Abstract
Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m−2) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log10 after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log10 after 6 h) was obtained only in the presence of riboflavin and 250 W m−2 irradiance.
Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).
The combination of UVA radiation and riboflavin (vitamin B2) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.
Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.
B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).
Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 106/ml) were suspended in 3 ml of one-quarter-strength Ringer's solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m−2) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.
At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).
The efficacies of simulated sunlight at an optical output irradiance of 150 W m−2 alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table 1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table 1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).
TABLE 1.
Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)
| Organism | Conditiona | Log10 reduction in viability at indicated h of exposureb |
|||
|---|---|---|---|---|---|
| 1 | 2 | 4 | 6 | ||
| E. coli | SODIS | 0.0 ± 0.0 | 0.2 ± 0.1 | 5.7 ± 0.0 | 5.7 ± 0.0 |
| SODIS-R | 1.1 ± 0.0 | 5.7 ± 0.0 | 5.7 ± 0.0 | 5.7 ± 0.0 | |
| L. pneumophila | SODIS | 0.7 ± 0.2 | 1.3 ± 0.3 | 4.8 ± 0.2 | 4.8 ± 0.2 |
| SODIS-R | 4.4 ± 0.0 | 4.4 ± 0.0 | 4.4 ± 0.0 | 4.4 ± 0.0 | |
| P. aeruginosa | SODIS | 0.7 ± 0.0 | 1.8 ± 0.0 | 4.9 ± 0.0 | 4.9 ± 0.0 |
| SODIS-R | 5.0 ± 0.0 | 5.0 ± 0.0 | 5.0 ± 0.0 | 5.0 ± 0.0 | |
| S. aureus | SODIS | 0.0 ± 0.0 | 0.0 ± 0.0 | 6.2 ± 0.0 | 6.2 ± 0.0 |
| SODIS-R | 0.2 ± 0.1 | 6.3 ± 0.0 | 6.3 ± 0.0 | 6.3 ± 0.0 | |
| C. albicans | SODIS | 0.2 ± 0.0 | 0.4 ± 0.1 | 0.5 ± 0.1 | 1.0 ± 0.1 |
| SODIS-R | 0.1 ± 0.0 | 0.7 ± 0.1 | 5.3 ± 0.0 | 5.3 ± 0.0 | |
| F. solani conidia | SODIS | 0.2 ± 0.1 | 0.3 ± 0.0 | 0.2 ± 0.0 | 0.7 ± 0.1 |
| SODIS-R | 0.3 ± 0.1 | 0.8 ± 0.1 | 1.3 ± 0.1 | 4.4 ± 0.0 | |
| B. subtilis spores | SODIS | 0.3 ± 0.0 | 0.2 ± 0.0 | 0.0 ± 0.0 | 0.1 ± 0.0 |
| SODIS-R | 0.1 ± 0.1 | 0.2 ± 0.1 | 0.3 ± 0.3 | 0.1 ± 0.0 | |
| SODIS (250 W m−2) | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.1 ± 0.1 | 0.0 ± 0.0 | |
| SODIS-R (250 W m−2) | 0.0 ± 0.0 | 0.0 ± 0.0 | 0.2 ± 0.0 | 0.4 ± 0.0 | |
| SODIS (320 W m−2) | 0.1 ± 0.1 | 0.1 ± 0.0 | 0.0 ± 0.1 | 4.3 ± 0.0 | |
| SODIS-R (320 W m−2) | 0.1 ± 0.0 | 0.1 ± 0.1 | 0.9 ± 0.0 | 4.3 ± 0.0 | |
| A. polyphaga trophozoites | SODIS | 0.4 ± 0.2 | 0.6 ± 0.1 | 0.6 ± 0.2 | 0.4 ± 0.1 |
| SODIS-R | 0.3 ± 0.1 | 1.3 ± 0.1 | 2.3 ± 0.4 | 3.1 ± 0.2 | |
| SODIS, naturalc | 0.3 ± 0.1 | 0.4 ± 0.1 | 0.5 ± 0.2 | 0.3 ± 0.2 | |
| SODIS-R, naturalc | 0.2 ± 0.1 | 1.0 ± 0.2 | 2.2 ± 0.3 | 2.9 ± 0.3 | |
| A. polyphaga cysts | SODIS | 0.4 ± 0.1 | 0.1 ± 0.3 | 0.3 ± 0.1 | 0.4 ± 0.2 |
| SODIS-R | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.5 ± 0.1 | 0.8 ± 0.3 | |
| SODIS (250 W m−2) | 0.0 ± 0.1 | 0.2 ± 0.3 | 0.2 ± 0.1 | 0.1 ± 0.2 | |
| SODIS-R (250 W m−2) | 0.4 ± 0.2 | 0.3 ± 0.2 | 0.8 ± 0.1 | 3.5 ± 0.3 | |
| SODIS (250 W m−2), naturalc | 0.0 ± 0.3 | 0.2 ± 0.1 | 0.1 ± 0.1 | 0.2 ± 0.1 | |
| SODIS-R (250 W m−2), naturalc | 0.1 ± 0.1 | 0.2 ± 0.2 | 0.6 ± 0.1 | 3.4 ± 0.2 | |
Conditions are at an intensity of 150 W m−2 unless otherwise indicated.
