Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hayes, S. L. Articles by Rodgers, M. R. Search for Related Content PubMed PubMed Citation Articles by Hayes, S. L. Articles by Rodgers, M. R. Agricola Articles by Hayes, S. L. Articles by Rodgers, M. R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, May 2006, p. 3763-3765, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3763-3765.2006

SHORT REPORT

Assessment of the Effectiveness of Low-Pressure UV Light for Inactivation of Helicobacter pylori

Samuel L. Hayes,^* Karen M. White, and Mark R. Rodgers

USEPA, National Risk Management Research Laboratory, Water Supply/Water Resources Division, Cincinnati, Ohio 45268

Received 15 December 2005/ Accepted 3 February 2006

Top Abstract Introduction References

 
Three strains of Helicobacter pylori were exposed to UV light from a low-pressure source to determine log inactivation versus applied fluence. Results indicate that H. pylori is readily inactivated at UV fluences typically used in water treatment regimens. Greater than 4-log₁₀ inactivation was demonstrated on all three strains at fluences of less than 8 mJ cm^–2.

Top Abstract Introduction References

 
Helicobacter pylori is a gram-negative, microaerophilic, spiral bacterium which infects the human stomach and upper gastrointestinal tract. The organism is implicated as a cause of peptic gastroduodenal ulcers, gastritis, and gastric carcinoma (4). H. pylori was designated a class 1 carcinogen for stomach cancer in 1994 by the International Agency for Research on Cancer, a subdivision within the World Health Organization (8). Certain virulence factors (vacuolating cytotoxin and cag pathogenicity island) have been associated with more severe symptoms (1). H. pylori is currently listed on the U.S. Environmental Protection Agency's Candidate Contaminant List (5) and was included because it is potentially a waterborne pathogen and has been detected in drinking water sources (8). Literature reviews estimate that the percentage of human carriers of H. pylori ranges from 70 to 90% in developing countries and from 25 to 50% in developed countries (6).

The U.S. drinking water industry is interested in including UV light treatment as an alternative to chemical disinfection treatment for disinfecting water supplies. The major driving force to include UV light treatment is the recent discovery of its effectiveness against protozoans, especially Cryptosporidium spp. The Long Term 2 Enhanced Surface Water Treatment Rule includes requirements for UV disinfection (3). Adding UV disinfection capability to existing treatment plants or including it in the construction of new plants can provide additional disinfection credit. Also, UV disinfection has the potential for reducing the use of chlorine and its associated disinfection by-products. Therefore, there is a need to increase current UV light dose-response databases for those known, suspected, or potential microorganisms (other than the protozoans) that cause waterborne disease.

H. pylori is difficult to culture, and this fact has resulted in limited analytical detection capabilities for this pathogen. Occurrence studies in the aquatic environment have relied upon molecular methods employing PCR amplification procedures (8). Recent studies using PCR-based methods have detected the presence of H. pylori in river water and ponds, although the viability of the organisms in these samples is uncertain (8, 10). Humans are considered the primary reservoir of H. pylori, and human fecal matter is the most likely source in the environment. The mode of transmission of H. pylori is not certain. Past studies provide various possibilities, with most oral routes of exposure being associated with H. pylori infection (6).

Three isolates of H. pylori were used in this study: one clinical (Texas isolate) and two from the American Type Culture Collection, Rockville, Md. (ATCC 43504 and ATCC 49503). The organisms were propagated on Wilkins-Chalgren agar plates supplemented with 5% horse serum and incubated at 37°C for 3 days under microaerophilic conditions. This medium provides good recovery of the organisms, as demonstrated from past research (9). Organisms were aseptically removed by scraping with a bacteriological loop and suspended in sterile 1x phosphate-buffered saline (PBS; pH 7.4). The use of PBS appeared to enhance the recovery of organisms compared to phosphate-buffered water, as reported also by West et al. (11). The suspensions were notably clumped in the initial suspension. In order to obtain monodispersion of organisms, the suspension was vortexed, sonicated for 30 seconds (setting 2) (model F60 sonic dismembrator; Fisher Scientific, Pittsburgh, PA), and then filtered through a sterile 5.0-µm syringe filter. This filtered suspension was adjusted to an optical density (600 nm) of approximately 0.200 as a stock solution. One milliliter of this stock was added to 9 ml of sterile 1x PBS for purposes of experimentation. Spread plates were completed immediately after UV light exposure to encourage high recovery.

