African Journal of Microbiology Research Vol. 6(30), pp. 5957-5964, 9 August, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.601 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

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

C. C. Ssemakalu1, 2, M. Pillay1,2 and E. Barros3*

1Department of Biosciences, Vaal University of Technology, Vanderbijlpark 1900, South Africa. 2Previous Address: Department of Life Sciences, College of Agriculture, University of South Africa, Florida Campus, South Africa. 3Council 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 like , the potential impact that this technology can have in resource poor areas is increasingly being considered as a potential component in for poor and rural communities and as a means to 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 ultraviolet radiation (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 to SUVR. 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 to render the bacteria unculturable, due to higher SUVR levels and temperature observed in these seasons. However, the results also indicated that in winter where the levels of SUVR were comparable to those in spring the extreme variation in the daily recorded ambient temperatures [± 3°C – ± 30°C] may have contributed to the observed disinfection.

Key words: Solar disinfection, solar ultraviolet radiation, Vibrio cholerae, cholera.

INTRODUCTION

Cholera, a disease well known for its life threatening pathogenesis of cholera. Both these serotypes have been secretory diarrhoea characterized by several, capacious shown to carry virulence factors (Aoki et al., 2009; watery stools, often accompanied by vomiting is a Hoshino et al., 1998) expressed by two genetic elements: waterborne disease that has infected thousands of CTXф which is responsible for the production of cholera people resulting in high mortality rates (Osei and Duker, toxin (CT) the causative agent of cholera and the VPI 2008; WHO, 2006, 2011). Vibrio cholerae the causative pathogenicity island required for the entry of CTXф. agent of cholera is a Gram negative micro-organism that The African continent is privileged with the availability exists naturally within the aquatic environment (Merrell et of freshwater sources such as lakes, rivers, ponds, al., 2000). To date, two serotypes of V. cholerae that is swamps, dams and boreholes which are essential in (O1 and O139) are known to play an important role in the meeting the basic needs of the people such as drinking, cooking and . However, the inability to protect these water sources have made them modes of transmission of waterborne diseases such as cholera in *Corresponding author. E-mail: [email protected]. Tel: (+27) various African communities (WHO, 2006, 2011). One of 12 841 3221. Fax: +27 12 841 3651. the millennium development goals is to provide resource 5958 Afr. J. Microbiol. Res.