The values reported are means ± standard errors of the means from triplicate experiments.
Additional experiments for this condition were performed using natural freshwater.
The highly resistant A. polyphaga cysts and B. subtilis spores were unaffected by SODIS or SODIS-R at an optical irradiance of 150 W m−2. However, a significant reduction in cyst viability was observed at 6 h when the optical irradiance was increased to 250 W m−2 for SODIS-R only (P < 0.001; Table 1). For spores, a kill was obtained only at 320 W m−2 after 6-h exposure, and no difference between SODIS and SODIS-R was observed (Table 1). Previously, we reported a >2-log kill at 6 h for Acanthamoeba cysts by using SODIS at the higher optical irradiance of 850 W m−2, compared to the 0.1-log10 kill observed here using the lower intensity of 250 W m−2 or the 3.5-log10 kill with SODIS-R.
Inactivation experiments performed with Acanthamoeba cysts and trophozoites suspended in natural freshwater gave results comparable to those obtained with Ringer's solution (P > 0.05; Table 1). However, it is acknowledged that the findings of this study are based on laboratory-grade water and freshwater and that differences in water quality through changes in turbidity, pH, and mineral composition may significantly affect the performance of SODIS (20). Accordingly, further studies are indicated to evaluate the enhanced efficacy of SODIS-R by using natural waters of varying composition in the areas where SODIS is to be employed.
Previous studies with SODIS under laboratory conditions have employed lamps delivering an optical irradiance of 850 W m−2 to reflect typical natural sunlight conditions (6, 11, 12, 15, 16). Here, we used an optical irradiance of 150 to 320 W m−2 to obtain slower organism inactivation and, hence, determine the potential enhancing effect of riboflavin on SODIS.
In conclusion, this study has shown that the addition of riboflavin significantly enhances the efficacy of simulated SODIS against a range of microorganisms. The precise mechanism by which photoactivated riboflavin enhances antimicrobial activity is unknown, but studies have indicated that the process may be due, in part, to the generation of singlet oxygen, H2O2, superoxide, and hydroxyl free radicals (10). Further studies are warranted to assess the potential benefits from riboflavin-enhanced SODIS in reducing the incidence of gastrointestinal infection in communities where potable water is not available.
Acknowledgments
This work was carried out as part of the European Union-funded SODIS water project, contract no. INCO-CT-2006-031650.
Footnotes
Published ahead of print on 16 July 2010.
REFERENCES
- 1.Blajchman, M. A., M. Goldman, and F. Baeza. 2004. Improving the bacteriological safety of platelet transfusions. Transfus. Med. Rev. 18:11-24. [DOI] [PubMed] [Google Scholar]
- 2.Conroy, R. M., M. Elmore-Meegan, T. Joyce, K. G. McGuigan, and J. Barnes. 1996. Solar disinfection of drinking water and diarrhoea in Maasai children: a controlled field trial. Lancet 348:1695-1697. [DOI] [PubMed] [Google Scholar]
- 3.Conroy, R. M., M. E. Meegan, T. Joyce, K. McGuigan, and J. Barnes. 2001. Solar disinfection of drinking water protects against cholera in children under 6 years of age. Arch. Dis. Child. 85:293-295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Conroy, R. M., M. E. Meegan, T. Joyce, K. McGuigan, and J. Barnes. 1999. Solar disinfection of water reduces diarrhoeal disease: an update. Arch. Dis. Child. 81:337-338. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Edelstein, P. H. 1981. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol. 14:298-303. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Heaselgrave, W., N. Patel, S. Kilvington, S. C. Kehoe, and K. G. McGuigan. 2006. Solar disinfection of poliovirus and Acanthamoeba polyphaga cysts in water—a laboratory study using simulated sunlight. Lett. Appl. Microbiol. 43:125-130. [DOI] [PubMed] [Google Scholar]
- 7.Hughes, R., J. Dart, and S. Kilvington. 2003. Activity of the amidoamine myristamidopropyl dimethylamine against keratitis pathogens. J. Antimicrob. Chemother. 51:1415-1418. [DOI] [PubMed] [Google Scholar]
- 8.Hughes, R., and S. Kilvington. 2001. Comparison of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga. Antimicrob. Agents Chemother. 45:2038-2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.International Organization for Standardization. 2001. Ophthalmic optics—contact lens care products—microbiological requirements and test methods for products and regimens for hygienic management of contact lenses. International Organization for Standardization, Geneva, Switzerland.
