceless component of thesun energy, has been used to treat water through aprocess known as solar disinfection (SODIS) (Berney etal., 2006b; Smith et al., 2000; Ubomba-Jaswa et al.,2008). During SODIS treatment, bacterial inactivation hasbeen shown to occur through a synergy between SUVRand an increase in water temperature (above 45°C)(Boyle et al., 2008; Navntoft et al., 2008; Ubomba-Jaswaet al., 2008). Clearly, the process through which SODISoccurs seems quite simple and straight forward.However, the underlying mechanisms are morecomplicated in that various factors such as SUVR,temperature, location and the type of container or vesselused are major determinants of the outcome. SUVR hasbeen shown to successfully inactivate the culturability of enteropathogenic Escherichia coli  , viruses such aspoliovirus and giardia cysts which are known to survive inaquatic environments (Heaselgrave et al., 2006;Ubomba-Jaswa et al., 2008).The consumption of pathogen free water throughoutthe year is critical in the fight against waterborne diseaseoutbreaks and epidemics. Therefore it is important toassess the applicability of using SODIS in Africancountries like South Africa, Lesotho and Swaziland thatexperience four defined seasons of the year. Theobjective of this study was to empirically determine thepertinence of using SUVR to disinfect V. cholerae  contaminated water during the different seasonsexperienced in South Africa. To achieve this objective aculture based method was used to provide insight into theextent of culturability changes of three V. cholerae strainswhen exposed to natural SUVR. These included twotoxigenic strains and one non-toxigenic strain of  V.cholerae. The integrity of the cell membrane of themicroorganisms was also assessed using a flowcytometer after the summer exposure. The use of flowcytometry was motivated by its ability to denote thedifferent cellular states of a bacterial culture and V.cholerae has been shown to exist in a viable but nonculturable (VBNC) state when stressed (Falcioni et al.,2008). The VBNC state that V. cholerae can exhibit hasbeen shown to play a role in the epidemiology of cholera(Chaiyanan et al., 2001). MATERIALS AND METHODSBacterial strains The two toxigenic V. cholerae strains used in this study wereserotypes O1 (NCTC 5941) and O139 (NCTC 12945) obtained fromthe national collection of type cultures. The non-toxigenicenvironmental V. cholerae strain 1009 was isolated from the VaalRiver in South Africa (Du Preez et al., 2010). All strains were storedat -80°C as bacterial stocks on beads. Growth media and growth conditions Bacterial suspensions were prepared by spreading 3 beads fromeach of the frozen bacterial stocks onto nutrient agar plates andincubating them for 18 h at 37°C. A colony of each strain was thenstreaked onto a fresh nutrient agar plate and incubated at 37°Covernight. Following this, each V. cholerae strain was inoculatedinto autoclaved Luria Broth (LB) at pH 8.5 and incubated at 37°Cwith agitation at 200 revolutions per minute (rpm) overnight till theyreached the stationary phase. Stationary phase cultures were usedfor solar exposures due to their resilience (Berney et al., 2006a).Bacterial suspensions were harvested by centrifugation at 7000 × gfor 10 min. The pelleted bacteria were re-suspended in 10 ml filter sterilized 1x phosphate buffer saline (PBS) at pH 7.5. Centrifugationand re-suspension was repeated three times to facilitate an almostcomplete removal of LB. The resultant bacterial suspensions werediluted in 15 ml of autoclaved ground water obtained fromSoshanguve, Pretoria (chemical analysis shown in Table 1), to anOD 600 of 0.01 corresponding to 7 or 8 Log colony forming units per millilitre (Log CFU/ml) before exposure to sunlight. Exposure to natural solar radiation Fifteen millilitres of each V. cholerae strain were transferred totransparent polystyrene 25 cm 3 unventilated tissue culture flasks.The samples were gently hand- shaken and allowed to stand for 10to 15 min to allow