poor communities such as those found in Africa with (Chaiyanan et al., 2001). access to clean potable water by the year 2015 (Rosellini and Pimple, 2010). Although a great effort has been geared towards achieving this goal its realization does MATERIALS AND METHODS not appear to be within reasonable reach (WHO, 2011) Bacterial strains as, more than half a billion people are still lacking access to and even more are without sanitation The two toxigenic V. cholerae strains used in this study were facilities (WHO, 2012). In South Africa as well as in other serotypes O1 (NCTC 5941) and O139 (NCTC 12945) obtained from African countries sporadic cholera outbreaks due to the the national collection of type cultures. The non-toxigenic consumption of untreated water have been reported in environmental V. cholerae strain 1009 was isolated from the Vaal rural and informal settlements (Mugero and Hoque, River in South Africa (Du Preez et al., 2010). All strains were stored at -80°C as bacterial stocks on beads. 2001). The problem is aggravated by the consumption of untreated microbiologically contaminated water or water that is treated and stored inappropriately (Firth et al., Growth media and growth conditions 2010; Rufener et al., 2010). As an intervention solar ultraviolet radiation (SUVR), a priceless component of the Bacterial suspensions were prepared by spreading 3 beads from each of the frozen bacterial stocks onto nutrient agar plates and sun energy, has been used to treat water through a incubating them for 18 h at 37°C. A colony of each strain was then process known as solar disinfection (SODIS) (Berney et streaked onto a fresh nutrient agar plate and incubated at 37°C al., 2006b; Smith et al., 2000; Ubomba-Jaswa et al., overnight. Following this, each V. cholerae strain was inoculated 2008). During SODIS treatment, bacterial inactivation has into autoclaved Luria Broth (LB) at pH 8.5 and incubated at 37°C been shown to occur through a synergy between SUVR with agitation at 200 revolutions per minute (rpm) overnight till they reached the stationary phase. Stationary phase cultures were used and an increase in water temperature (above 45°C) for solar exposures due to their resilience (Berney et al., 2006a). (Boyle et al., 2008; Navntoft et al., 2008; Ubomba-Jaswa Bacterial suspensions were harvested by centrifugation at 7000 × g et al., 2008). Clearly, the process through which SODIS for 10 min. The pelleted bacteria were re-suspended in 10 ml filter occurs seems quite simple and straight forward. sterilized 1x phosphate buffer saline (PBS) at pH 7.5. Centrifugation However, the underlying mechanisms are more and re-suspension was repeated three times to facilitate an almost complicated in that various factors such as SUVR, complete removal of LB. The resultant bacterial suspensions were diluted in 15 ml of autoclaved ground water obtained from temperature, location and the type of container or vessel Soshanguve, Pretoria (chemical analysis shown in Table 1), to an used are major determinants of the outcome. SUVR has OD600 of 0.01 corresponding to 7 or 8 Log colony forming units per been shown to successfully inactivate the culturability of millilitre (Log CFU/ml) before exposure to . enteropathogenic Escherichia coli, viruses such as poliovirus and giardia cysts which are known to survive in Exposure to natural solar radiation aquatic environments (Heaselgrave et al., 2006; Ubomba-Jaswa et al., 2008). Fifteen millilitres of each V. cholerae strain were transferred to The consumption of pathogen free water throughout transparent polystyrene 25 cm3 unventilated tissue culture flasks. the year is critical in the fight against waterborne disease The samples were gently hand- shaken and allowed to stand for 10 outbreaks and epidemics. Therefore it is important to to 15 min to allow the bacterial cells to adapt to the water. The samples were then exposed to natural sunlight by placing them on assess the applicability of using SODIS in African the roof top of the Council for Scientific and Industrial Research countries like South Africa, Lesotho and Swaziland that (CSIR) building in Pretoria (lat. 25°44’50.40”S; long. 28°16’50.50”E) experience four defined seasons of the year. The at an elevation of 1.4 km above sea level. The control samples objective of this study was to empirically determine the were prepared in a similar manner, placed on the roof top and pertinence of using SUVR to disinfect V. cholerae protected from direct sunlight by covering them with an opaque contaminated water during the different seasons ventilated cardboard box. All the samples were exposed for a 24 h period from 6:00 am to 6:00 am the following day. SUVR was experienced in South Africa. To achieve this objective a measured with two UV meters (Solar Light Co., Philadelphia, PA, culture based method was used to provide insight into the USA) that were placed on the rooftop next to the samples. One of extent of culturability changes of three V. cholerae strains UV meters (model 10, serial number 14056) measured radiance when exposed to natural SUVR. These included two due to UVA (wavelength range 320 to 400 nm), while the other toxigenic strains and one non-toxigenic strain of V. (model 4, serial number 14085) measured radiance due to UVB cholerae. The integrity of the cell membrane of the (wavelength range 290 320 nm). The UV data was recorded hourly per day for each season by each UV probe and downloaded from microorganisms was also assessed using a flow the PMA-2100 data logger via a computer (Solar Light Co.). The cytometer after the summer exposure. The use of flow UVA data was recorded in W/m2 while the UVB data was recorded cytometry was motivated by its ability to denote the as μW/cm2 and then converted to W/m2. In addition, the cumulative different cellular states of a bacterial culture and V. UVA and UVB doses (radiation) received after 7 and 24 h were cholerae has been shown to exist in a viable but non recorded from the PMA data logger. The hourly ambient temperature data was acquired from a weather station located in culturable (VBNC) state when stressed (Falcioni et al., Meyers Park (less than 6 km from CSIR) (Weather Underground, 2008). The VBNC state that V. cholerae can exhibit has 2010). A temperature probe was also placed next to the point of been shown to play a role in the epidemiology of cholera exposure and the ambient air temperature was taken each time a Ssemakalu et al. 5959

Table 1. The physiochemical properties of ground water from Soshanguve.

