WO2002090904A2

WO2002090904A2 - Uv sensitive bacillus subtilis spores and biodosimetry applications

Info

Publication number: WO2002090904A2
Application number: PCT/US2002/013917
Authority: WO
Inventors: Wayne L. Nicholson
Original assignee: The Arizona Board Of Regents On Behalf Of The University Of Arizona
Priority date: 2001-05-03
Filing date: 2002-05-03
Publication date: 2002-11-14
Also published as: AU2002257240A1; AU2002257240A8; WO2002090904A9

Abstract

Methods and materials for biodosimetry in radiant energy disinfection applications are disclosed (Figure 1). Isolated strains of B. subtilis bacteria spores sensitive or resistant to radiant energy (such as ultra violet light) relative to a predetermined dose response of pathogenic organisms (protozoon, bacterial, and/or viral) are provided as surrogates for the pathogenic organism of interest in measuring the efficacy of a particular disinfection protocol.

Description

UV SENSITIVE BACILLUS SUBTILIS SPORES AND BIODOSIMETRY

APPLICATIONS

U.S. GOVERNMENT RIGHTS

[001 ] This work was supported by USDA Hatch Funds ARZT 136753-

H-02-116. The government has certain rights in the invention.

REFERENCE TO RELATED APPLICATIONS

[002] This application claims the filing date benefit of U.S. Provisional

Patent Application Serial No. 60/288,474, filed May 3, 2001 , which is incorporated by reference in its entirety for any purpose.

DESCRIPTION OF THE INVENTION

Field of the Invention

[003] The invention is directed generally to methods of biodosimetry and in particular to the measurement of radiant energy, and especially ultraviolet (UV) light, dosage using novel spores of the bacterial organism B. subtilis.

Background of the Invention

[004] Ultraviolet light (UV) currently is used in many commercial applications, such as UV disinfection of air, UV curing of inks and coatings, UV disinfection of foods, and UV-based oxidation destruction of pollutants. Physically, UV is that portion of the electromagnetic spectrum that lies beyond the "purple" edge of the visible light spectrum and has wavelengths between 100 and 400 nanometers (nm).

[005] UV treatment of drinking water is rapidly becoming an attractive and accepted method for disinfection of viable oocysts, cysts and spores of a number of pathogens. In fact, ultraviolet (UV) technologies for disinfection of municipal drinking water have been in use for several years in Europe, and interest in UV is rapidly gaining momentum as a choice for North American water treatment. Accordingly, field testing, installation and actual operation of UV systems has already begun at selected North American sites. One important reason why UV disinfection of municipal water has become increasingly attractive are recent findings that oocysts of protozoan intestinal parasites such as Cryptosporidium parvum, which are resistant to chlorination, are readily inactivated by relatively low doses of UV (Clancy, J.L. et al. J. American Water Works Assoc. 92(9):97-104 [2000]; Korich et al. Inter- laboratory comparison of the CD-1 neonatal mouse dose response model for Cryptosporidium parvum oocysts. J. Euk. Microbiol.47: 294-298 [2000]).

[006] Cryptosporidium are microscopic parasites that are responsible for an illness called cryptosporidiosis. When ingested, these parasites germinate, reproduce, and cause gastrointestinal distress, such as diarrhea, abdominal cramps, and nausea, as well as headaches. Cattle feces appear to be the primary source of Cryptosporidium, although these parasites have also been found in humans and other animals. Drinking water sources become contaminated when feces containing the parasites are deposited or flushed into water. If treatment is inadequate, drinking water may contain sufficient numbers of parasites to cause illness. Although municipal drinking water treatment providing filtration and disinfection with chlorine can reduce the risk of contracting cryptosporidiosis, chlorine by itself is not effective against Cryptosporidium.

[007] The microsporidian intestinal parasite Encephalitozoon intestinalis has also been placed on the first Contaminant Candidate List [CCL] (see Federal Register: March 2, Vol. 63, Num. 40 pp. 10273-10287 [1998]) under the Safe Drinking Water Act [SDWA], and two other microsporidian species, E. cuniculi and E. hellem, are known to be pathogenic for humans. Recently, the sensitivities of these microsporidial species to UV disinfection have been demonstrated by the inventor as described in example 2 below.

[008] The United States Environmental Protection Agency (USEPA) regulators are currently assessing UV disinfection as a key component of the Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR). Under the LT2ESWTR, validation tests will be necessary to prove that any given UV reactor conforms to the standards of drinking water disinfection, which will be determined by the USEPA. UV reactor validation testing is used to determine conditions that can be monitored to ensure delivery in routine operation of the proper UV fluence required for adequate water disinfection.

