NZ549568A - Bacteriophage composition useful in treating food products to prevent bacterial contamination - Google Patents

Abstract

A method for preventing the growth of E. coli microorganisms of a food product comprising contacting the food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolate 119U, wherein the application time of said bacteriophage composition is for at least of fraction of a second to prevent the growth of microorganisms on the food product

Description

*10052440967* 54 95 6 8 PATENTS FORM NO. 5 Our ref: JP803054NZPR NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION DIVISIONAL APPLICATION OUT OF NEW ZEALAND PATENT APPLICATION NO. 538985 FILED ON 15 November 2001 Bacteriophage composition useful in treating food products to prevent bacterial contamination We, Nymox Pharmaceutical Corporation of 9900 Cavendish Boulevard, Suite 306, St. Laurent, Quebec, H4M 2V2, Canada hereby declare the invention, for which we pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: 1 INTELLECTUAL PROPERTY OFFICE OF N.Z. 3 t AUG 2006 RECEIV E D -tern p. doc: J P: q wg tn WO 02/44322 PCT/IB01/02836 BACTERIOPHAGE COMPOSITION USEFUL IN TREATING FOOD PRODUCTS TO PREVENT BACTERIAL CONTAMINATION FIELD OF THE INVENTION The present invention is directed to a bacteriophage composition useful in treating food products to prevent bacterial contamination by Escherichia coli bacteria.

BACKGROUND OF THE INVENTION The Centers for Disease Control and Prevention (CDC) estimates that as many as 33 million people develop food poisoning each year - about one out of every ten Americans - and about 9,000 die. Since many cases of food poisoning are mistaken for a 24-hour "stomach flu," the actual number of people with foodborn illness is probably much 10 higher. Cerrato, P., "When food is the culprit; food poisoning," RN, 62(6):52 (Jun. 1, 1999). The presence of foodborn pathogens in a country's food supply not only affects the health of the local population, but also represents a potential for spread of pathogens to visitors to the country and to consumers in countries which import food products. Buzby et al., "Economic costs and trade impacts of microbial foodbome illness," World Health Stat. Q., 50(l-2):57-66 15 (1997).

Prevention of foodborne illnesses by microbial contamination is of major concern to the food processing industry, regulatory agencies, and consumers. Foodborne microbial contamination occurs both prior to entry into the processing facility, and by cross-contamination in the processing environment. The Food Safety & Inspection Service (FSIS) 20 of the United States Department of Agriculture has instituted new Hazard Analysis and Critical Control Point (HACCP) requirements to reduce the occurrence and number of foodbome pathogens. These regulations must be met by food processors. Although the means of achieving this microbial reduction is left to the discretion of the processor, FSIS expects that antimicrobial treatments will be an important component of HACCP plans. The 25 treatment methods of the present invention, which employ formulations of the bacteriophage compositions of the invention, are useful in meeting the HACCP requirements.

In their efforts to provide a product completely free of microbial contamination, poultry and meat processors have encountered major difficulties in removing microorganisms that infect poultry and meat tissues intended as food products.

WO 02/44322 PCT/IB01/02836 E. coli Bacteria E. coli bacteria are short Gram-negative rods that are part of the normal flora of the intestines of most warm-blooded animals. These organisms are the most common 5 facultative anaerobe in the large bowel and provide protection against colonization by other harmful microbes. There are, though, five distinct groups of E. coli that can cause enteric disease: (1) enteroinvasive, (2) enteropathogenic, (3) enterotoxigenic, (4) enteroadherent, and (5) enterohemorrhagic. Pilot et al., "Threats from the food we eat; includes related articles; New and Emerging Pathogens, part 3," Medical Laboratory Observer, 28:42 (Apr. 1996). 10 E. coli 0157:H7 bacteria, first described in 1982, is a subset of the enterohemorrhagic variety that produces Shiga-like toxins (verotoxins) 1 and 2. Pilot et al, (Apr. 1996); and "Medical Experts Urge Radiation of Beef to Kill Deadly Bacteria," The New York Times, July 14, 1994, at page A15.

