1. is an efficient means of disinfecting drinking water contaminated with Shigella Dysenteriae type I (Figure 1. )

2. Goal of the Study  This paper investigates the possibility of using batch process solar disinfection (SODIS) as an effective means of disinfecting drinking water contaminated with Shigella dysenteriae type I (Figure 2.)

3. Motivation for This Work (People Problem)  The mortality and morbidity rate caused by Shigella dysenteriae type I infection is increasing in the developing countries each year.  Today, bacillary dysentery is endemic throughout the world with 150 million cases, and almost 600,000 deaths annually. About 95% of these cases occurred in developing countries.  Low infective dose (as little as 10 cells ) together with emergence of antimicrobial resistance make it more difficult to control the spread and treatment.  Provision of good quality drinking water does not influence the incidence of bacillary dysentery. (Figure 3.)

4. Motivation for This Work (technical problem)  To control all potential routes of transmission of Sh. Dysenteriae type I is desirable.  SODIS may be an effective means of disinfecting drinking water contaminated with Sh. dysenteriae type I.  SODIS takes advantage of one of the most abundant source of energy, natural sunlight: - Environmental friendly; - Economical; - Possible to control bacillary dysentery - Bactericidal effect due to optical and thermal process and syner gistic effect (Figure 4.)

5. More about Sunlight’s Bactericidal Effect  In addition to direct u.v. killing, sunlight is absorbed by endogenous (e.g. cytochromes) and exogenous (e.g. humic substances) photosensitizers that then react with oxygen, producing highly reactive oxygen molecules such as hydrogen peroxide (H2O2), singlet oxygen and superoxides which exert a bactericidal effect  Most bacterial strains produce catalase in response to hydrogen peroxide. However, Sh. dysenteriae type I does not produce a catalase that is detected by standard methods and thus may be more sensitive to batch process SODIS. (Figure 5.)

6. Materials and Methods  4 different types of strains were used: Sh. dysenteriae type I, V. cholerae 8021, Salmonella typhimurium, and Sh. Flexneri.  Each strain was collected in a 100ml sterile nutrient broth, and then incubated to 37c for 18h to obtain a culture.  Cells were then extracted by centrifugation for 10min. And washed 3 time with sterile water to remove nutrients.  The remaining substance was re-suspended in sterile phosphate- buffered saline (PBS), pH7.3, to a final concentration of 10^6 CFU/ml.  The authors chose to conduct all tests with the cells suspended in PBS because the organisms were found to be more unstable in H2O than PBS.

7. Methods Continued… The authors chose to conduct all tests with the cells suspended in PBS because the organisms were found to be more unstable in H2O than PBS. A 150W Xenon arc lamp and heat-absorbing filter were used to simulate solar irradiation. First all four strains were exposed to irradiance of 87 mW/cm^2, which simulates conditions at the equator at sea level. But the two Shagella strains inactivated too quickly for calculation of the kinetics, so the irradiance for those two were reduced to 42 mW/cm^2, similar to an overcast day in Kenya. In the field trials of a previous study of SODIS, bottles of water were placed on the roof and reached temperatures of 40-55C,while a control group was kept in a dark room. So in this study, test samples were kept at 42C (Shagella strains) and 45C (other strains) and a control solution was kept in a dark room.

8. Methods continued… Vol. of 100ul were taken from each test and control samples. These samples were diluted in 10 fold dilutions and placed on either standard plate count agar (SPCA) or agar plates supplemented with catalase or pyruvate. Following incubation, solar decay constants were calculated from the slope of the regression line vs cumulative dose. Plotting this gave us the differences between the 4 organisms… (Figure 6.)

9. Catalase Preparation  Catalase solutions were prepared by diluting in ice cold phosphate buffer solution  Solutions were filter sterilized with 0.2um membrane filters, and 0.5ml samples were transferred to the surface of standard agar plates for each organism.  Different species of catalase were applied to the plates, and the plate with the greatest efficiency was determined.  A solution of catalase which had been boiled for 10min acted as a control…

10. Pyruvate Preparation Pyruvate plates were prepared by direct application of Sodium Pyruvate prior to autoclaving. Different concentrations were observed for plating efficiency, and the greatest one determined. Glacial acetic acid acted as the control. Pyruvate usually acts an important energy source for cells, whereas catalase is not thought of as an energy source.

11. Assumptions and Uncertainties in methods.  The uv rays used from the Xenon arc lamp via the heated solar filter to simulate solar irradiation conditions present uncertainties, as we do not know the accurate output from it. We do not know if it will replicate the real conditions.  Also how much water can be treated this way is still undetermined, whether a large quantity or a small quantity.

