WO2009075884A1 - Nouveaux bactériophages d'e. coli et leurs utilisations - Google Patents

Nouveaux bactériophages d'e. coli et leurs utilisations Download PDF

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Publication number
WO2009075884A1
WO2009075884A1 PCT/US2008/013659 US2008013659W WO2009075884A1 WO 2009075884 A1 WO2009075884 A1 WO 2009075884A1 US 2008013659 W US2008013659 W US 2008013659W WO 2009075884 A1 WO2009075884 A1 WO 2009075884A1
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Prior art keywords
bacteriophage
ecml
pta
variants
strains
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PCT/US2008/013659
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English (en)
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Gary R. Pasternack
Alexander Sulakvelidze
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Intralytix, Inc.
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Priority claimed from US11/955,145 external-priority patent/US7625556B2/en
Priority claimed from US11/955,176 external-priority patent/US7625741B2/en
Application filed by Intralytix, Inc. filed Critical Intralytix, Inc.
Publication of WO2009075884A1 publication Critical patent/WO2009075884A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the present invention relates to novel bacteriophages designated ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML-134 (the "Deposited Bacteriophage"), progeny, variants, derivatives, compositions, and preparations comprising the same.
  • isolated bacteriophage preparations possessing lytic activity against strains of Escherichia coli including but not limited to Escherichia coli O157:H7 strains (the "Targeted Bacteria") are provided in order to control the growth of the Targeted Bacteria, which will reduce their ability to contaminate and colonize various environments, including but not limited to: (i) raw, unprocessed food products; (ii) equipment used to process or manufacture various food products; (iii) various food products processed or manufactured with equipment contaminated with the Targeted Bacteria; (iv) animals contaminated with the Targeted Bacteria; (v) animal environments contaminated with the Targeted Bacteria; (vi) humans or animals colonized with the Targeted Bacteria; and (vii) wounds in animals or humans colonized or infected by the Targeted Bacteria.
  • the invention also provides methods for detecting the presence of the Targeted Bacteria in processed or unprocessed food products, and in equipment used to process or manufacture the food products.
  • the invention provides methods of using the Deposited Bacteriophage to remove the Targeted Bacteria from medical, veterinary, animal husbandry, and other environments where they may be passed to humans or animals.
  • the invention additionally provides methods of using the bacteriophage to prevent and treat animal and human diseases caused by the Targeted Bacteria.
  • Bacteriophages are bacterial viruses that attach to their specific hosts and kill them by internal replication and bacterial lysis involving a complex lytic cycle involving several structural and regulatory genes. Phages are very specific in that they only attack their targeted bacterial hosts. They cannot infect human or other eukaryotic cells. Bacteriophages were first identified, in the early part of the 20th century by Frederick Twort and Felix D'Herelle who called them bacteriophages or bacteria-eaters (from the Greek phago meaning to eat or devour). Duckworth (1976) "Who discovered bacteriophage?" Bacterid Rev 40(4): 793-802; Summers (1999) Bacteriophage discovered. Felix d'Herelle and the origins of molecular biology.
  • phage therapy continued to be utilized in the former Soviet Union and Eastern Europe, where phage therapy still is being used to treat a wide range of bacterial diseases ranging from intestinal infections to septicemia.
  • Comprehensive information about human and veterinary applications of bacteriophages has been recently reviewed by several investigators.
  • Bacteriophages have a lytic cycle or a lysogenic cycle, but few bacteriophages are capable of carrying out both. With lytic phages such as the T4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the new bacteriophage viruses can find new hosts. Kutter and Sulakvelidze (2005) Bacteriophages: Biology and Application. Boca Raton, FL, CRC Press: 381-436, herein incorporated by reference in its entirety.
  • the lysogenic cycle does not result in immediate lysing of the host cell.
  • Those phages able to undergo lysogeny are known as temperate phages.
  • Their viral genome will integrate with host DNA and replicate along with it fairly harmlessly, or may even become established as a plasmid.