Physiochemical property (Units) Value

Alkalinity (mg/L CaCO3) 25 Ammonia nitrogen (mg/L N) <0.1 Calcium (mg/L Ca) 29 Chloride (mg/L Cℓ) 70 COD (mg/L COD) <10 Elect Conductivity (mS/m [25°C]) 56.9 Iron (mg/L Fe) <0.06 Magnesium (mg/L Mg) 16 pH (pH units [25°C]) 6.1 Sodium (mg/L Na) 43

Total hardness (mg/L CaCO3) 137 Turbidity (NTU) 0.1

set of samples was taken for further analysis. transfer) typical of the presence or entry of PI (Berney et al., 2006a, 2007). Flow cytometric data was collected using the CytoSoft Data Bacterial enumeration Acquisition and Analysis Software (Guava software) (Version 3.6) and analysed with the same software or WinMDI software (version Bacterial samples were taken from each flask after 7 and 24 h of 2.9) where necessary. exposure to SUVR. The 7 h exposure time was selected because it is the optimal time for the SODIS technique. The 24 h period was used to assess for bacterial regrowth or increase in bacterial load. The samples were serially diluted in sterile 1x PBS and plated on RESULTS nutrient agar using a slightly modified version of the Miles and Misra drop count technique (Miles et al., 1938). Briefly 10 µl of the Seasonal temperature and SUVR appropriate dilution was dropped onto sterile nutrient agar plates in quadruplicate. The plates were then incubated at 37°C for 18 to 20 The temperature and solar radiation due to UVA and h and those plates with less than 50 discrete colonies per drop were selected and counted. The total count was divided by the UVB was captured between the 19th of May 2009 and number of drops, multiplied by 100 to convert to 1 ml, and then 31st of July 2010. The daily maximum temperature and divided by the dilution factor to give the number of CFU/ml. solar UVA and UVB were extracted from this data and grouped according to the four seasons as experienced in South Africa and defined according to South Africa Info Flow cytometric measurements (2012); spring (August to mid-October), summer (mid-

The effect of a 7 and 24 h exposure on the integrity of V. cholerae October to mid-February), autumn (mid-February to April) cells was assessed only on samples exposed on the 24th of and winter (May to July). The average maximum November 2009 during summer using a combination of two temperature and irradiance due to UVA and UVB was fluorescent dyes: Sybr Green I (SGI) with a final concentration of 1x then calculated for each season (Table 2). The results (S940 Sigma-Aldrich, St. Louis, USA) and Propidium Iodide (PI) showed that the amount of SUVR and temperature was with final concentration of 3 μM (81845 Fluka Sigma-Aldrich, St. primarily dependent on the season of the year followed Louis, USA). All the samples where incubated in the dark at 37°C for 45 min before analysis on the Easy-Cyte Plus flow cytometer by the solar conditions of the day. A higher SUVR and (Guava Technologies, Billerica, MA, USA) with an excitation of 488 temperature was recorded during summer and autumn as nm from a laser. Prior to flow cytometric analysis all the microbial opposed to that recorded during spring and winter (Figure samples were diluted to 10-5 CFU/ml with filter (0.22 μm pore) 1). The highest amount of solar UVA and UVB was sterilised 1x PBS (in house) and the flow cytometer was set to generally recorded at midday or 1:00 pm and radiance acquire ten thousand events per sample; each sample was done in duplicate. The optical filters of the flow cytometer were set such that due to UVA was always greater than that due to UVB PI was measured at 590 nm while SGI at 520 nm. Green (Figure 1). fluorescence histograms were used to ascertain the effect of SUVR on the integrity of the cell membrane. This criterion was used because red fluorescence intensity could only account for PI Exposure of V. cholerae to SUVR positive events (cells with ruptured cell walls) while the rest of the events (PI negative) would have been registered as background regardless of the physiological state of the cells. Thus by using In spring, SODIS was done on three different days (Table green fluorescent intensity (due to SGI) it was possible to observe 3) during which the average maximum SUVR fluence due 2 the quenching effect (due to fluorescence resonance energy to UVA was 594 kJ/m while the temperature ranged 5960 Afr. J. Microbiol. Res.

Table 2. Mean and standard deviations of the maximum UVA, UVB and ambient temperature for the different seasons for the period between 19 May 2009 and 31 July 2010.