The toxin produced by E. coli 0157:H7 in the intestines can cause anything 15 from a mild diarrhea to severe hemorrhagic colitis, where the cells of the intestinal lining are damaged, allowing blood to pass into stool. In as many as 16% of all cases, the infection progresses to a more serious condition - hemolytic-uremic syndrome (HUS). Mackenzie, D.L., "When E. coli turns deadly," RN, <52(7):28 (Jul. 1, 1999). HUS, which occurs when the bacterial toxin enters the bloodstream through the damaged intestinafwall and travels to the 20 smaller arteries that supply the kidneys, going on to damage those vessels, is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. In addition, anywhere from 10% - 50% of patients with HUS develop long-term sequelae, including cardiomyopathy, pulmonary problems, pericardial effusions, end-stage renal disease, chronic hypertension, hyperglycemia, and encephalopathy. Mackenzie, D.L. (Jul. 1, 1999). 25 E. coli 0157:H7 is a leading cause of HUS in the United States and a leading cause of acute renal failure in children. Although the overall incidence of Is. coli 0157:H7 infection is unknown, estimates based on a 1994 outbreak in the Seattle area suggest that more than 20,000 cases occur in this country each year, and that 250 of them result in death. Mackenzie, D.L. (Jul. 1,1999); and "Ban the 0157:H7 Bomb," Nutrition Action Healthletter, 30 22:3 (Jan. 1995/Feb. 1995). Young children, the elderly, and people with weakened immune systems are most susceptible to infection and resulting complications. 2 WO 02/44322 PCT/IB01/02836 There is no known treatment for is. coli 0157:H7 infection. Nutrition Action Healthletter, 22:3 (Jan. 1995/Feb. 1995). Antibiotics do little to deter the infection.

' "Medical Experts Urge Radiation of Beef to Kill Deadly Bacteria," The New York Times, July 14,1994, at page A15; and Cerrato, P. (Jun. 1,1999) One reason this strain of E. coli is so dangerous is that it can cause disease in such low doses - ingestion of less than 1,000 organisms compared to more than the 10,000,000 needed before the bacterium that causes cholera causes disease. Cerrato, P. (Jun. 1, 1999). Some reports state that E. coli 0157:H7 can cause illness with as few as 69. The New York Times, July 14, 1994, at page A15. As little as one-half cup of E. coli-10 contaminated cider can cause abdominal pain, bloody diarrhea, vomiting and fever.

"Roadside cider may be risky; may be contaminated with E. coli," Environmental Nutrition, 16:8 (Sept. 1993). Such small amounts of disease-causing bacteria cannot be detected by routine meat inspection practices, and the presence of contaminated food products cannot be identified by sight, taste or smell. The New York Times, July 14, 1994, at page A15. 15 Similarly, the pathogen's acid resistance gives it an advantage over other pathogens. Gastric acid, one of the gastrointestinal tract's first lines of defense against foodborne illness, has little effect on£. coli 0157:H7. Cerrato, P. (Jun. 1, 1999).

E. coli 0157 is found regularly in the feces of healthy cattle, and is transmitted to humans through contaminated food, water, and direct contact witKlnfected people or 20 animals. Mead et al., "Escherichia coli 0157:H7," The Lancet, 352(9135): 1207-1212 (Oct. 10, 1998). Transmission of the infection is primarily linked to consumption of undercooked ground beef, contaminated drinking water, and unpasteurized milk. Hamburger is a major vehicle of foodborne outbreaks of E. coli 0157:H7 infection. Koutkia et al., "Enterohemorrhagic Escherichia coli 0157:H7," American Family Physician, 56:853 (Sept. 25 1, 1997).

. During the slaughter process, intestinal fluid or feces of infected cattle can drip onto the surface of the meat, contaminating it. It is theorized the harmful bacteria on the surface of the raw meat become mixed throughout the meat during the grinding process, where it can better survive the heat of cooking. One hamburger patty can contain the meat 30 from many cows.

Currently, E. coli 0157:H7 is the most common of verotoxin-producing E. coli serotypes. Various sources indicate it comprises from about 60% to more than 90% of all 3 WO 02/44322 PCT/IB01/02836 toxin-producing isolates and has been responsible for most outbreaks when food sources have been implicated. Pilot et al. (Apr. 1996). Distribution of E. coli 0157:H7 probably is worldwide; the majority of cases have been noted in North America and Europe. Pilot et al. (Apr. 1996).

Current Methods of Treating Food Products to Eliminate Bacterial Contamination A. Irradiation Food irradiation is the treatment of foods by subjecting them to ionizing radiation, also called ionizing energy. The radiation used in the process comes either from radioactive isotopes of cobalt or cesium or from devices that produce controlled amounts of high-energy electrons, gamma rays, or X rays. The process does not and cannot make the food radioactive. Greenberg et al., "Irradiated Foods," American Counsil on Sci. and Health 15 Booklets, 1-28 (April 30, 1996).

The radiation used to treat foods is called "ionizing radiation" because it produces ions — electrically charged particles. Ionizing radiation — including X rays, gamma rays, and beams of high-energy electrons produced by electron accelerators - has a higher energy than other, nonionizing radiation such as visible light, television waves, radio waves 20 and microwaves. Greenberg et al. (April 30, 1996).