[009] The two current methods of measuring UV radiation are spectroradiometry and biological dosimetry (or biodosimetry). Spectroradiometers provide the most detailed spectrally resolved information on solar UV in physical units, but they are expensive bulky instruments requiring regular calibration. Moreover, physical monitoring of applied irradiation using electronic UV sensors can be problematic and not yield an accurate picture of the biologically-effective UV fluence in large-scale municipal settings. Therefore to determine the biologically-effective fluence delivered by a UV reactor, one must rely on more direct measures such as biodosimetry, an in situ assessment of UV biocidal effectiveness during operation of UV reactors (see Quails, R. G. and J. D. Johnson. Appl. Environ. Microbiol. 45(3):872-877 [1983]). Essentially, biodosimetry involves the inactivation of a non-pathogenic test organism (i.e. a "surrogate" of the pathogenic organism) whose UV dose- response characteristics have been precisely quantified (Bolton et al., Pure Appl. Chem. (in press) [2002]; Hoyer, O. IUVA News 2: 22-27 [2000]).

[010] Biodosimeters are inexpensive, small devices requiring no external power, and, thus, are widely accepted for both natural and artificial UV dose control. Escherichia coli bacteriophage MS-2 and spores of Bacillus subtilis strain ATCC 6633 have been used extensively as UV biodosimetry reference organisms. The 254-nm UV fluences required to cause 4-log inactivation of the bacteriophage MS-2 are approximately 800 Joules(J)/m² (Hoyer, O. IUVA News 2: 22-27 [2000]), which greatly exceeds the 4-log inactivation fluence for C. parvum oocysts which is only approximately 80 J/m² (Clancy, J.L. et al. J. American Water Works Assoc. 92(9):97-104 [2000]; Korich et al. Inter-laboratory comparison of the CD-1 neonatal mouse dose response model for Cryptosporidium parvum oocysts. J. Euk. Microbiol. 47: 294- 298 [2000]).

[011] Bacillus subtilis spores also have been routinely used as UV biodosimeters in monitoring UV disinfection of water (Nicholson. W.L. et al. Microbiol. Mol. Biol.Rev. 64:548-572 [2000]; Hoyer. O. IUVA News 2:22-27 [2000]). Although β. subtilis spores have the advantages of non-pathogenicity and ease of use and cultivation, a major drawback of the commonly used strains of B. subtilis is that the spores of these strains exhibit UV inactivation profiles that are dramatically different from those of target microorganisms such as C. parvum oocysts. Thus, the response of C. parvum oocysts to low pressure UV has been shown to result in a 2 log inactivation at 20 J/m² and 3.5 to 4 log inactivation at 90 J/m² (Shin, G. et al. Proc, Water Quality Technology Conference, AWWA, Tampa Bay, FL, Nov. 1-3, 1999; Clancy, J.L et al. J. American Water Works Assoc. 92(9):97-104 [2000]), while wild strains of B. subtilis spores require on the order of 350 J/m² for 2 log inactivation, and 600 J/cm² for a 4 log inactivation (Hoyer. O. IUVA News 2:22-27 [2000]).

[012] The shortcomings of using a surrogate more UV resistant than the target organism have been described by Wright and Lawryshyn in a presentation entitled Disinfection 2000: Disinfection of Wastes in the New Millennium, New Orleans, LA, March 2000. Where log inactivation is limited by reactor hydraulic characteristics rather than theoretical irradiance and residence time, a reactor might achieve inactivation of UV sensitive organisms only to the extent demonstrated for the resistant surrogate. These researchers recommend dual microbe challenges, using both a resistant organism, to measure applied dose, and one with a UV dose similar to that of the target pathogen, to identify any limitations due to the influence of hydraulic conditions. In other words, if information is required as to how closely a UV reactor system approaches the performance of an ideal system, reactor validation should include at least two biodosimeters of widely differing UV sensitivities, one of which exhibits a UV sensitivity similar to or greater than that of the target pathogen, and the other exhibiting greater UV resistance than the target pathogen (Bolton et al., Pure Appl. Chem. (in press) [2002]). [013] Therefore, although recent research indicates that ultraviolet light will inactivate Encephalitozoon intestinalis, E.cuniculi, E. hellem and Cryptosporidium parvum, no reliable methods are currently available to detect these parasites on a routine basis. This is largely because proposed methods underestimate the number of organisms present and do not provide any information on their capacity to cause illness in humans. Moreover, the tests that do exist take at least a few days to come up with results, which means they are unsuitable for day-to-day monitoring. More generally, other forms of radiant energy are being increasingly used to disinfect (e.g. x-ray bombardment of mail). Thus, there is a continuing need to for improved biodosimetry materials and methods.

SUMMARY OF THE INVENTION

[014] The invention relates in general to novel and improved methods and materials with which biodosimetry of radiant energy dose is performed. In terms of biodosimetry materials, three novel strains of UV-sensitive B. subtilis spores created by the inventor for use in biodosimetry applications are described. For example, the determination of the effective dose delivered by a UV reactor using spores UV sensitive B. subtilis in a biodosimetrical assay is carried out by irradiating the spores and performing growth assays is accomplished as described in detail below. More particularly, methods for monitoring the efficacy of water UV disinfection methods for various pathogenic organisms are also disclosed.