1 2 2 Seasons Year N X Maximum UVA (W/m ) and σ Maximum UVB (W/m ) and σ Maximum temp (°C) and σ Winter 2009 63 24.67 ± 3.86 0.08 ± 0.02 16.62 ± 2.67 Spring 2009 78 30.92 ± 5.15 0.10 ± 0.02 21.90 ± 4.95 Summer 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 ± 2.98 Winter 2010 71 23.02 ± 4.41 0.05 ± 0.01 18.36 ± 3.07

1Number of days analyzed during the season.

C)

°

) and temperature ( temperature ) and

2 UVA (W/m UVA

Number of days /seasons of the year

Figure 1. Daily maximum levels of UVA (black line), UVB (green line) solar radiance (W/m2) and temperature (Red line) (°C) for the period between the 19th of May 2009 and 31st of July 2010.

from 8 to 36°C. On the two sunny days, the pathogenic 30°C. It was noticed that SUVR due to UVA was at its strains showed a different culturability response after 7 h lowest regardless of the solar conditions (Figure 1) in of exposure (Table 3). On the cloudy day, a 7 h exposure comparison to the other seasons. However, 7 h of did not result in a total loss in the culturability of any of exposure was sufficient to render the pathogenic strains the V. cholerae strains such as that seen during the of V. cholerae non-culturable without regrowth (Table 3). sunny days. Although the loss of culturability during The environmental strain unlike the pathogenic serotypes spring was strain specific, the trend suggests that it was was not totally inactivated on two of the three days (Table dependent on UVA fluence and temperature. During 3). Total loss of culturability in the pathogenic strains summer and autumn the inactivation of all three V. during winter could have probably been enhanced by the cholerae strains followed a similar trend where 7 h of low temperatures. solar exposure was sufficient to bring about the total loss in culturability of all the strains without regrowth (Table 3). This inactivation is perhaps due to the high UVA radiation Flow cytometric analysis and higher minimum temperatures (18 to 21°C) that were received during both seasons of the year. In winter, the Flow cytometric results showed two major peaks in all the maximum UVA fluence ranged from 512.87 to 686.71 histograms of all the three V. cholerae strains; the peaks KJ/m2 on the three different days of exposure while the were more pronounced on cultures that were exposed minimum and maximum temperatures ranged from 3 to to SUVR (Figure 2). The first peak (Figure 2) with Ssemakalu et al. 5961

Table 3. Culturability of V. cholerae after exposure to SUVR during the four seasons of the year in Pretoria, South Africa.

2 Solar UVA radiation (kJ/m )* Season Date of exposure Min and Max temp (°C) Exposure conditions 7 h 24 h Sample 25 August 2009 Sunny 301.41 675.22 8 -- 26 Control

Sample Spring 15 September 2009 Sunny 314.93 668.19 17 - 30 Control

Sample 29 September 2009 Cloudya 189.72 438.55 11-- 25 Control

Sample 24 November 2010 Sunny 814.77 1347.74 21-- 35 Control Summer Sample 02 February 2010 Sunny 671.25 1261.7 25 -- 37 Control

Sample 16 March 2010 Sunny 524.05 1163.74 23 -- 37 Control Autumn Sample 13 April 2010 Sunny 460.98 903.46 18 -- 34 Control

Sample 18 May 2010 Sunny 376.71 686.71 7 -- 30 Control

Partiallyb Sample 08 June 2010 314.59 506.82 7 -- 26 sunny Control Winter Sample Partially 20 July 2010 241.42 512.87 3 -- 24 sunny Control

Table 3. Continued.

Resultant Log10 CFU/ml for each strain and duration of solar exposure Season O1 O139 1009 0 h 7 h 24 h 0 h 7 h 24 h 0 h 7 h 24 h

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 ± 0.17

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 ± 0.05 Spring

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 ± 0.04

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 ± 0.12

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 ± 0.71 5962 Afr. J. Microbiol. Res.

Table 3. Continued.