Food irradiation has been proposed as one solution to the food safety problem. Food irradiation was first approved by the EDA in 1963 for the control of insects in wheat flour and again in 1964 to prevent sprouting in potatoes. Later, it was approved for use in spices, produce, and poultry, and on December 3,1998, it was approved for beef, lamb, and 25 pork to control disease-causing microorganisms. Klausner, A., "Food Irradiation: We May Be Zapping Up The Wrong Tree," Environmental Nutrition, / 7(12): 1 (Dec. 31,1994).

Two radiation sources are practical for food treatment. The first is a tightly sealed metal container of radioactive elements - cobalt 60 or cesium 137 — that produce gamma rays. The rays are directed onto the food being irradiated, with the food itself never 30 being touched by the cobalt or cesium. The second type of radiation source is a machine that produces X rays and high-energy electrons. Neither of these sources has enough energy to make the irradiated foods radioactive. Greenberg et al. (April 30,1996). 4 WO 02/44322 PCT/IBO1/02836 During the irradiation process, food is carried on a conveyor belt into a sealed chamber where it is hit with large doses of energy from a radiation source (usually gamma rays from radio-active cobalt). The energy disrupts molecules in the food, killing insects, molds, fungi and bacteria that cause spoilage, as well as pathogens that cause foodborne 5 illnesses. Klausner, A. (Dec. 31,1994) Consumer advocates warn that food irradiation is not a panacea. In fact, they say, it poses safety risks of its own. For example, food irradiation opponents cite research showing the process depletes nutrients (vitamins A, C, and the B vitamin, thiamin). The greater the radiation dose, the greater the losses. In addition, an irradiated food must still 10 undergo normal nutrient-depleting processing, compounding the losses. Klausner, A. (Dec. 31, 1994) Food irradiation also forms residual substances such as benzene and formaldehyde — both known carcinogens. And it produces free radicals and chemically altered food components called radiolytic products, whose long-term health effects are 15 unknown. Klausner, A. (Dec. 31,1994). It is hypothesized that changes from such food irradiation could lead to more cancers. Id. Moreover, reports on consumer acceptance are mixed. Taste tests for irradiated beef first reported in The Economist failed miserably, as consumers described a "burnt-hair" taste. Klausner, A. (Dec. 31, 1994).

Another drawback of irradiation as an bacterial-control process is that 20 irradiation, like heat treatment, does not leave any active agent in an unpackaged food to protect it against reinfestation. Greenberg et al. (April 30, 1996).

B. Chemical Treatments of Meat Several chemical and physical methods have been proposed to reduce 25 microorganisms in meat products, such as the use of chlorine or chlorine dioxide, ozone, hydrogen peroxide, lactic acid, sodium carbonate, trisodium phosphate, and electrical stimulation. Generally, these methods have shown limited effectiveness in reducing microbial contamination and may affect the physical appearance of the meat products. In addition, such studies show that frequently unacceptably high levels of chemicals are 30 required to kill pathogens such as E. coli 0157:H7. Id.

For example, U.S. Patent No. 5,366,983, incorporated herein by reference, discloses a method for removing or preventing Salmonella contamination of meat products by WO 02/44322 PCT/IB01/02836 treatment with an effective amount of an aqueous solution of a quaternary ammonium cationic surfactant, such as alkylpyridinium, particularly cetylpyridinium chloride (CPC) and cetylpyridinium bromide (CPB).

Disinfestation by irradiation can substitute for some of the former uses of the 5 now-banned fumigant ethylene dibromide (EDB). The possibility of such a replacement is one of the major reasons for renewed interest in irradiation in the United States, as the chemical fumigants being used currently in place of EDB have serious disadvantages, including increased hazards for the workers who must apply them. Greenberg et al. (April 30,1996).

Another disadvantage of chemical disinfectants is that residues of such chemicals invariably remain on the foodstuffs. Greenberg et al. (April 30, 1996).

Treatment with chemical disinfectants can also produce resistant bacteria. "Growing Menace: Antibiotic-Resistant 'Supergerms'," Thelnt'l. Council for Health Freedom Newsletter, 77(3-4): 18 (Fall, 1998); Freeman, C., "Antimicrobial Resistance: 15 Implications for the Clinician," Critical Care Nursing Q., 20(3):21 (Nov. 1997).