the bacterial cells to adapt to the water. Thesamples were then exposed to natural sunlight by placing them onthe roof top of the Council for Scientific and Industrial Research(CSIR) building in Pretoria (lat. 25° 44’50.40”S; long. 28 ° 16’50.50”E) at an elevation of 1.4 km above sea level. The control sampleswere prepared in a similar manner, placed on the roof top andprotected from direct sunlight by covering them with an opaqueventilated cardboard box. All the samples were exposed for a 24 hperiod from 6:00 a.m to 6:00 a.m the following day. SUVR wasmeasured with two UV meters (Solar Light Co., Philadelphia, PA,USA) that were placed on the rooftop next to the samples. One of UV meters (model 10, serial number 14056) measured radiancedue to UVA (wavelength range 320 to 400 nm), while the other (model 4, serial number 14085) measured radiance due to UVB(wavelength range 290 320 nm). The UV data was recorded hourlyper day for each season by each UV probe and downloaded fromthe PMA-2100 data logger via a computer (Solar Light Co.). TheUVA data was recorded in W/m 2 while the UVB data was recordedas μW/cm 2 and then converted to W/m 2 . In addition, the cumulativeUVA and UVB doses (radiation) received after 7 and 24 h wererecorded from the PMA data logger. The hourly ambienttemperature data was acquired from a weather station located inMeyers Park (less than 6 km from CSIR) (Weather Underground,2010). A temperature probe was also placed next to the point of exposure and the ambient air temperature was taken each time a    Table 1. The physiochemical properties of ground water from Soshanguve. Physiochemical property (Units) Value  Alkalinity (mg/L CaCO 3 )   25 Ammonia nitrogen (mg/L N)   <0.1Calcium (mg/L Ca)   29 Chloride (mg/L Cℓ)   70COD (mg/L COD)   <10Elect Conductivity (mS/m [25°C])   56.9Iron (mg/L Fe)   <0.06Magnesium (mg/L Mg)   16pH (pH units [25°C])   6.1Sodium (mg/L Na)   43Total hardness (mg/L CaCO 3 )   137Turbidity (NTU)   0.1 set of samples was taken for further analysis. Bacterial enumeration Bacterial samples were taken from each flask after 7 and 24 h of exposure to SUVR. The 7 h exposure time was selected because itis the optimal time for the SODIS technique. The 24 h period wasused to assess for bacterial regrowth or increase in bacterial load.The samples were serially diluted in sterile 1x PBS and plated onnutrient agar using a slightly modified version of the Miles and Misradrop count technique (Miles et al., 1938). Briefly 10 µl of theappropriate dilution was dropped onto sterile nutrient agar plates inquadruplicate. The plates were then incubated at 37°C for 18 to 20h and those plates with less than 50 discrete colonies per dropwere selected and counted. The total count was divided by thenumber of drops, multiplied by 100 to convert to 1 ml, and thendivided by the dilution factor to give the number of CFU/ml. Flow cytometric measurements The effect of a 7 and 24 h exposure on the integrity of  V. cholerae cells was assessed only on samples exposed on the 24th of November 2009 during summer using a combination of twofluorescent dyes: Sybr Green I (SGI) with a final concentration of 1x(S940 Sigma-Aldrich, St. Louis, USA) and Propidium Iodide (PI)with final concentration of 3 μM (81845 Fluka Sigma -Aldrich, St.Louis, USA). All the samples where incubated in the dark at 37°Cfor 45 min before analysis on the Easy-Cyte Plus flow cytometer (Guava Technologies, Billerica, MA, USA) with an excitation of 488nm from a laser. Prior to flow cytometric analysis all the microbialsamples were diluted to 10 -5 CFU/ml with filter (0.22 μm pore) sterilised 1x PBS (in house) and the flow cytometer was set toacquire ten thousand events per sample; each sample was done induplicate. The optical filters of the flow cytometer were set such thatPI was measured at 590 nm while SGI at 520 nm. Greenfluorescence histograms were used to ascertain the effect of SUVRon the integrity of the cell membrane. This criterion was usedbecause red fluorescence