  • the virus remains dormant until host conditions deteriorate (e.g., due to depletion of nutrients) then the endogenous phages (known as prophages) become active. At this point they initiate the reproductive cycle resulting in lysis of the host cell.
  • the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is reproduced in all of the host cell's offspring. See Kutter and Sulakvelidze (2005) Bacteriophages: Biology and Application, herein incorporated by reference in its entirety.
  • nucleic acid can be either DNA or RNA but not both, and it can exist in various forms.
  • the nucleic acids of phages often contain unusual or modified bases. These modified bases protect phage nucleic acid from nucleases that break down host nucleic acids during phage infection.
  • the size of the nucleic acid varies depending upon the phage. The simplest phages only have enough nucleic acid to code for 3-5 average size gene products while the more complex phages may code for over 100 gene products.
  • the number of different kinds of protein and the amount of each kind of protein in the phage particle will vary depending upon the phage.
  • the simplest phage have many copies of only one or two different proteins while more complex phages may have many different kinds.
  • the proteins function in infection and to protect the nucleic acid from nucleases in the environment. See also McGrath and van Sinderen (2007) Bacteriophage: Genetics and Molecular Biology, herein incorporated by reference in its entirety.
  • Bacteriophage come in many different sizes and shapes.
  • the basic structural features of bacteriophages include their size, head or capsid, tail.
  • T4 a common phage is among the largest phages; it is approximately 200 run long and 80-100 nm wide. Other phages are smaller. Most phages range in size from 24-200 nm in length. All phages contain a head structure which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous.
  • the head or capsid is composed of many copies of one or more different proteins. Inside the head is found the nucleic acid. The head acts as the protective covering for the nucleic acid.
  • phages have tails attached to the phage head.
  • the tail is a hollow tube through which the nucleic acid passes during infection.
  • the size of the tail can vary, and some phages do not even have a tail structure.
  • the tail is surrounded by a contractile sheath which contracts during infection of the bacterium.
  • the more complex phages like T4 have a base plate and one or more tail fibers attached to it.
  • the base plate and tail fibers are involved in the binding of the phage to the bacterial cell. Not all phages have base plates and tail fibers. In these instances, other structures are involved in binding of the phage particle to the bacterium. See Kutter and Sulakvelidze (2005) Bacteriophages: Biology and Application, herein incorporated by reference in its entirety.
  • the first step in the infection process is the adsorption of the phage to the bacterial cell.
  • This step is mediated by the tail fibers or by some analogous structure on those phages that lack tail fibers, and it is reversible.
  • the tail fibers attach to specific receptors on the bacterial cell, and the host specificity of the phage ⁇ i.e., the bacteria that it is able to infect) is usually determined by the type of tail fibers that a phage has.
  • the nature of the bacterial receptor varies for different bacteria ⁇ e.g., proteins on the outer surface of the bacterium, LPS, pili, and lipoprotein). These receptors are on the bacteria for other purposes, and phage have evolved to use these receptors for infection. See Kutter and Sulakvelidze (2005) Bacteriophages: Biology and Application, herein incorporated by reference in its entirety.
  • the attachment of the phage to the bacterium via the tail fibers is a weak one and is reversible. Irreversible binding of phage to a bacterium is mediated by one or more of the components of the base plate. Phages lacking base plates have other ways of becoming tightly bound to the bacterial cell.
  • Lytic or virulent phages are phages which can only multiply on bacteria and kill the cell by lysis at the end of the life cycle.
  • phage nucleic acid takes over the host biosynthetic machinery, and phage specified m-RNA's and proteins are made. There is an orderly expression of phage directed macromolecular synthesis, just as one sees in animal virus infections.
  • Early m-RNA's code for early proteins which are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. After phage DNA is made, late m-RNA's and late proteins are made. The late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of the bacterial cell.
  • the bacteria begin to lyse due to the accumulation of the phage lysis protein, and intracellular phage are released into the medium.
  • the number of particles released per infected bacteria may be as high as 1000.