C. Pulsed Light Yet another method for treating food products to eliminate bacterial contamination is treatment with high intensity light emissions, produced by a pulsed power 20 energization technique (PPET). MacGregor et al., "Light inactivation of food-related pathogenic bacteria using a pulsed power source," Letters in Applied Microbiology, 27(2):67-70 (1998). This process comprises dissipating many megawatts (MW) of peak electrical power in the light source in an extremely short energization time (about 1 (mu)s). The light source is subjected to electric field levels greater than could be achieved under conventional 25 continuous operation, which leads to a greater production of the shorter bacteriocidal wavelengths of light.

The usefulness of this process is limited, as the pulsed light emissions can only significantly reduce bacterial populations on exposed surfaces. Such a process is not useful for treating, for example, ground hamburger. This is significant as recent studies indicate that 30 E. coli bacteria can be present on the interior of foodstuffs such as apples, which would not be treatable using a pulsed light process. Buchanan et al., "Contamination of intact apples after 6 immersion in an aqueous environment containing Escherichia coli 0157:H7," J. Food Prot., 62(5):444-50 (May, 1999).

***** It is an object of the invention to go some way in providing a superior composition and method for treating food stuffs to eliminate bacterial contamination by E. coli bacteria, particularly toxin-producing E. coli bacteria, or to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION The present invention is directed to novel phage compositions useful in treating food products to minimize or eliminate bacterial contamination by E. coli bacteria, particularly toxin-producing E. coli bacteria. The phage compositions can be formulated with suitable carriers.

Also encompassed by the invention are methods of treating food products, such as meat (including but not limited to poultry, beef, lamb, and pork), juices, spices, and produce.

The invention provides a purified bacteriophage composition comprising the bacteriophage isolate 119U. (ATCC No. 209998).

The invention describes a purified bacteriophage composition comprising the bacteriophage isolate B3. (ATCC No. 209999).

The invention also describes a purified bacteriophage composition comprising the bacteriophage isolate 146A. (ATCC No. 55950).

The invention also provides a purified bacteriophage composition comprising at least two bacteriophage isolates selected from the group consisting of Bl, 119U and 146A.

The invention provides a method for preventing the growth of E. coli microorganisms on a food product comprising: contacting said food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolate 119U, wherein the application time of said bacteriophage composition is for at least a (Followed by page 7a) 7 wfELLECTUAL property OFFICII OF N.I. 2 5 JAM 2007 nrrkCI\/P.n fraction of a second to prevent the growth of microorganisms on the food product.

The invention also describes a method for preventing the growth of E. coli microorganisms on a food product comprising: contacting said food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolate B3, wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent the growth of microorganisms on the food product.

The invention also describes a method for preventing the growth of E. coli microorganisms on a food product comprising: contacting said food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolate 146A, wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent the growth of microorganisms on the food product.

The invention also describes a method for preventing the growth of E. coli microorganisms on a food product comprising: contacting said food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolates selected from the group consisting of Bl, B3, 119U and 146A, wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent the growth of microorganisms on the food product.

The invention also describes a method for preventing the growth of E. coli' microorganisms on a surface which comes in contact with food comprising: contacting said surface with a microbial growth inhibiting effective amount of a bacteriophage composition comprising at least two bacteriophage isolates selected from the group consisting of Bl, 119U and 146A wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent (Followed by page 7b) 7a intellectual property office of N.z. 2 5 JAN 2007 received the growth of microorganisms on the food product.

Both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide farther explanation of the invention as claimed. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including, but not limited to".

The applicant also refers to its parent applications NZ 526537 and NZ538985.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Provides a description of the open reading frames (ORFs) of Phage 119U; Figure 2: Shows the DNA sequence of the head of Phage Bl; Figure 3: Shows the DNA sequence of the tail of Phage Bl; Figure 4: Shows the amino acid sequence of the tail of Phage Bl; Figure 5: Shows the DNA sequence of the head of Phage B3; Figure 6: Shows the DNA sequence of the tail of Phage B3; Figure 7: Shows the amino acid sequence of the tail of Phage B3; Figure 8: Shows the host ranges for the bacteriophages Bl, B3, 146A, and 119U against a large number ofi?. coli isolates; (Followed by page 8) 7b WO 02/44322 PCT/1B01/02836 Figure 9: Shows the growth of bacteria over time as compared to the concentration of Phage 119U; Figure 10: Shows the growth of bacteria over time as compared to the concentration of Phage B3; and Figure 11: Shows the host range of Phages Bl arid B3 against thirty-three different strains, and fifteen different serotypes, of human and porcine E. coli, including the-toxin producing E. coli strain 0157.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed to novel phage compositions useful in 10 treating food products to minimis or eliminate bacterial contamination by E. coli bacteria, particularly toxin-producing E. coli bacteria. The phage compositions can be formulated with suitable carriers.

