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And Ionizing (Gamma) Radiation: a Comparative Approach Wat. Res. Vol. 35, No. 13, pp. 3109–3116, 2001 # 2001 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0043-1354(01)00030-6 0043-1354/01/$ - see front matter INACTIVATION OF BACTERIOPHAGES IN WATER BY MEANS OF NON-IONIZING(UV-253.7 nm) AND IONIZING (GAMMA) RADIATION: A COMPARATIVE APPROACH REGINA SOMMER1*, WALTER PRIBIL1, SILVIA APPELT1, PETER GEHRINGER2, HELMUT ESCHWEILER2, HERMANN LETH2, ALEXANDER CABAJ3 and THOMAS HAIDER4 1 Hygiene Institute University of Vienna, Kinderspitalgasse 15, 1095, Vienna, Austria; 2 Austrian Research Center Seibersdorf, Austria; 3 Institute of Medical Physics and Biostatistics, University of Veterinary Medicine Vienna, Austria and 4 Institute of Environmental Hygiene, University of Vienna, Austria (First received 14 March 2000; accepted in revised form 9 January 2001) Abstract}The inactivation behaviour of the bacteriophages PHI X 174 (ssDNA virus), MS2 (ssRNA virus) and B40-8 (dsDNA) toward non-ionizing (UV-253.7 nm) as well as to ionizing radiation (gamma radiation) was studied in order to evaluate their potential as viral indicators for water disinfection by irradiation. Previous findings of the high UV-253.7 nm resistance of MS2 were confirmed whereas an unexpected high sensitivity to gamma radiation compared to the two other phages was found. On the other hand, PHI X 174 revealed an enhanced UV sensitivity but a high resistance to ionizing radiation. B40-8 had an intermediate position between the other two bacteriophages relative to both types of radiation. As expected, the data of E. coli reconfirmed the unreliability of fecal indicator bacteria for the purpose of predicting responses of viruses to water treatment. In UV disinfection the influence of water matrix may be adequately controlled by considering the UV (253.7 nm) absorption of the water whereas so far no such parameter has existed for the influence of the water quality on ionizing irradiation with respect to the scavenger concentration. # 2001 Elsevier Science Ltd. All rights reserved Key words}UV radiation, ionizing radiation, inactivation, bacteriophages, disinfection, water INTRODUCTION formation of photoproducts. The most prominent Irradiation treatment has been proved to be a photoproducts are the dimers of thymine, a powerful tool in inactivating human pathogenic pyrimidine base. Another very clearly defined UV- microorganisms in water, waste water and sludge, induced damage in the DNA is the bacterial spore in food and medical products (Lagunas-Solar, 1995; photoproduct. The changes in the nucleic acid Farooq et al., 1993). One of the main advantages of lead to the so-called reproductive cell death of the such treatment consists of the fact that since microorganisms (Harm, 1980). The individual UV irradiation is a physical process, no chemicals have resistance of a microorganism depends on the UV to be added. In order to understand these techniques fluence applied and its ability to protect from and to repair damages. The unit of the UV fluence we have to consider that microbial inactivation by À2 means of non-ionizing radiation (e.g. UV-253.7 nm) is J m . compared to ionizing radiation (e.g. gamma rays, The microbicidal mechanism of ionizing radiation electron beams), respectively, are each based on is not yet fully understood. The nucleic acids are entirely different mechanisms. likely to be the main target, but in viruses the coat UV plants, equipped with low pressure mercury also plays an important role (von Sonntag, 1987; lamps, emitting mainly UV radiation with a Mahnel et al., 1980). The microorganisms may be wavelength of 253.7 nm, are widely used in the affected either by direct (single and double strand disinfection of both drinking and waste water. UV- breaks of the nucleic acids) or by indirect reaction 253.7 nm is predominately absorbed by the nucleic (radicals originating from water, mainly OH free À acids as the main target and causes damage by the radicals, H atoms and solvated electrons eaqu, generated by radiolysis either intra- or extra cellu- larly). The effect of the indirect attack by radicals *Author to whom all correspondence should be addressed. may be reduced by the presence of scavengers such as Tel.: +43-1-40490-79452; fax: +43-1-40490-9794; bicarbonate, but can also be enhanced by the e-mail: [email protected] presence of oxygen (von Sonntag, 1987). The direct 3109 3110 Regina Sommer et al. effect is not scavengeable. The unit for the absorbed of 4 log of microbial pathogens is demanded to dose is Gy (J kgÀ1). ensure safe drinking water (US EPA, 1989). Bacterial viruses such as somatic coliphages, F-specific coliphages and phages infecting Bacter- oides fragilis have been proposed as indicators for MATERIALS AND METHODS human pathogenic, water-transmittable viruses to Bacteriophages}propagation and enumeration monitor the quality of water (drinking, recreational MS2 (F-specific bacteriophage), PHI