UV inactivation experiments were conducted at room temperature with continuous stirring in a 60-mm petri dish reactor under a collimated beam apparatus. A manually operated shutter controlled the length of UV exposure. Two 15-watt low-pressure UV lamps (model G15T8; American UV Co., Lebanon, IN) were used as the light source and were part of a UV reflector assembly (model XX-15S; UVP, Inc., Upland, CA). A radiometer (model IL-700; International Light, Inc., Newburyport, MA) was used to measure UV irradiance. The detector was model SED240, fitted with an NS254 filter and wide-eye diffuser (International Light, Inc.). The entire unit was factory calibrated to a U.S. National Institute of Standards and Technology standard. The absorbance (245 nm) of the bacterial suspensions was measured by using a Lambda 20 UV/VIS Spectrometer (Perkin Elmer, Boston, MA). The UV fluence was calculated as described by Blatchley et al. (2).

Initial suspensions were exposed to six fluences, ranging from 0 to 8 mJ cm^–2. Each exposure was tested in triplicate. Dilutions were also plated in triplicate, and the average of the nine results (except where noted) was used to calculate the log reduction. Wilkins-Chalgren agar plates with 5% horse serum were inoculated at appropriate dilutions for each exposure time. A volume of 0.1 ml was inoculated per plate. Plates were incubated in a microaerophilic environment for 3 days at 37°C.

The results for UV inactivation are presented in Table 1 and graphically in Fig. 1. The graph displays individual inactivation experiments with trend lines added, while the table summarizes the UV fluences used and associated colony counts. As can be seen, there was a moderate amount of variability in the testing. A higher standard error is present in replicates of higher colony counts in some Table 1 entries, and this is atypical. This was due to the fastidious growth conditions for the organism. Also, obtaining a monodispersed stock solution was difficult. Bacterial suspensions that exhibit significant clumping produce variable data sets. Covariance analysis was performed to compare the slopes of the inactivation curves. A significant difference (P = 0.0192) is seen among the three slopes. Nonetheless, the results demonstrate that H. pylori is effectively inactivated with UV light doses typically used in water treatment plants and point-of-use devices (i.e., 40 mJ cm^–2), as >4-log₁₀ inactivation was demonstrated on all three strains at fluences of less than 8 mJ cm^–2.

View this table: [in this window] [in a new window]

TABLE 1. Inactivation of Helicobacter pylori by UV irradiation

View larger version (18K): [in this window] [in a new window]

FIG. 1. UV inactivation plot for H. pylori. 49503, ATCC 49503; 43504, ATCC 43504; N, CFU per milliliter in unexposed initial solution; No, CFU per milliliter in UV-exposed solution.

The method used to produce a monodispersed suspension proved successful. Initially, test suspensions were prepared by vortexing until clumps were undectectable upon macroscopic examination. However, even after extensive vortexing and settling, large microorganism clumps were observed microscopically. Experimentation using this preparation method showed significant tailing of colony counts at the higher fluences. Detectable and highly variable numbers of colonies remained even at fluences of greater than 10 mJ cm^–2 (data not shown). After sonication and filtering, few visible clumps remained, and those that were observed were small. Exposures at the highest fluence tested produced consistent and low colony counts, with many plates having no growth. This experience supports the need for standardization of UV disinfection experiments. Also, recognition needs to be made that H. pylori and other organisms likely occur in the environment as biofilm or in association with particulates. Penetration of UV light into these matrices and conditions has not been quantified. However, obtaining baseline UV light dose responses on microorganisms is a necessary first step before embarking on research into real-world conditions.