Bacteriophages Bl and B3 were isolated from raw sewage using an E. coli 0157:H7 strain as a host. Phages 146A and 119U were isolated against a urinaiy tract 15 infection (UTI) isolate. Phages Bl and B3 were deposited at the American Type Culture Collection (Manassas, VA) under Accession Nos. 203000 and 209999 on June 24, 1998. Phage ]46A was deposited under Accession No. 55950 on April 14, 1997, and Phage 119U was deposited under Accession No. 209998 on June 24, 1998, both a? the American Type Culture Collection. The bacteriophage compositions can be in an aqueous or non-aqueous 20 medium.

The present invention provides a method for preventing growth of microorganisms on food products comprising contacting a food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolate B1, B3,119U, 146A, or a combination thereof, for the prevention of 25 growth of E. coli microorganisms on food products. The prevention of growth of microorganisms on food products is intended to provide a food product that is devoid of or contains minimal numbers of viable microorganisms that could cause illness in humans or animals or spoilage of the food product prior to ingestion.

The prevention of growth of microorganisms on food products is intended to 30 include but is not limited to the following mechanisms: (1) removal of attached microorganisms from the food products; (2) inhibition of attachment of microorganisms to 8 WO 02/44322 PCT/IB01/02836 the food products; (3) killing or inactivation of attached microorganisms on the food products; and (4) killing or inactivation of microorganisms which are not attached to the food product but which are present in liquids associated with the food products during processing; such as in chill tanks, or which are present on surfaces associated with food preparation, 5 liquids remaining on such surfaces, such as countertops, cutting boards and sinks, and equipment used in food preparation and sanitization of the food.

The present invention has an important application in the food processing industry, as well as for home and institutional food preparation. The bacteriophage compositions of the invention are readily available and the cost of carrying out the method of 10 the present invention is not expensive as compared to existing antimicrobial processes.

Unlike existing treatments using, for example, trisodium phosphate, the use of the bacteriophage compositions of the invention does not alter the appearance, color, taste, or texture of the food product. Moreover, the bacteriophage compositions of the invention are non-toxic.

The bacteriophage composition is applied for a period of time sufficient to kill E. coli bacteria present on the food product. It is important that the application time of the bacteriophage compositions is for a sufficient time to result in significant prevention of growth of E. coli microorganisms on the food product.

The present invention also includes methods of contacting the bacteriophage 20 compositions of the invention with food products, including but not limited to, spraying or misting the compound on the food product, or by immersing the food product in a composition comprising one or more bacteriophages of the invention.

The present invention is intended to encompass any method that contacts the bacteriophage compositions of the invention with a food product by any direct means, 25 including spraying, misting, dipping, or soaking. But the present invention also is intended to include contact of the bacteriophage compositions of the invention with the food by indirect means, such as applying the bacteriophage compositions of the invention to equipment or food product processing or preparation surfaces in which the food product is contacted during processing, preparation, storage, and/or packaging.

Any type of method of contact of the bacteriophage compositions with the food product is useful in the present method as long as it is capable of allowing a short application time.- A method that utilizes a cabinet that provides spraying or misting of the 9 WO 02/44322 PCT7IB01/02836 food product is useful in the present invention. Machineiy for use in such cabinets on a processing line 121 a food processing plant are adaptable for reducing the application time to a minimum while still obtaining efficacious antimicrobial effects on the food.

The present method is useful, for example, in a poultry processing plant for 5 treating post-chilled chickens that have been immersed in a chill bath of cold water. The chickens are removed from the chill bath and treated with the bacteriophage compositions of the invention for an application time sufficient to result in significant prevention of growth of microorganisms on the chickens. The treated chickens are subsequently packaged without further washing or rinsing. However, the method optionally may include, if deemed 10 necessary, at least one washing step of the chickens prior to packaging. The optional washing step may include spraying or misting the food product with water or immersing the food product in a container or tank of water.

Further, the method of the present invention can optionally include a determination step prior to contacting the food product with the bacteriophage compositions 15 of the invention to determine the presence of microorganisms on the food before treatment. Any conventional methods for rapidly determining the presence of microorganisms can be utilized as the determination step, which for example, includes PCR and immunoassays.

Additionally, the method of the present invention optionally includes a step to determine the presence of the bacteriophage compositions of the invention on the surface of 20 the food product after contact with the bacteriophage compositions. This determination is performed immediately after the contacting step or after several washing steps. For example, the bacteriophage compositions of the invention is extracted from the tissues of the food in a form suitable for high performance liquid chromatography (HPLC) analysis.