[015] In certain embodiments, the invention comprises B. subtilis strains designated WN624 (trpC2, a yEv.spc); WN625 (uvrB42, amyEv.cat; and WN626 (uvrB42, AsplB::ermC1 , amyEv.tet), wherein the designation "WNxxx" (e.g. WN624) refers to strain accession numbers in the inventor's strain collection; italicized phrases are gene designations as follows: amyE, amylase gene; cat, chloramphenicol resistance gene; ermC, erythromycin- lincomycin (MLS) resistance gene; spc, spectinomycin resistance gene; splB, spore photoproduct lyase gene; tet, tetracycline resistance gene; trp, tryptophan biosynthetic gene; and uvrB, nucleotide excision repair gene; Δ indicates a genetic deletion; and :: indicates a genetic insertion.

[016] In certain embodiments, the invention comprises the use of spores derived from B. subtilis strains WN624, WN625, and/or WN626 as surrogates for the protozoan pathogen in biodosimetry studies of UV disinfection.

[017] In certain embodiments, the invention comprises the use of spores derived from B. subtilis strains WN624, WN625, WN626 and/or WN333 as surrogates for bacterial, protozoon, and/or viral pathogenic organisms in the measurement of a biologically effective dose of radiant energy.

[018] Thus, it is an object of the invention to provide new B. subtilis strains to be used for measurement of radiant energy dosage.

[019] Another object of the invention is to provide new and improved methods of biodosimetry for monitoring the effectiveness of radiant-energy based disinfection protocols.

[020] A further object of the invention is to provide a method of UV reactor validation in drinking water applications where control of protozoan pathogens such as C. parvum and Encepalitozoan species is a priority.

[021] Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose only some of the various ways in which the invention may be practiced.

BRIEF DESCRIPTION OF THE DRAWINGS

[022] Figure 1 depicts the pedigree of the strains constructed for use in spore radiant energy dosimetry. WNxxx numbers refer to strain accession numbers in the inventor's strain collection; Numbers beginning with pWNxxx refer to plasmids; Italicized phrases are gene designations: amyE, amylase gene; cat, chloramphenicol resistance gene; ermC, erythromycin-lincomycin (MLS) resistance gene; spc, spectinomycin resistance gene; splB, spore photoproduct lyase gene; tet, tetracycline resistance gene; trp, tryptophan biosynthetic gene; uvrB, nucleotide excision repair gene; Δ indicates a genetic deletion; :: indicates a genetic insertion; tf = transformation; Straight vertical arrows denote construction of a strain from the previous strain; Merging lines indicate the DNA introduced during construction; Super script symbols indicate as follows: R= resistant; S= sensitive; - = deficient.

[023] Figure 2 depicts UV inactivation curves for DNA repair-mutant B. subtilis spores (irradiated at the University of Arizona).

[024] Figure 3 depicts the UV inactivation response of spores of β. subtilis strains WN333 and WN626 using CEC collimated beam apparatus, compared with C. parvum data from Shin, G. et al. Proc, Water Quality Technology Conference, AWWA, Tampa Bay, FL, Nov. 1-3, 1999; and Clancy, J.L. et al. J. American Water Works Assoc. 92(9):97-104, 2000.

[025] Figure 4 depicts a summary of spore inactivation by 254-nm UV. Bacillus subtilis dosimetry strains WN333 (circles) and WN626 (triangles) at EPA (open symbols) or at UA (hatched symbols). For comparison, the UV inactivation kinetics from Fig. 5 are plotted for spores of E. intestinalis (solid squares), E. cuniculi (hatched squares) and E. hellem (open squares).

[026] Figure 5 depicts a table of data showing the UV inactivation kinetics for spores of the protozoans E. intestinalis , E. cuniculi, and E. hellem.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

[027] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are expressly incorporated by reference for any purpose.

[028] The term "water" means water in any condition and fit for any purpose including, but not limited to, drinking water, waste water, and reclaimed or recycled water. [029] The term "pathogenic organism" means a bacterial, viral, or protozoon agent cable of causing symptoms or disease in animals, especially humans. Exemplary pathogenic bacteria include, but are not limited to, Escherichia coli ATCC 11, E. coli ATCC 23958, E. coli NCTC 5934, E. coli NCIB 9481, E. coli wild isolate, Enterobacter cloaca, Klebsiella pneumoniae, Citrobacter freundii, Yersinia enterocolitica, Salmonella Typhi, Salmonella Typhimurium, Serratia marcescens, Enterocolitica faecium, Vibrio cholerae wild isolate, Pseudomonas aeruginosa, Mycobacterium smegmatis, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, C. perfringens, C. tetani, and C. difficile. Exemplary pathogenic protozoon organisms include, but are not limited to Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon hellem, Cryptosporidium parvum, Giardia lamblia, or species of Cyclospora. Exemplary pathogenic viral organisms include, but are not limited to Polio virus (Mahoney), Rotavirus SA 11 , or Staphylococcus aureus phage A994.