12. Results  Solar inactivation of the four bacteria differed significantly  Sh. Dysenteriae was more sensitive to SODIS (batch process solar disinfection) than the other three bacteria  P value of experiment was 0.015  There was a 6 log reduction in CFUs after just a 1.5 hour exposure to simulated overcast conditions  Six hour exposure is required to inactivate Shigella flexneri (Figure 7.)

13. Results  When Sh. Dysenteriae was grown on SPCA ( standard plate count agar) 6 log units were completely inactivated after 1.5 hours  When the plate was supplemented with 0.05% pyruvate or 406 units of catalase, there was around 10 4 CFU ml -1 of Sh. Dysenteriae still active  For the other three bacteria, supplementation with pyruvate or catalase had little effect on inactivating the bacteria  Recovery of injured Sh Dysenteriae may be improved by supplementing the medium with pyruvate or catalase when plating. (Figure 8.)

14.  The results show that Shigella dysenteriae type I is sensitive to batch process SODIS, much more sensitive than the other bacteria.  A study done in Kenya reported that children under age 6 who drank solar disinfected water were protected from V. cholerae infection during an outbreak (Conroy et al. 2001).  The authors suggest that drinking solar disinfected water during a Sh. dysenteriae outbreak would protect against infection. Results show (Figure 9.)

15.  Figure 2 shows that it takes almost three times longer for SODIS to inactivate Sh. dysenteriae when it is grown on agar supplemented with pyruvate or catalase.  Table 1 shows that during the trials with pyruvate and catalase, Sh. dysenterae in simulated overcast conditions is still inactivated much faster than Salm. typhimurium and V. cholerae in simulated equatorial conditions. Effects of Medium Supplementation (Figure 10.)

16. Pyruvate vs. Catalase  SODIS causes photosynthesizers to produce peroxides, which are highly reactive and kill bacteria.  Pyruvate is an acid that neutralizes peroxides and it is also a source of energy for cells.  Catalase is an enzyme that breaks down hydrogen peroxide, but it is not an energy source.  Since both pyruvate and catalase produce similar increases in the bacteria’s resistance to SODIS, the authors conclude that pyruvate’s mechanism is peroxide neutralization, not energy reserve.  For future testing of SODIS, the authors recommend medium supplementation with pyruvate, and not catalase. They have similar effects and catalase is very unstable at room temperature, making it difficult to work with. catalase (Figure 11.) (Figure 12.) (Figure 13.)

17. SODIS Proven Effective in Reducing Rate of Infection  Studies have shown that other species of solar treated bacteria which remained culturable on standard agar plates were still less infective than cells that had not been exposed to UV light (Smith et. al 2000 and Kehoe 2001).  The authors conclude that SODIS is an appropriate intervention during Sh. dysenteriae type I endemics in developing countries. While improvements in water quality and sanitation have not helped the spread of dysentery because of the low infective dose, SODIS appears to be a more effective approach. (Figure 14.) (Figure 15.)

18. Figures References (1). www.sodis.ch/Text2002/T-EducationMaterials.htm (2). http://www.sodis.ch/files/MaterialsIndonesia/SODISPoster.jpg. (3). http://knol.google.com/k/william-marler/about-hemolytic-uremic-syndrome/3474z7yug2xwi/17#. (4). www.envirotherm.ie/site/solar.php. (5). http://www.thewaterschool.org/images/15.jpg. (6). Batch process solar disinfection is an efficient means of disinfecting drinking water contaminated with Shigella dysenteriae type I by Kehoe, S., Barer, M., Devlin, R, and McGuigan, K. 2003 (7). Batch process solar disinfection is an efficient means of disinfecting drinking water contaminated with Shigella dysenteriae type I by Kehoe, S., Barer, M., Devlin, R, and McGuigan, K. 2003 (9). http://www.thewaterschool.org/blog/wp-content/uploads/2009/01/chilrentable.jpg (10). Batch process solar disinfection is an efficient means of disinfecting drinking water contaminated with Shigella dysenteriae type I by Kehoe, S., Barer, M., Devlin, R, and McGuigan, K. 2003 (11).http://bioengineering.ucsd.edu/seniordesign/Past_Designs/studentpages/2006/Project8/pyruvatese nsors-background_files/pyruvate.gif. (12).http://www.scripps.edu/news/sr/sr2005/images/olson.mb.fig5.gif (13).http://www.umesc.usgs.gov/aquatic/drug_research/hydrogen_peroxide/hydrogen_peroxide_structu re_400.gif. (14). http://www.motherearthnews.com/uploadedImages/Blogs/Energy_Matters/Sun.jpg