The food processing industry, as well as home, restaurant or institutional food 25 preparation, is in need of more effective products and processes for the prevention of growth of a broad range of contaminating microorganisms on many different food products and/or surfaces that the food products and juices or liquids from the food come in contact. This is especially true for microorganisms which are attached to the surfaces of food. As a result of increasing numbers of illnesses caused by foodbome pathogenic microorganisms, the food 30 processing industry now requires more effective processes for the removal and prevention of a broader spectrum of microorganisms, and particularly for pathogenic microorganisms, such as, toxin-producing Escherichia coli, such as E. coli 0157:H7, which are known to cause WO 02/44322 PCT/IB01/02836 serious human diseases as a result of food contamination. The present invention provides a composition comprising at least one bacteriophage of the invention and methods of preventing the growth of microorganisms on and in the food, as well as in liquids and on surfaces associated with food products and their preparation. This method of prevention is an important goal in preventing cross-contamination from infected food products; in removing attached microorganisms from food products; in inhibiting the attachment of microorganisms to the food products; and in preventing the growth of microorganisms that remain attached to the food products. Further, the method of the present invention can easily be adapted for use in a food processing plant.

* * * * * The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all 15 references to a publicly available document, including a U.S. patent, are specifically incorporated by reference.

Example 1 The purpose of this example was to describe the isolation and characterization of the novel bacteriophages Bl, B3, and 119U.

Bacteriophages B1, B3, and 119U were isolated from raw sewage using an E. coli 0157:H7 strain as a host. The strains were isolated according to the method described in U.S. Patent No. 6,121,036, which is specifically incorporated by reference.

The method comprised (a) obtaining a sample containing a bacteriophage to at least one bacterial organism selected from the group consisting of staphylococci, hemophilii, 25 helicobacter, mycobacterium, mycoplasmi, streptococci, neisserii, klebsiella, enterobacter, proteus, bacteriodes, pseudomonas, borrelii, citrobacter, escherichia, salmonella, propionibacterium, treponema, shigella, enterococci and leptospirex; (b) dispersing the sample in phosphate buffered saline; (c) filtering the dispersed sample through a filter that will retain the bacterial organism and allow the bacteriophage to pass; (d) purifying the 30 bacteriophage that passes through the filter; (e) growing bacteriophage in medium containing at least one of the bacterial organisms; (f) selecting and isolating bacteriophage preparations li WO 1)2/44322 achieving titers higher than about 10s to 109 bacteriophages per plaque after about eight hours to provide an isolated bacteriophage; (g) purifying the isolated bacteriophage; and (h) repeating steps (e)-(g) at least 5 times using the purified isolated bacteriophage of step (g) in step (e) to prepare the bacteriophage preparation.

All phage isolates were plaque purified at least 3 times to obtain a pure culture. Large scale preparations were done using CsCl equilibrium gradient centrifiigation. (Gilakjan et al, J. Bacteriol., 181:1221-1 (1999).) There are six major families of bacteriophages, one of which is Myoviridae, also known as T-even bacteriophages. (U.S. Patent No. 6,090,541.) Phages Bl and B3 are 10 similar in moiphology to the T-even phages (i.e., T2, T4, and T6), which includes the well characterized T4.

Phage 119U is unique in its genome (sequence), host range, and virulence; it has both strain K1 and K5 antigen lysase genes; and it is non-toxic and efficacious. Phage 119U, having a linear double-strand DNA genome of 44 kb is highly effective against the 15 highly virulent K1 and K5 strains of E. coli, as its sequence codes for both the Kl- and K5-specific proteins. Figure 1 provides a description of the open reading frames (ORFs) of Phage 119U. Phage 119U shows no evidence of lysogeny, horizontal gene transfer, virulence genes (conferring virulence to a bacterial host), or toxin genes (conferring toxicity to a sty bactenal host).

Example 2 The purpose of this example was to isolate and characterize bacteriophage 146A.

Phage 146A was isolated against a urinary tract infection (UTI) isolate using 25 the method described in Example 1.

Phage 146A is also similar to the T-even phages but appears to have a slightly smaller head structure than the others. (Aebi et al., "Comparison of the Structural and Chemical Composition of Giant T-even Phage Heads," J. Supramol. Struct., J(4):475-495 (1977); Russell, R.L., "Comparative Genetics of the T Even Bacteriophages," Genetics, 30 7<5(4):967-988 (1978); and Kutter et al., "Evolution of Tr-related Phages," Virus Genes, 11(2-3):285-297 (1995).) 12 Example 3 The purpose of this example was to prepare restriction digests and partial sequences for the bacteriophages Bl and B3 to further characterize the phages.