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 ± 0.02

7.48 ± 0.10 0 0 8.09 ± 0.12 0 0 7.59 ± 0.16 0 0 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 ± 0.19 Summer 8.67 ± 0.06 0 0 8.16 ± 0.02 0 0 8.66 ± 0.04 0 0

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

7.11 ± 0.05 0 0 7.48 ± 0.03 0 0 7.53 ± 0.02 0 0

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 ± 0.12

Autumn 3.45 ± 0.05 0 0 4.70 ± 0.03 0 0 6.81 ± 0.05 0 0 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 ± 0.28

7.43 ± 0.07 0 0 7.51 ± 0.10 0 0 7.56 ± 0.02 2.60 ± 0.27 0

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 ± 0.01

7.38 ± 0.03 0 0 6.93 ± 0.04 0 0 7.63 ± 0.02 3.26 ± 0.00 3.16 ± 0.29

7.38 ± 0.03 Winter 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 ± 0.02

7.35 ± 0.04 0 0 7.44 ± 0.01 0 0 7.45 ± 0.02 2.60 ± 0.06 3.16 ± 0.29 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/m2) 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 full sunshine.

fluorescence intensity between 100 and 101 from the second peak (Figure 2). corresponded to the peak represented by unstained microbial cells or cellular debris. The second peak was dependent on whether the sample was exposed to SUVR DISCUSSION (Figure 2). All solar exposed samples exhibited a second peak between 101 and 102 of fluorescence intensity corresponding to partially damaged microbial cells In this study the effect of SUVR on the viability of both (Figure 2). On the other hand all the non-exposed toxigenic and non-toxigenic strains of V. cholerae was samples had their second peaks between 102 and 103 of evaluated during the different seasons of the year in fluorescence intensity. After 24 h of exposure there Pretoria, South Africa. The data was correlated to the was a great increase in the number of events in the mean of UVA and UVB radiation as well as to the first peak that corresponded to the reduction in events average temperature. Ssemakalu et al. 5963

Figure 2. Superimposed histograms of green fluorescence intensity (GRN-HLog) resulting from the flow cytometric analysis of cultures of V. cholerae serotypes O1, O139 and 1009 grown in ground water and exposed to 7 and 24 h SUVR (solid line) and non-SUVR exposed cultures (dotted line). The cells were stained with a mixture of SGI and PI fluorescent dyes.

Seasonal temperature and SUVR Exposure of V. cholerae to SUVR

This study showed that the amount of SUVR and the The effect of the weather conditions played an important ambient temperature recorded at the point of exposure role in the inactivation of V. cholerae (Table 3). Although were dependent on the season of the year (Figure 1) and in spring the conditions were ineffective in inducing the were influenced by the solar conditions. Cloudy total loss of culturability in both the pathogenic and (overcast) days as well as partially cloudy (intermitted environmental strains of V. cholerae (Table 3) our results sunshine) days shielded the amount of SUVR reaching showed that there was a 3 to 5 Log10 CFU/ml reduction in the point of exposure. The results demonstrated that the number of V. cholerae. In their natural habitats these reduced SUVR and temperature did not result in the total microorganisms exist in concentrations less than 4 logs loss of culturability of the V. cholerae strains (Table 3). It before a waterborne disease outbreak (Boyle et al., appears that total inactivation of V. cholerae under these 2008). One of the ways of increasing the loss of conditions could be attained by increasing the exposure culturability of these microorganisms is to perhaps times. Besides the amount of SUVR, ambient increase the time of exposure to SUVR. Therefore for temperature also influenced the viability of the solar safety reasons if the bacterial load is as high as that used exposed cultures. There was enhanced inactivation of the in this study it would be advisable to expose the water for toxigenic strains of V. cholerae during winter than in a minimum of 36 h during spring as well as in winter. spring. Although similar SUVR fluencies were recorded in During summer and autumn, 7 h of SUVR on a sunny spring and winter, the only observable difference day was sufficient for the total inactivation of all the V. between these two seasons was the lower temperature cholerae strains. This was perhaps due to the synergistic recorded in the winter (Table 3). This suggests that either effect between the high SUVR and temperatures that the lower temperatures could act in synergy with SUVR in were recorded for these seasons. Similar findings were bringing about inactivation of the microorganisms or the reported in Boyle et al. (2008) and Ubomba-Jaswa et al. extreme variation in the daily temperature from ±3 and ± (2008). Besides climatic factors, perhaps other inherent 25°C could also impact on the survival of V. cholerae. factors within the toxigenic strains made them more 5964 Afr. J. Microbiol. Res.

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