  • a common assay for lytic phage is the plaque assay where lytic phage are enumerated by a plaque assay.
  • a plaque is a clear area which results from the lysis of bacteria. Each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a pfu (plaque forming unit). See Kutter and Sulakvelidze
  • Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are not transcribed; the phage genome exists in a repressed state. The phage DNA in this repressed state is called a prophage because it is not a phage but it has the potential to produce phage. In most cases the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The cell harboring a prophage is not adversely affected by the presence of the prophage, and the lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen. See also McGrath and van Sinderen (2007) Bacteriophage: Genetics and Molecular Biology, herein incorporated by reference in its entirety.
  • a lysogenic bacterium is exposed to adverse conditions, the lysogenic state can be terminated. This process is called induction.
  • Adverse conditions which favor the termination of the lysogenic state include desiccation, exposure to UV or ionizing radiation, and exposure to mutagenic chemicals. This leads to the expression of the phage genes, reversal of the integration process, and lytic multiplication. See Kutter and Sulakvelidze (2005) Bacteriophages: Biology and Application, herein incorporated by reference in its entirety.
  • bacteriophage in various practical settings, including the treatment of diseases in various animals, there remains in the art a need for the discovery of novel bacteriophages, selection of optimal bacteriophages for specific practical applications, and identifying methods for using these bacteriophages in several critical areas, including clinical applications, food safety-related uses and environmental decontamination.
  • one significant need concerns the treatment of processed or unprocessed food products to reduce, eliminate or prevent colonization with undesirable bacteria such as pathogens responsible for food-borne illness and food spoilage organisms.
  • a second critical area of need concerns the removal of undesirable bacteria from industrial environments such as food processing facilities to prevent colonization thereof.
  • a third critical area of need concerns the removal of antibiotic resistant organisms from environments where they may be passed to susceptible humans and animals, such as hospitals, nursing homes, veterinary facilities, and other such environments. Additionally, new bacteriophage and methods of using the same are needed for the prevention or treatment of animal and human bacterial disease, particularly those diseases caused by antibiotic-resistant organisms.
  • compositions comprising at least one of the novel ECTA-47, ECML-83, ECML-117, ECML-119,
  • the invention additionally provides methods of using the Deposited
  • the invention additionally provides methods of using the Deposited Bacteriophage to prevent, eradicate, or reduce the levels of colonization of various animals (including humans) with Targeted Bacteria.
  • the invention also provides methods of detecting the presence of Targeted Bacteria cells on processed or unprocessed food products, or equipment involved in the processing of the same food products.
  • the invention additionally provides methods of using the Deposited Bacteriophage for the removal of antibiotic-resistant or other undesirable pathogens from medical, veterinary, animal husbandry, and other environments where they may be passed to humans or animals.
  • the invention additionally provides for methods of using the Deposited Bacteriophage to prevent or treat human and/or other animal diseases caused by Targeted Bacteria.
  • the Deposited Bacteriophage has binding specificity for Targeted Bacteria, and is capable of lysing Targeted Bacteria (i.e., lytic bacteriophage) — only lytic phages are suitable for phage therapy.
  • the invention also contemplates progeny, variants, substantially equivalent bacteriophages, and bacteriophage derivative(s) of the Deposited Bacteriophage.
  • the invention provides progeny of the Deposited Bacteriophage having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and/or phenotypic characteristics as the Deposited Bacteriophage.
  • these progeny are the result of successive passaging of the Deposited Bacteriophage where the variants accumulate silent mutations, conservative mutations, minor deletions, and/or minor replications of genetic material.
  • the progeny described herein of the Deposited Bacteriophage retain the phenotypic characteristics of the Deposited Bacteriophage, in a preferred embodiment, the progeny retain lytic activity against the Target Bacteria.
  • the invention provides variants of the Deposited Bacteriophage having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and/or phenotypic characteristics as the Deposited Bacteriophage.
  • these variants can be the result of successive passaging of the Deposited Bacteriophage where the variants accumulate silent mutations, conservative mutations, minor deletions, and/or minor replications of genetic material.