[030] The term "radiant energy" means forms of electromagnetic radiation or particle bombardment including, but not limited to, ultra violet light, X-rays, γ-rays, and electron beams.

[031] The terms ATCC, NCTC, and NCIB are acronyms for repositories of microbial organisms and indicate American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA; National Collection of Type Cultures, Central Public Health Laboratory, London, UK; and National Collection of Industrial Bacteria; now NCIMB Ltd, National Collections of Industrial and Marine Bacteria Ltd, Aberdeen, Scotland, respectively. [032] The term "dose response" or variations thereof means a measurement of microbial growth versus dose of radiant energy and is synonymous with the term "inactivation kinetics." Thus, in this context, the terms "dosimetry" or "biodosimetry" mean the measurement of a biologically effective dose of radiant energy or the dose needed to inactivate an organism to a particular degree.

[033] The invention includes strains of β. subtilis bacteria specially constructed for dosimetry applications. Turning to Fig. 1, these strains were constructed as follows: All constructed strains were derived from parental strain WN131, which is the "wild-type" laboratory strain of β. subtilis commonly referred to as strain 168 (stock number 1A1 deposited at the Bacillus Genetic Stock Center, Ohio State University, Columbus, OH). Strain WN624 was constructed by transforming competent cells of strain WN131 with chromosomal DNA isolated from strain WN513 and selecting for spectinomycin-resistant, amylase-negative transformants. One of these was designated strain WN624. Strain WN625 was constructed in two steps as follows. First, competent cells of strain WN131 were transformed with a combination of chromosomal DNA from strain WN623 and plasmid pWN161. Tryptophan prototrophs were selected and screened for mitomycin-C sensitivity; mitomycin-sensitive transformants were then screened for UV- sensitive vegetative cells, resulting in strain WN538. Second, competent cells of WN538 were transformed with chromosomal DNA isolated from strain WN512, and a chloramphenicol-resistant, amylase-negative transformant from this cross was designated WN625. Strain WN626 was also constructed in two steps as follows. First, competent cells of strain WN538 were transformed to MLS resistance with DNA from plasmid pWN403, resulting in strain WN542. Second, strain WN542 was transformed with chromosomal DNA from strain WN514, selecting for tetracycline resistance and screening for amylase- negative transformants, one of which was designated strain WN626. Construction of strain WN333 is outlined in detail in Nicholson et al. (Mol. Gen. Genet. 255: 587-594 [1997]). Detailed protocols of all of the techniques used above are known to one skilled in the art as can be found in: Cutting, S.M and P.B. Vander Horn (eds.) Molecular Biological Methods for Bacillus, John Wiley & Sons, Sussex, England, 1990; and Sambrook et al. (eds.) Molecular Cloning: a Laboratory Manual, 2nd. Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.

[034] Essentially, the inventive strains of B. subtilis are used in biodosimetry applications to monitor the effectiveness of radiant energy-based disinfection protocols. A particularly preferred use of the invention is in biodosimetry application involving disinfection with UV light. Such protocols could include UV dose monitoring during the disinfection of air, water, or food. Moreover, spores of the invention may be used in biodosimetry applications as surrogates for a wide variety of pathogens, such as bacteria, protozoa, and viruses, upon routine testing for radiant energy, and especially, UV sensitivity. [035] To further disclose the invention in its most practical and preferred embodiments, the following non-limited examples are provided.

EXAMPLES Example 1 UV Sensitive Spores of B. subtilis as a Biodosimetry Surrogate for Cryptosporidium In Water Disinfected by UV.

Materials and Methods

[036] β. subtilis strains from the strain collection of the inventor were used for this example. The genotype of these strains included: WN 333 (trpC2, AsρlB::ermC1; see Nicholson et al. Molec. Gen. Genet. 255:587-594 [1997]); WN624 (trpC2, amyE::spc); WN625 (uvrB42, amyEv.cat, and WN626 (uvrB42, AsplB::ermC1 , amyEv.tet). All strains were routinely cultivated at 37°C on either Trypticase Soy Agar (TSA; Difco) or Luria-Bertani (LB) agar (Miller, J. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1972]) containing the appropriate antibiotics at a final concentration of: 1 μg erythromycin/mL and 25 μg lincomycin/mL (WN333); 100μg spectinomycin/mL (WN624); 3 μg chloramphenicol/mL (WN625); and 10 μg tetracycline/mL (WN626).