Attempts to demonstrate light and dark repair were unsuccessful. Colony counts observed after 4 hours of incubation in either light or dark conditions were lower than those obtained after plating immediately after exposure. This was possibly due to the organisms converting to the viable but not culturable (VBNC) state or the reclumping of organisms in a static solution.

These results indicate that a monodispersed culture of H. pylori is inactivated by UV light to approximately the same degree as other tested gram-negative organisms (7). A cautionary note associated with this conclusion is that testing was performed on laboratory strains that experienced multiple passages. H. pylori likely occurs in the VBNC state in the environment. Little is known about the survivability of the organism in this state. If a procedure to assess live versus dead organisms in this VBNC state is developed, additional testing with UV light needs to be performed.

 
* Corresponding author. Mailing address: United States Environmental Protection Agency, 26 W. Martin Luther King Dr., MS387, Cincinnati, OH 45268. Phone: (513) 569-7514. Fax: (513) 569-7328. E-mail: hayes.sam{at}epa.gov.

Top Abstract Introduction References

 

1
1. Atherton, J. C. 1998. Helicobacter pylori virulence factors. Br. Med. Bull. 54:105-120.[Abstract/Free Full Text]
2
2. Blatchley, E. R., R. W. Emerick, T. Hargy, O. Hoyer, R. H. Hultquist, R. H. Sakaji, D. C. Schmelling, F. Soroushian, and G. Tchobanoglous. 2000. Chapter 3: protocols, p. 37-56. In G. Melin (ed.), Ultraviolet disinfection, guidelines for drinking water and water reuse. National Water Research Institute, Fountain Valley, Calif.
3
3. Cotton, C., and L. Passantino. 2005. Regulations in the United States: requirements and guidance for ultraviolet disinfection of drinking water. J. Environ. Eng. Sci. 4:S57-S63.[CrossRef]
4
4. Dunn, B. E., H. Cohen, and M. J. Blaser. 1997. Helicobacter pylori. Clin. Microbiol. Rev. 10:720-741.[Abstract]
5
5. Federal Register. 1998. Announcement of the Drinking Water Contaminant Candidate List. Fed. Regist. 63:10273-10287.
6
6. Gomes, B. C., and E. C. P. De Martinis. 2004. The significance of Helicobacter pylori in water, food and environmental samples. Food Control 15:397-403.[CrossRef]
7
7. Roessler, P. F., and B. F. Severin. 1996. Ultraviolet light disinfection of water and wastewater, p. 313-366. In C. Hurst (ed.), Modeling disease transmission and its prevention by disinfection. Cambridge Press, Cambridge, United Kingdom.
8
8. Sasaki, K., Y. Tajui, M. Sata, Y. Fuji, F. Matsubara, M. Zhao, S. Shimizu, A. Toyonaga, and K. Tanikawa. 1999. Helicobacter pylori in the natural environment. Scand. J. Infect. Dis. 31:275-279.[CrossRef][Medline]
9
9. Velázquez, M., and J. M. Feirtag. 1999. Helicobacter pylori: characteristics, pathogenicity, detection methods and mode of transmission implicating food and water. Int. J. Food Microbiol. 53:95-104.[CrossRef][Medline]
10
10. Voytek, M. A., J. B. Ashen, J. D. Kirshtein, E. R. Landa, and L. R. Fogarty. 2005. Detection of Helicobacter pylori and fecal indicator bacteria in five North American rivers. J. Water Health 3:405-422.[Medline]
11
11. West, A. P., M. R. Milliar, and D. S. Tompkins. 1992. Effect of physical environment on survival of Helicobacter pylori. J. Clin. Pathol. 45:228-231.[Abstract/Free Full Text]

---

Applied and Environmental Microbiology, May 2006, p. 3763-3765, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3763-3765.2006

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hayes, S. L. Articles by Rodgers, M. R. Search for Related Content PubMed PubMed Citation Articles by Hayes, S. L. Articles by Rodgers, M. R. Agricola Articles by Hayes, S. L. Articles by Rodgers, M. R.