X 174 (somatic and waste water) as well as to evaluate water coliphage) and B40-8 (phage infecting Bacteriodes fragilis) treatment and disinfection (Anon, 1991; Havelaar and the bacterial host strains (Salmonella typhimurium WG et al., 1991; Jofre et al., 1986). The search for viral 49, E. coli WG5 and Bacteroides fragilis HSP-40) were provided by the National Institute of Health and Environ- indicators became necessary, since numerous studies ment (Bilthoven, Netherlands), the Institute Pasteur de Lille showed that viruses are fairly resistant both to (France) and the University of Barcelona (Spain). natural factors in the environment (sunlight, tem- Bacteriophage MS2 was propagated in the host strain perature, antagonists) and to chemical and physical E. coli K-12 Hfr and enumerated by the double agar layer treatment. The bacterial indicators usually applied procedure according to ISO/CD 10705-1/1997, using the strain Salmonella typhimurium WG49. Bacteriophage PHI are not appropriate for predicting the viral quality of X 174 was grown in the bacterial host strain E. coli WG5 water (Tree et al., 1997; Dizer et al., 1993; Havelaar and analysed by the double agar layer technique, following et al., 1991; Anon, 1991; Sommer et al., 1989). the protocol of ISO 10705-2/1997. The propagation and Therefore the fecal indicator bacteria have to be detection of phage B40-8 by the double agar layer method was carried out in host strain B. fragilis HSP-40 under regarded as inadequate for predicting viral responses anaerobic culture conditions according to ISO/WD to treatment. 10705-4/1997. The compositions of the nutrient broths used However, the accurate knowledge of the charac- for the propagation of the bacteriophages are shown in teristics of possible indicators is a prerequisite for Table 1. After propagation of the phages in the bacterial their reliable application in water hygiene. Therefore hosts, suspended in the respective broth, bacteria were inactivated by adding chloroform (5 ml CHCl3 per 50 ml we chose representatives of the three phage groups broth) and removed by centrifugation. The supernatant proposed as viral indicators as mentioned above. representing the phage stock solution was stored at 4–68C. Moreover, these phages were subject of an extensive Immediately before use, phage stock solutions were passed international study about the feasibility of phages through a 0.2 mm membrane filter (Millex-GV, Millipore) to remove virus aggregates and were then diluted in tap water for the monitoring of the bathing water quality (drinking water, City of Vienna). The chemical main (European Community, 1999). characteristics of the tap water are indicated in Table 2. The aim of our study was to investigate the The final concentrations of the phage suspensions after 6 7 inactivation behaviour of three selected bacterio- dilution (1 ml phage stock to 1 l tap water) were 10 to 10 phages, namely PHI X 174 (somatic coliphage), MS2 plaque forming units (PFU) per ml. (F-specific coliphage) and B40-8 (phage infecting UV irradiation device and performance of UV-253.7 nm Bacteroides fragilis) regarding non-ionizing (UV- inactivation experiments 253.7 nm) as well as ionizing radiation (gamma A board mounted with 10 low-pressure mercury UV radiation) in order to evaluate their potential as viral lamps (EK 36; length 500 mm, wavelength 253.7 nm, ozone indicators for water disinfection by irradiation free, Katadyn) was horizontally suspended over the treatment. Since the dose of ionizing and the fluence irradiation vessel. An aperture (100 mm  150 mm) was of UV radiation cannot be directly compared due to fixed directly below the UV lamps, providing a bundle of quasi parallel radiation to the irradiation vessel. The test their entirely different inactivation mechanism, we suspensions (volume 25 ml) were irradiated in sterile petri chose a 4 log reduction of the phages as a measure for dishes (diameter 90 mm) and placed on a magnetic stirrer comparison. In drinking water treatment a reduction during permanent mixing. The depth of the layer was 4 mm. Table 1. Composition of the nutrient broths used for phage propagation (according to ISO/CD 10705-1/1997; ISO/CD 10705-2/1997 and ISO/WD 10705-4/1997)a Components of the nutrient broth for the propagation of phage MS2 PHI X 174 B40-8 Yeast extract (1 g lÀ1) Yeast extract (3 g lÀ1) Yeast extract (2 g lÀ1) Trypticase peptone (10 g lÀ1) Peptone (10 g lÀ1) Casein peptone (10 g lÀ1) Meat extract (12 g lÀ1) Meat peptone (10 g lÀ1) Glucose (1 g lÀ1) Glucose (1.8 g lÀ1) Hemin (0.01 g lÀ1) l-Cystein (0.5 g lÀ1) NaCl (8 g lÀ1) NaCl (3 g lÀ1) NaCl (5 g lÀ1) À1 À1 CaCl2 (0.3 g l ) CaCl2 (0.05 g l ) À1 À1 Na2CO3 (0.75 g l )Na2CO3 (2.65 g l ) À1 À1 MgCl2 (0.60 g l ) MgSO4Á7H2O (0.12 g l ) pH: 7.2 Æ 0.2 pH: 7.2 Æ 0.2 pH: 6.8 Æ 0.5 a During the preparation of the test suspensions of the phages, 1 ml of the respective phage stock solution is added to 1 l of tap water.
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