[037] To prepare spores, β. subtilis strains were cultivated in liquid Nutrient Sporulation Medium (NSM) (Schaeffer et al. Pro. Natl. Acad. Sci. USA 54:704-711 [1965]) lacking selective antibiotics for 48 hours at 37°C with vigorous aeration, and production of spores was confirmed by appearance of refractile bodies by phase-contrast microscopy. Spores were harvested by centrifugation, purified as described in detail previously (Nicholson, W.L. and P. Setlow. Molecular biological methods for Bacillus. John Wiley and Sons,

Sussex, England, pp. 391-450 [1990]), heat-shocked (80°C, 10min), and stored protected form light at 4°C as a suspension in deionized water. Spore suspensions were enumerated by serial dilution and viable colony counts on TSA or LB plates containing the appropriate selective antibiotic for a given strain.

[038] Irradiation of spores was then performed either at the University of Arizona (UA) with a standard low-pressure mercury vapor lamp (Model UVGL-25, UV Products, Upland, CA) or at Clancy Environmental Consultants, Inc. (CEC) using a low pressure UV collimated beam apparatus. UV doses were determined using an International Light Model IL-1400 Radiometer (CEC) or a UV Products UVX radiometer (UA) fitted with the appropriate 254 nm sensors. To ensure that radiometry at the 2 sites were equivalent, the response of the UVX radiometer was calibrated to that of the IL-1400 radiometer using the collimated beam apparatus at CEC.

[039] Spores were diluted from stock suspensions into sterile deionized water to a final concentration of either 1x10 ⁶ spores/mL (UA) or 5 x 10⁶ spores/mL (CEC). Either 10 mL (UA) or 15 mL (CEC) of the diluted spore suspensions were pipetted into 6-cm diameter Petri dishes with stir bars, placed on stir plates in the center of the UV beams with the lids removed, and exposed to the indicated UV doses. Average irradiance over the surface of the Petri dish and through the suspension volume had been determined as described by Bukhari, et al. (J. American Water Works Assoc.91 (3):86-94

[1999]) using the formulas prepared by Bolton (UV Dose Protocol posted on the iuva.org website). Exposures were performed on duplicate suspensions. Samples of irradiated spores were diluted serially tenfold in sterile deionized water, plated in quadruplicate on TSA or LB containing the appropriate antibiotics, and incubated overnight at 37°C for viable colony counts.

[040] Based on the UV inactivation profiles of the 4 strains developed at UA (Fig. 2), and the previous observation of the 3.5 log inactivation of C. parvum oocysts irradiated with 90 J/m² using low pressure UV at CEC, strains WN333 and WN626 were chosen for further analysis and comparison with C. parvum oocysts using irradiation and dosimetry equipment at CEC. Comparison of B. subtilis spores and C. parvum oocysts

[041 ] UV inactivation data for spores of β. subtilis strains WN333 and WN626 are shown in Fig. 3. These data indicate the UV dose required for 3.5 log inactivation of B. subtilis spores was approximately 20 J/m² for WN626 spores and 85 J/m² for WN333 spores. The dose response curves of these strains are compared in Fig. 2 with existing C. parvum data. The WN626 compares closely at very low doses, and WN333 compares well at 80 J/m². Further definition of the C. parvum dose response curve will help establish the correlation of these surrogates and pathogens. However, either of these strains would provide a suitable surrogate for assurance of high log inactivation at relatively low UV doses. Thus, these non-pathogenic surrogates that mimic C. parvum dose response and which afford results in 24 hours (as opposed to the weeks required for C. parvum infectivity analyses) are of great utility, especially to the drinking water industry. More generally, given the ease and speed of manipulation and culture, the B. subtilis strains of the invention show strong potential for use as biodosimetry surrogates in a broad variety of UV testing and verification applications.

Example 2: UV Sensitive Spores of B. subtilis as a Biodosimetry Surrogate for Encephalitozoon species

[042] Spores of three common Encephalitozoon species are responsible for infection in humans. Thus, this protocol is designed to define the sensitivity of E. intestinalis, E. cuniculi, and E. hellem to 254-nm UV radiation. The second objective is to investigate the use of spores of Bacillus subtilis DNA repair-deficient strains as biodosimeters that would exhibit inactivation kinetics similar to those of the microsporidial spores when irradiated with UV under identical conditions. For comparative purposes, experiments were performed in parallel at the EPA facility in Cincinnati (called hereafter "EPA") and at the University of Arizona (hereafter called "UA"). Materials and Methods

[043] Spores of Encephalitozoon cuniculi, ATCC #50502, E. hellem, ATCC # 50451 , and a duodenal isolate of E. intestinalis, ATCC #50603, were purified weekly from stock flasks, enumerated by hemocytometry ,(Wolk, et al 2000), and stored in phosphate-buffered saline (PBS) or sterile deionized water at 4°C (UA). βac/7/us subtilis strains WN333 (trpC2, ΔsplABxermCI) (Nicholson, W.L. et al. Mol. Gen. Genet. 255:587-594 [1997]) and WN626 (uvrB42, AsplAB::ermC1, amyEr.tet) (this study) were used for biodosimetry. B. subtilis strains were routinely cultivated at 37°C on Luria-Bertani (LB) agar containing the appropriate antibiotics at a final concentration of : 1 μg erythromycin/mL and 25 μg lincomycin /mL (for WN333); and 10 μg tetracycline/ mL (for WN 626). Spores were produced in Schaeffer's Sporulation medium (SSM) (Schaeffer et al. Pro. Natl. Acad. Sci. USA 54:704- 711 [1965]) and purified by lysozyme treatment and buffer washing as described previously by Nicholson and Setiow (Molecular biological methods for Bacillus. John Wiley and Sons, Sussex, England, pp. 391-450 [1990])) (UA).