- 10.Joshi, P. C. 1985. Comparison of the DNA-damaging property of photosensitised riboflavin via singlet oxygen (1O2) and superoxide radical O2−· mechanisms. Toxicol. Lett. 26:211-217. [DOI] [PubMed] [Google Scholar]
- 11.Joyce, T. M., K. G. McGuigan, M. Elmore-Meegan, and R. M. Conroy. 1996. Inactivation of fecal bacteria in drinking water by solar heating. Appl. Environ. Microbiol. 62:399-402. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Lonnen, J., S. Kilvington, S. C. Kehoe, F. Al-Touati, and K. G. McGuigan. 2005. Solar and photocatalytic disinfection of protozoan, fungal and bacterial microbes in drinking water. Water Res. 39:877-883. [DOI] [PubMed] [Google Scholar]
- 13.Martins, S. A., J. C. Combs, G. Noguera, W. Camacho, P. Wittmann, R. Walther, M. Cano, J. Dick, and A. Behrens. 2008. Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest. Ophthalmol. Vis. Sci. 49:3402-3408. [DOI] [PubMed] [Google Scholar]
- 14.McGuigan, K. G., T. M. Joyce, R. M. Conroy, J. B. Gillespie, and M. Elmore-Meegan. 1998. Solar disinfection of drinking water contained in transparent plastic bottles: characterizing the bacterial inactivation process. J. Appl. Microbiol. 84:1138-1148. [DOI] [PubMed] [Google Scholar]
- 15.McGuigan, K. G., F. Mendez-Hermida, J. A. Castro-Hermida, E. Ares-Mazas, S. C. Kehoe, M. Boyle, C. Sichel, P. Fernandez-Ibanez, B. P. Meyer, S. Ramalingham, and E. A. Meyer. 2006. Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water. J. Appl. Microbiol. 101:453-463. [DOI] [PubMed] [Google Scholar]
- 16.Méndez-Hermida, F., J. A. Castro-Hermida, E. Ares-Mazas, S. C. Kehoe, and K. G. McGuigan. 2005. Effect of batch-process solar disinfection on survival of Cryptosporidium parvum oocysts in drinking water. Appl. Environ. Microbiol. 71:1653-1654. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Micelli Ferrari, T., M. Leozappa, M. Lorusso, E. Epifani, and L. Micelli Ferrari. 2009. Escherichia coli keratitis treated with ultraviolet A/riboflavin corneal cross-linking: a case report. Eur. J. Ophthalmol. 19:295-297. [DOI] [PubMed] [Google Scholar]
- 18.Neff, R. J., S. A. Ray, W. F. Benton, and M. Wilborn. 1964. Induction of synchronous encystment in Acanthamoeba sp. Methods Cell Physiol. 1:55-83. [Google Scholar]
- 19.Penna, T. C., P. G. Mazzola, and A. M. Silva Martins. 2001. The efficacy of chemical agents in cleaning and disinfection programs. BMC Infect. Dis. 1:16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Rainey, R. C., and A. K. Harding. 2005. Drinking water quality and solar disinfection: effectiveness in peri-urban households in Nepal. J. Water Health 3:239-248. [DOI] [PubMed] [Google Scholar]
- 21.Wegelin, M., S. Canonica, K. Mechsner, T. Fleischmann, F. Pesara, and A. Metzler. 1994. Solar water disinfection: scope of the process and analysis of radiation experiments. J. Water Supply Res. Technol. Aqua 43:154-169. [Google Scholar]