intensity could only account for PIpositive events (cells with ruptured cell walls) while the rest of theevents (PI negative) would have been registered as backgroundregardless of the physiological state of the cells. Thus by usinggreen fluorescent intensity (due to SGI) it was possible to observethe quenching effect (due to fluorescence resonance energytransfer) typical of the presence or entry of PI (Berney et al., 2006a,2007).Flow cytometric data was collected using the CytoSoft Data Acquisition and Analysis Software (Guava software) (Version 3.6)and analysed with the same software or WinMDI software (version2.9) where necessary . RESULTSSeasonal temperature and SUVR The temperature and solar radiation due to UVA andUVB was captured between the 19th of May 2009 and31st of July 2010. The daily maximum temperature andsolar UVA and UVB were extracted from this data andgrouped according to the four seasons as experienced inSouth Africa and defined according to South Africa Info(2012); spring (August to mid-October), summer (mid-October to mid-February), autumn (mid-February to April)and winter (May to July). The average maximumtemperature and irradiance due to UVA and UVB wasthen calculated for each season (Table 2). The resultsshowed that the amount of SUVR and temperature wasprimarily dependent on the season of the year followedby the solar conditions of the day. A higher SUVR andtemperature was recorded during summer and autumn asopposed to that recorded during spring and winter (Figure1). The highest amount of solar UVA and UVB wasgenerally recorded at midday or 1:00 pm and radiancedue to UVA was always greater than that due to UVB(Figure 1). Exposure of  V. cholerae  to SUVR In spring, SODIS was done on three different days (Table3) during which the average maximum SUVR fluence dueto UVA was 594 KJ/m 2 while the temperature ranged    Table 2. Mean and standard deviations of the maximum UVA, UVB and ambient temperature for the different seasons for the periodbetween 19 May 2009 and 31 July 2010. Seasons Year N 1  Maximum UVA (W/m 2 ) & σ Maximum UVB (W/m 2 ) & σ Maximum temp (° C) & σ   Winter    2009 63 24.67 ± 3.86 0.08 ± 0.02 16.62 ± 2.67Spring   2009 78 30.92 ± 5.15 0.10 ± 0.02 21.90 ± 4.95Summer    2009/2010 57 45.79 ± 10.89 0.14 ± 0.04 27.04 ± 5.23 Autumn   2010 24 36.94 ± 6.78 0.09 ± 0.03 25.46 ± 2.98Winter    2010 71 23.02 ± 4.41 0.05 ± 0.01 18.36 ± 3.07 1 Number of days analyzed during the season.      U   V   A   (   W   /  m    2    )  a  n   d   t  e  m  p  e  r  a   t  u  r  e   (   °   C   ) Number of days /seasons of the year  Figure 1. Daily maximum levels of UVA (black line), UVB (green line) solar radiance (W/m 2 ) andtemperature (Red line) (°C) for the period between the 19 th of May 2009 and 31 st of July 2010. from 8 to 36°C. On the two sunny days, the pathogenicstrains showed a different culturability response after 7 hof exposure (Table 3). On the cloudy day, a 7 h exposuredid not result in a total loss in the culturability of any of the V. cholerae strains such as that seen during thesunny days. Although the loss of culturability duringspring was strain specific, the trend suggests that it wasdependent on UVA fluence and temperature. Duringsummer and autumn the inactivation of all three V.cholerae strains followed a similar trend where 7 h of solar exposure was sufficient to bring about the total lossin culturability of all the strains without regrowth (Table 3).This inactivation is perhaps due to the high UVA radiationand higher minimum temperatures (18 to 21°C) that werereceived during both seasons of the year. In winter, themaximum UVA fluence ranged from 512.87 to 686.71KJ/m 2 on the three different days of exposure while theminimum and maximum temperatures ranged from 3 to30°C. It was noticed that SUVR due to UVA was at itslowest regardless of the solar conditions (Figure 1) incomparison to the other seasons. However, 7 h of exposure was sufficient to render the pathogenic strainsof  V. cholerae non-culturable