[044] UV irradiation was performed with a standard low-pressure mercury vapor lamp (Model UVGL-25, UVP, Upland, CA) (UA) or with a collimated beam apparatus containing two 15-watt low pressure UV lamps, Model G15T8 (American Ultraviolet Co, Lebanon, IN) as the light source and a UV reflector assembly, Model XX-15S (UVP, Inc., Upland, CA) (EPA). UV fluences were measured using: a UVX radiometer (UVP, Upland, CA) fitted with the appropriate calibrated probe for low-pressure UV (Model UVX-25) (UA); or a model IL-1700 radiometer fitted with detector model SED240, an NS254 filter and wide eye diffuser (International Light, Inc. Newburyport, MA)(EPA). Both UA and EPA radiometers units were calibrated by their respective manufacturers traceable to a U.S. National Institutes of Standards and Technology standard. [045] Furthermore, both instruments were directly compared to one another at UA using the same UV source and were found to be in agreement. The UV irradiance distribution across the surface to be treated was predetermined by measuring the 254 nm irradiance at 0.5 cm intervals along the x-y axis of a 6-cm diameter grid originating at the center of the UV beam. The average incident irradiance over the entire 6-cm diameter Petri dish was calculated using UVCalc, a Microsoft Excel worksheet devised by Dr. James Bolton and kindly posted on the International Ultraviolet Association (IUVA) website (iuva.org). β. subtilis and Encephalitozoon species spores were diluted from stock suspensions into sterile deionized water to a final concentration of 1 x 10⁷ spores per 10 mL. The absorbances at 254 nm of the spore suspensions were measured in a UV spectrophotometer and entered into UVCalc to derive the exposure time needed for each final UV dose used. Spore suspensions were pipetted into an open 6-cm diameter Petri dish set atop a rotating platform and exposed to the indicated UV doses. Serial tenfold dilutions of β. subtilis spores were plated on SSM containing the appropriate antibiotics and incubated overnight at 37°C for viable colony counts. B. subtilis spore inactivation was calculated using the formula Log₁₀ inactivation = logio (No / N_t) where No and N_t stand for Colony Forming Units (CFU) of the suspension at the exposure time 0 and time t, respectively. The E. intestinalis, E. cuniculi and E. hellem spores were inoculated onto RK-13 seeded 15mm Thermanox coverslips of a 24 well plate as previously described and incubated for 7 days (Wolk, D.M. et al. Appl.Environ. Microbiol 66: 1266-1273 [2000]).

[046] Coverslips showing one or more infected cells were scored as positive and those without infection were scored as negative. The percent infection was calculated by dividing the number of positive wells infected by the number of wells inoculated. The response logit (Korich et al, 2000) was calculated for a given spore dose as the natural logarithm (In) of the proportion of wells infected divided by one minus the proportion of wells inoculated. Therefore the response logit = ln[P/(1-P)]. The response logit values obtained experimentally were treated as the dependent (Y) variable for regression analysis with the log₁₀ of the number of spores in each dose as the independent (X) variable. The regression analysis tool of Microsoft Excel 97^® was used to perform the least squares regression, the regression equation parameters (b,m,) and to test the validity of the resulting regression model equation. The results of 145 coverslips from the in vitro assay showed that the most sensitive of the three microsporidian spores tested to low pressure UV light was E. intestinalis, which exhibited 3.2-log inactivation at 60 J/m². E. cuniculi and E. hellem spores showed 3.2-log inactivation at 140 J/m2 and 190 J/m² respectively (Figure 5).

[047] Thus, E. intestinalis spores exhibited a degree of UV sensitivity comparable to that of C. parvum oocysts, and spores of E. cuniculi and E. hellem are roughly twice as resistant to UV as C. parvum oocysts. In all cases, a UV dose of approximately 200 J/m² or higher would inactive the three pathogenic microsporidian species tested.

