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 ultraviolet radiation and ambient temperature on the culturability of toxigenic and non- toxigenic Vibrio cholerae in Pretoria, South Africa 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 cholera, the potential impact that this technology can have in resource poor areas is increasingly being considered as a potential component in water treatment 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 hygiene. 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 drinking water 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 sunlight. 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 (wavelength range 290 320 nm). The UV data was recorded hourly cholerae. The integrity of the cell membrane of the 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).