without regrowth (Table 3).The environmental strain unlike the pathogenic serotypeswas not totally inactivated on two of the three days (Table3). Total loss of culturability in the pathogenic strainsduring winter could have probably been enhanced by thelow temperatures. Flow cytometric analysis Flow cytometric results showed two major peaks in all thehistograms of all the three V. cholerae strains; the peakswere more pronounced on cultures that were exposedto SUVR (Figure 2). The first peak (Figure 2) with    Table 3. Culturability of  V. cholerae after exposure to SUVR during the four seasons of the year in Pretoria, South Africa. Resultant Log 10 CFU/ml for each strain and duration of solar exposureSeason Date of exposureSolar conditionsUVA radiation(KJ/m 2 )*Min and Maxtemp (°C)ExposureO1 O139 10097 h   24 h   0 h 7 h 24 h 0 h 7 h 24 h 0 h 7 h 24 hSpring25 August 2009 Sunny 301.41 675.22 8 -- 26Sample 7.43 ± 0.07 4.15 ± 0.21 0 6.78 ± 0.09 0 0 7.61 ± 0.13 2.59 ± 0.16 2.30 ± 0.17Control 7.43 ± 0.07 7.42 ± 0.02 4.70 ± 0.04 6.78 ± 0.10 5.83 ± 0.04 3.11 ± 0.05 7.61 ± 0.13 7.75 ± 0.01 5.08 ± 0.0515 September 2009 Sunny 314.93 668.19 17 -- 30Sample 7.52 ± 0.01 0 0 7.71 ± 0.02 2.00 ± 0.06 1.14 ± 0.06 7.83 ± 0.04 2.60 ± 0.02 2.00 ± 0.04Control 7.52 ± 0.01 7.42 ± 0.02 7.20 ± 0.10 7.71 ± 0.02 7.43 ± 0.02 8.20 ± 0.12 7.83 ± 0.04 7.79 ± 0.01 7.57 ± 0.1229 September 2009 Cloudy a 189.72 438.55 11-- 25Sample 7.57 ± 0.01 4.30 ± 0.21 2.74± 0.06 7.36 ± 0.03 3.22 ± 0.02 2.15± 0.21 7.70 ± 0.01 3.40 ± 0.06 4.14 ± 0.71Control 7.57 ± 0.01 7.60 ± 0.02 7.57 ± 0.16 7.36 ± 0.04 7.35 ± 0.04 8.59 ± 0.16 7.70 ± 0.01 7.57 ± 0.00 7.60 ± 0.02Summer 24 November 2010 Sunny 814.77 1347.74 21-- 35Sample 7.48 ± 0.10 0 0 8.09 ± 0.12 0 0 7.59 ± 0.16 0 0Control 7.48 ± 0.10 7.74 ± 0.37 7.00 ± 0.12 8.09 ± 0.12 8.54 ± 0.07 7.85 ± 0.00 7.59 ± 0.16 8.66 ± 0.07 8.25 ± 0.1902 February 2010 Sunny 671.25 1261.7 25 -- 37Sample 8.67 ± 0.06 0 0 8.16 ± 0.02 0 0 8.66 ± 0.04 0 0Control 8.67 ± 0.06 8.82 ± 0.02 6.65 ± 0.03 8.16 ± 0.02 8.51 ± 0.03 8.43 ± 0.09 8.66 ± 0.04 8.84 ± 0.05 8.58 ± 0.05 Autumn16 March 2010 Sunny 524.05 1163.74 23 -- 37Sample 7.11 ± 0.05 0 0 7.48 ± 0.03 0 0 7.53 ± 0.02 0 0Control 7.11 ± 0.06 6.39 ± 0.12 6.48 ± 0.16 7.48 ± 0.03 4.80 ± 0.04 4.95 ± 0.07 7.53 ± 0.02 6.84 ± 0.09 7.00 ± 0.1213 April 2010 Sunny 460.98 903.46 18 -- 34Sample 3.45 ± 0.05 0 0 4.70 ± 0.03 0 0 6.81 ± 0.05 0 0Control 3.45 ± 0.05 2.95 ± 0.06 5.02 ± 0.25 4.70 ± 0.03 2.54 ± 0.38 5.50 ± 0.16 6.81 ± 0.05 6.60 ± 0.00 6.50 ± 0.28Winter 18 May 2010 Sunny 376.71 686.71 7 -- 30Sample 7.43 ± 0.07 0 0 7.51 ± 0.10 0 0 7.56 ± 0.02 2.60 ± 0.27 0Control 7.43 ± 0.07 6.70 ± 0.02 7.13 ± 0.02 7.51 ± 0.10 6.70 ± 0.00 6.93 ± 0.04 7.56 ± 0.02 7.40 ± 0.05 7.35 ± 0.0108 June 2010Partially b  sunny314.59 506.82 7 -- 26Sample 7.38 ± 0.03 0 0 6.93 ± 0.04 0 0 7.63 ± 0.02 3.26 ± 0.00 3.16 ± 0.29Control 7.38 ± 0.03 7.02 ± 0.09 7.19 ± 0.02 6.93 ± 0.04 6.39 ± 0.12 6.77 ± 0.30 7.63 ± 0.02 6.87 ± 0.04 7.45 ± 0.0220 July 2010Partiallysunny241.42 512.87 3 -- 24Sample 7.35 ± 0.04 0 0 7.44 ± 0.01 0 0 7.45 ± 0.02 2.60 ± 0.06 3.16 ± 0.29Control 7.35 ± 0.04 7.19 ± 0.06 7.04 ± 0.06 7.44 ± 0.01 7.40 ± 0.04 7.36 ± 0.03 7.45 ± 0.02 9.04 ± 0.06 8.35± 0.49 * Radiation (KJ/m 2 ) here is the cumulative SUVR dose the exposed samples received throughout the noted duration of exposure. a Fully cloudy day with no clear sunshine. b Day with intermittent fullsunshine. fluorescence intensity between 10 0 and 10 1 corresponded to the peak represented byunstained microbial cells or cellular debris. Thesecond peak was dependent on whether thesample was exposed to SUVR (Figure 2). All solar exposed samples exhibited a second peakbetween 10 1 and 10 2 of fluorescence intensitycorresponding to partially damaged microbial cells(Figure 2). On the other hand all the non-exposedsamples had their second peaks between 10 2 and10 3 of fluorescence intensity. After 24 h of exposure there was a great increase in the
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