[048] The UV inactivation kinetics of two Bacillus subtilis dosimetry strains, WN333 and WN626, were determined in parallel at EPA and at UA. Spores of strain WN626 were observed to be extremely sensitive to UV, exhibiting 3.5-4 log reduction at only 20 J / m² (Fig. 4). The data from the two labs concerning UV sensitivity of WN626 spores were in excellent agreement (Fig. 4). The UV sensitivity of spores of B. subtilis strain WN333 was found to vary by roughly a factor of 2 between the determinations at EPA vs. UA (Fig. 4). At EPA and UA, the UV fluence required for 4-log inactivation of WN333 spores was found to be 80-90 J / m² and 180-190 J / m², respectively (Fig. 4), which we attribute to minor variations in the execution of the experiment at the two locations. At both locations, however, WN333 spores exhibited low pressure UV inactivation kinetics similar to spores of the three Encephalitozoon species tested (Fig. 4).

[049] The above experiments were performed at room temperature (ca. 25°C). However, the water temperature in many municipal water treatment situations can be considerably lower, which we thought might affect the UV inactivation kinetics of microsporidial spores. In order to test this notion, spores of E. intestinalis were irradiated at UA to a final fluence of 140 J/ m² at either 25°C or 5°C, and exhibited >3.2 log inactivation at both temperatures (Table 1 ). Therefore, spores irradiated at 5°C were not more resistant to UV. In addition, the UV inactivation kinetics of β. subtilis WN333 and WN626 spores at UA was found to be the same at 25°C and 5°C (data not shown). [050] In conclusion, the data presented here indicate that spores of the microsporidial species E. intestestinalis, E. cuniculi and E. hellem exhibit UV inactivation kinetics similar to those of C. parvum oocysts, and that both microsporidian spores and oocysts would be inactivated by a low-pressure UV fluence of 200 J/m². Spores of Bacillus subtilis biodosimetry strain WN333 closely mimic the Encephalitozoon dose-response curves, and thus provide a suitable surrogate for Encephalitozoon spp. in UV reactor validation studies, β. subtilis spores have several advantages for use as a biodosimetry surrogate. They: (i) are non-pathogenic; (ii) do not require eukaryotic cell culture or animal facilities; (iii) afford testing results in <_24 hours (as opposed to 6-10 days required for the Encephalitozoon in vitro cell culture assay); and (iv) can be used in-house by any municipal water testing facility equipped to perform basic microbiology. The ease and speed of manipulation and the potential production capability makes WN333 a strong potential candidate for use as a biodosimetry surrogate for the Encephalitozoon spores in a broad variety of UV testing and verification applications.

[051] For example, because the log 3 inactivation ranges from about 15 to over 800 J/m², the spores described in this application can be used as biodosimetry surrogates not only for intestinal parasites described in the examples, but for many pathogenic bacterial and viral species which are known to pose risks for water- air- or food-borne contamination, such as those disclosed by Hoyer, O. (1998) Water Supply 16(1 /2):419-442. In addition, the inventive spores described herein can be used as a surrogate for bacterial spores of pathogenic sporeforming species such as Bacillus anthracis to verify screening procedures for mail and the like.

[052] Although the invention has been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications may be made without departing from the invention. The foregoing examples are provided to better illustrate the invention and are not intended to limit the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. An isolated B. subtilis bacterium having the identification characteristics of strain WN624, WN625, or WN626.

2. The bacterium of claim 1 , wherein said β. subtilis bacterium comprises a spore.

3. A surrogate for measuring a dose of radiant energy administered to a pathogenic organism, said surrogate comprising an isolated B. subtilis bacterium having the identification characteristics of strain WN624, WN625, or WN626.

4. The surrogate of claim 3, wherein said bacterium comprises a spore.

5. The surrogate of claim 3, wherein said radiant energy comprises ultra violet light.

6. The surrogate of claim 3, wherein said radiant energy is selected from the group consisting of electromagnetic radiation, X-rays, γ-rays, electron beams or combinations thereof.

7. The surrogate of claim 3, wherein said pathogenic organism comprises a protozoan organism or spore thereof.

8. The surrogate of claim 7, wherein said protozoan organism or spore thereof is selected from the group consisting of Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon hellem,Cryptosporidium parvum, or combinations thereof.

9. The surrogate of claim 7, wherein said protozoan organism or spore thereof is selected from the group consisting of Giardia lamblia, Cyclospora, or combinations thereof.

10. The surrogate of claim 3, wherein said pathogenic organism comprises a bacterial organism or spore thereof.

11. The surrogate of claim 10, wherein said bacterial organism or spore thereof is selected from the group consisting of Escherichia coliATCC 11, E. coli ATCC 23958, E. coli NCTC 5934, E. coli NCIB 9481 , E. coli wild isolate, Enterobacter cloaca, Klebsiella pneumoniae, Citrobacter freundii, Yersinia enterocolitica, Salmonella Typhi, Salmonella Typhimurium, Serratia marcescens, Enterocolitica faecium, Vibrio cholerae wild isolate, Pseudomonas aeruginosa, Mycobacterium smegmatis, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, C. perfringens, C. tetani, and C. difficile.