Restriction Digests DNA preparations for Bl and B3 were made from CsCl stocks using the phenol/chloroform extraction method. (Chomczynski et al., Analyt. Biochem., 162:156-159 Restriction digests Phages Bl andB3 were performed. Digested DNA was run on 0.6-0.7% TAE (Tris-acetate-ethylenediamine tetraacetic acid (EDTA)] running buffer 10 agarose gels with a X HinDITT marker. The restriction enzymes EcoRI, BamHI, HinDIE, PstI, Xhol, and Bgll all failed to cut either Bl or B3 DNA. The only two enzymes that did cut were Dral and Vspl.

These results are typical of the T-even phages; the DNA of T-even phages contains D-glucosyl hydroxymethyl cytidine instead of cytidine, which blocks cleavage by 15 most restriction enzymes. Dral and Vspl are able to cut because there are no G-C base pairs in their recognition sequences. The restriction patterns for Dral and Vspl digests of Bl, B3, and T4 are all different.

Phages Bl and B2 were restriction mapped, with the resulting maps being unique as compared to all other published DNA enzyme restriction maps. 3, respectively, and the amino acid sequence of the tail of Phage Bl is shown in Figure 4. In addition, the DNA sequence of the head and tail of Phage B3 is shown in Figures 5 and 6, 25 respectively, and the amino acid sequence of the tail of Phage B3 is shown in Figure 7. Phages Bl and B3 are estimated to have genomes of about 150,000 bp. (1987).) Partial Sequencing The DNA sequence of the head and tail of Phage Bl is shown in Figures 2 and Example 4 The purpose of this example was to determine the host ranges for the bacteriophages Bl, B3,146A, and 119U against a large number of urinary tract infection 13 WO 02/44322 PCT/IB01/02836 (UTI) E. coli isolates. Bacteriophage 119U is specific to E. coli strain Kl, and probably E. coli strain K5.

Host Ranges of Bl, B3,146A, and 119U 104 pfu of each phage was spotted on a lawn for each UTI bacterial isolate and 5 the plate was incubated overnight at 37°C. A plus (+) indicates complete clearing, a plus(t) (+(t)) indicates a turbid spot, a plus(p) (+(p)) indicates a spot that has small individual plaques, and a minus (-) indicates that the phage had no effect on the bacterial isolate.

All four phages showed fairly broad host ranges. Individually, the host range of the four phage preparations was 30 to 60%, and collectively the host range was 84%. Each 10 isolate lysed at least 40% of the E. coli isolates, as shown in Figure 8. In addition, the phages complement each other as they overlap in their ability to clear the bacterial isolates.

Specifically, Phages Bl and B3 lysed 57% and 49%, respectively, of the 61 is. coli UTI isolates, and additionally lysed E. coli strain K12; and Phages 146A and 119U lysed 31% and 30%, respectively, of the 61 E. coli UTI isolates. See Figure 8. 15 Phage T4 did not lyse any of the UTI isolates, as it only grew on E. coli K12 (ATCC Deposit No. 29425) (data not-shown).

Example 5 The purpose of this example was to analyze the efficacy of phages 119U and 20 B3 by determining the minimal amount of phage that will lyse a susceptible bacteria grown in urine.

The urine used was collected from a healthy adult male, and was sterilized by passing through a 0.45 jiM filter. 50 ml flasks of urine were inoculated with 100 p.1 of an LB overnight culture of E. coli 119U. The innoculum was spun down and washed in urine to 25 remove any residual LB media. 102,104, and 106 pfu of Phage 119U or B3 was added at the beginning, one flask had zero phage. The cultures were incubated at 37°C with moderate shaking (200 rpm in New Brunswick incubator). OD 600 was measured to monitor bacterial growth.

The results showed that as little as 102 pfu of Phage 119U completely lysed the 30 culture. See Figures 9 (Phage 119U) and 10 (Phage B3), showing the growth of bacteria over time as compared to the concentration of phage. With no phage, bacterial growth (correlated 14 WO 02/44322 PCT/IB01/02836 to absorbance) dramatically increased, whereas with the presence of phage, bacterial growth rapidly declined to approximately 0.

Example 6 The purpose of this example was to determine the toxicity of Phages 119U and Bl.

No acute toxicity was observed following injecting a large dose of Phage 119U in mice (up to 106 times of efficacy level), while Phage Bl showed no toxicity in animals.