12. The surrogate of claim 3, wherein said pathogenic organism comprises a viral organism.

13. The surrogate of claim 12, wherein said viral organism is selected from the group consisting of Polio virus (Mahoney), Rotavirus SA 11, or Staphylococcus aureus phage A994.

14. A method of biodosimetry, comprising the steps of: a. providing spores isolated from B. subtilis bacteria having the identification characteristics of strain WN624, WN625, WN626, WN333 or combinations thereof, b. irradiating said spores with a source of radiant energy; and c. determining a number of active spores remaining post irradiation.

15. The method of claim 14, wherein said source of radiant energy comprises ultra violet light.

16. The method of claim 14, wherein said source of radiant energy is selected from the group consisting of electromagnetic radiation, X-rays, γ-rays, electron beams or combinations thereof.

17. The method of claim 14, further comprising the step of comparing the number of active spores to a known radiant energy dose response value for a pathogenic organism of interest.

18. The method of claim 17, wherein said pathogenic organism comprises a protozoan organism or spore thereof.

19. The method of claim 18, wherein said protozoan organism or spore thereof is selected from the group consisting of Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon hellem, Cryptosporidium parvum, or combinations thereof.

20. The method of claim 18, wherein said protozoan organism or spore thereof is selected from the group consisting of Giardia lamblia, Cyclospora, or combinations thereof.

21. The method of claim 17, wherein said pathogenic organism comprises a bacterial organism or spore thereof.

22. The method of claim 21 , wherein said bacterial organism or spore thereof is selected from the group consisting of Escherichia coliATCC 11, E. coli ATCC 23958, E. coli NCTC 5934, E. coli NCIB 9481, E. coli wild isolate, Enterobacter cloaca, Klebsiella pneumoniae, Citrobacter freundii, Yersinia enterocolitica, Salmonella Typhi, Salmonella Typhimurium, Serratia marcescens, Enterocolitica faecium, Vibrio cholerae wild isolate, Pseudomonas aeruginosa, Mycobacterium smegmatis, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, C. perfringens, C. tetani, and C. difficile.

23. The method of claim 17, wherein said pathogenic organism comprises a viral organism.

24. The method of claim 23, wherein said viral organism is selected from the group consisting of Polio virus (Mahoney), Rotavirus SA 11 , or Staphylococcus aureus phage A994.

25. A method of biodosimetry, comprising the steps of: a. determining a radiant energy dose response for a pathogenic organism, b. selecting, based upon the dose response measured in step (a), spores isolated from B. subtilis bacteria having the identification characteristics of strain WN624, WN625, WN626, WN333 or combinations thereof, c. irradiating said spores isolated from B. subtilis with a source of radiant energy; and d. determining a number of active spores remaining post irradiation.

26. The method of claim 25, wherein said source of radiant energy comprises ultra violet light.

27. The method of claim 25, wherein said source of radiant energy is selected from the group consisting of electromagnetic radiation, X-rays, γ-rays, electron beams or combinations thereof.

28. The method of claim 25, further comprising the step of comparing the number of active spores isolated from B. subtilis to the radiant energy dose response for the pathogenic organism of step (a).

29. The method of claim 25, wherein said pathogenic organism comprises a protozoan organism or spores thereof.

30. The method of claim 29, wherein said protozoan organism or spores thereof is selected from the group consisting of Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon hetlem,Cryptosporidium parvum, or combinations thereof.

31. The method of claim 29, wherein said protozoan organism or spores thereof is selected from the group consisting of Giardia lamblia, Cyclospora, or combinations thereof.

32. The method of claim 25, wherein said pathogenic organism comprises a bacterial organism or spores thereof.

33. The method of claim 32, wherein said bacterial organism or spores thereof is selected from the group consisting of Escherichia coli ATCC 11, E. coli ATCC 23958, E. coli NCTC 5934, E coli NCIB 9481 , E. coli wild isolate, Enterobacter cloaca, Klebsiella pneumoniae, Citrobacter freundii, Yersinia enterocolitica, Salmonella Typhi, Salmonella Typhimurium, Serratia marcescens, Enterocolitica faecium, Vibrio cholerae wild isolate, Pseudomonas aeruginosa, Mycobacterium smegmatis, Bacillus anthracis, Bacillus cereus, Clostridium botulinum, C. perfringens, C. tetani, and C. difficile.

34. The method of claim 25, wherein said pathogenic organism comprises a viral organism.

35. The method of claim 34, wherein said viral organism is selected from the group consisting of Polio virus (Mahoney), Rotavirus SA 11 , or Staphylococcus aureus phage A994.

36. A method for validating a water disinfection protocol, comprising the steps of: a. providing spores isolated from B. subtilis bacteria having the identification characteristics of strain WN624, WN625, WN626, WN333 or combinations thereof, b. irradiating said spores with a source of radiant energy, c. determining a number of active spores remaining post irradiation; and d. comparing said number of active spores to a known radiant energy dose response value for a pathogenic organism of interest.