To test for toxicity of Phages 119U and B1, aliquots of each phage of 3.4 x " pfu/ml (Phage 119U) and 3.2 x 10n pfu/ml (Phage Bl) were prepared for acute toxicity studies by diluting samples in PBS to produce final titers of 1010 and 106 pfu/ml. The Limulus amebocyte lysate (LAL) test was performed on the 10'° pfu/ml stock of each phage. The endotoxin levels for the samples of Phage Bl and 119U were determined to be ~6 EU/ml and 15 <6 EU/ml, respectively.

The acute toxicity study was conducted using 30 CD-I female mice (19-21 g). The mice were divided into six groups of five, and treated as follows: Group 1: no injection Group 2: intraperitoneal injection with 100 p.1 sterile saline 20 Group 3: intraperitoneal injection with 100 (J.1 phage Bl (109 pfu) Group 4: intraperitoneal injection with 100 |J.l phage Bl (10s pfu) Group 5: intraperitoneal injection with 100 (J.1 phage 119U (109 pfu) Group 6: intraperitoneal injection with 100 |_il phage 119U (10s pfu) Mice were monitored (visual observation, random temperatures) over the three days following the injections.- All mice in the experimental groups appeared active and healthy, identical to the control groups. This demonstrates that phage obtained according to the claimed invention are non-toxic.

Example 7 The purpose of this example was to determine the host range of Phages Bl and B3 against thirty-three different strains, and fifteen different serotypes, of human and porcine WO 02/44322 PCT/IB01/02836 E. coli, including the toxin producing E. coli strain 0157. The strains were obtained from a research collection. 104 pfu of each phage was spotted on a lawn for each bacterial isolate and the plate was incubated overnight at 37°C. A plus (+) indicates complete clearing, a minus (-) indicates that the phage had no effect on the bacterial isolate, and a plus/minus (+/-) indicates that the bacteriophage had partial efficacy against the E. coli isolate.

Both Phage Bl and B3 showed broad host ranges, including against the toxin-producing E. coli 0157 strains. See Figure 11. Phage B1 lysed or had activity against 26 of the 33 strains (79%), and lysed or had activity against 10 of the 15 serotypes of E. coli (67%). Phage B3 lysed or had activity against 27 of the 33 strains (82%), and lysed or had activity against 11 of the 15 serotypes of E. coli (73%).

These results demonstrate the usefulness of the bacteriophage compositions of the invention for preventing or treating contamination of food products by toxic and nontoxic E. coli strains.

* * * * It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. 16

Claims (12)

We claim:

1. A purified bacteriophage composition comprising the bacteriophage isolate 119U (ATCC No. 209998).

2. A bacteriophage composition comprising at least two bacteriophage isolates, the first bacteriophage being 119U and the second bacteriophage being selected from the group consisting of Bl (ATCC No. 203000), 119U, and 146A.

3. A method for preventing the growth of E. coli microorganisms on a food product comprising contacting said food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the bacteriophage isolate 119U, wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent the growth of microorganisms on the food product.

4. A method for preventing the growth of E. coli microorganisms on a food product comprising contacting said food product with a microbial growth inhibiting effective amount of a bacteriophage composition comprising at least two bacteriophage according to claim 2, wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent the growth of microorganisms on the food product.

5. A method for preventing the growth of E. coli microorganisms on a surface which comes in contact with food comprising contacting said surface with a microbial growth inhibiting effective amount of a bacteriophage composition comprising at least two bacteriophage isolates according to claim 2, wherein the application time of said bacteriophage composition is for at least a fraction of a second to prevent the growth of microorganisms on the food product.

6. The method of claim 5, wherein the surface comes in contact with one or more food products selected from the group consisting of fruit juices, vegetable juices, produce, poultry, beef, lamb, and pork.

7. The method of any one of claims 3 to 6, wherein said contact is by spraying, misting, dipping, or soaking. 17 intellectual property office of n.z. 2 5 JAN 2007 received

8. The method of any one of claims 3, 4 or 7, additionally comprising a washing step in which the food product is contacted with an aqueous medium to remove the bacteriophage composition.

9. The method of any one of claims 3, 4 or 7-9, wherein the food product is selected from the group consisting of fruit juices, vegetable juices, produce, poultry, beef; lamb, and pork.

10. The method of any one of claims 3, 4 or 7-9, wherein the E. coli microorganism is a toxin-producing E. coli strain 0157.

11. A purified bacteriophage as claimed in claim 1 or claim 2 and substantially as herein described with reference to the accompanying Examples.

12. A method according to claim 3 and substantially as herein described with reference to the accompanying Examples. 18 intellectual property office OF Ml. 25 JAN 2007 RFP.FIVED