  • the variants described herein of the Deposited Bacteriophage retain the phenotypic characteristics of the Deposited Bacteriophage, in a preferred embodiment, the variants retain lytic activity against the Target Bacteria.
  • the invention provides derivatives of the Deposited Bacteriophage comprising substances that constitute subunits or expression products of the Deposited bacteriophage or its progeny, including (but not limited to) phage nucleic acids, partial or complete phage genes, gene expression products, and structural components (e.g., polyribonucleotide(s) and polydeoxyribonucleotide(s), including modified or unmodified bacteriophage DNA, cDNA, mRNA and synthetic polynucleotide sequences, as well as DNA/RNA hybrids.)
  • the invention provides modified polynucleotides (e.g., phosphorylated DNAs) of the Deposited Bacteriophages.
  • the invention provides the use of the Deposited Bacteriophage, and its progeny and derivatives, to control the growth on, or colonization of, processed and unprocessed food products by Targeted Bacteria, or the colonization of buildings and equipment, particularly those associated with the processing of the same food product.
  • the invention also provides methods of identifying Targeted Bacteria as a bacterial diagnostic and/or detecting the presence of Targeted Bacteria on processed or unprocessed food products, or equipment or buildings such as those involved in the processing of the same food products.
  • the invention further provides methods of using the Deposited Bacteriophage for the removal of antibiotic-resistant or other undesirable pathogens from medical, veterinary, animal husbandry, or any additional environments where they may be passed to humans or animals.
  • the invention additionally provides for methods of using the Deposited Bacteriophage to prevent and/or treat human and animal diseases caused by Targeted Bacteria.
  • the Deposited Bacteriophage is administered for the methods of the invention as a homogenous phage administration, or alternatively as a component of a multi-phage composition comprising several bacteriophages. These methods of use are provided with greater particularity infra.
  • the invention comprises bacteriophages substantially equivalent to the Deposited Bacteriophages — bacteriophages that are "indistinguishable” from or “closely related” to the Deposited Bacteriophages as these terms are defined in Tenover.
  • One embodiment of the invention comprises at least one of the isolated bacteriophages ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA- 8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • Another embodiment of the invention comprises at least one isolated progeny of bacteriophage ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA- 8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • compositions comprises at least one isolated bacteriophage ECTA-47, ECML-83, ECML-117, ECML- 119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA- 8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • compositions comprises at least one progeny of bacteriophage ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA- 8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • Still another embodiment comprises at least one derivative of the bacteriophage of isolated bacteriophage ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains, said derivative comprising nucleic acids, partial or complete genes, gene expression products, structural components, or one or more combinations thereof.
  • Another embodiment comprises at least one derivative of the progeny bacteriophage of isolated bacteriophage ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains, said derivative comprising nucleic acids, partial or complete genes, gene expression products, structural components, or one or more combinations thereof.
  • One embodiment comprises a method for the prevention of food borne illnesses caused by E. coli strains, comprising contacting a food product or products with a microbial growth inhibiting effective amount of a bacteriophage composition comprising at least one of the isolated bacteriophages ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • One embodiment comprising a method for the reduction of the incidence of food borne illnesses caused by E. coli strains, comprising contacting a food product or products with a microbial growth inhibiting effective amount of a bacteriophage composition comprising at least one of the isolated bacteriophages ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, or ECML-134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E.
  • the contacting described in the methods herein comprises spraying or misting the bacteriophage composition on the food product(s), by dipping or soaking the food product(s) in a solution containing a concentration of the bacteriophage composition sufficiently high to inhibit the growth of E. coli strains, or adding, injecting or inserting the bacteriophage composition into the food product(s).
  • a method for reducing the risk of bacterial infection or sepsis in a person colonized with bacteria comprising treating the colonized person with a pharmaceutical composition containing bacteriophage of one or more strains of the Deposited Bacteriophage which produce lyric infections in said bacteria, wherein said treatment occurs prior to said colonized person developing an illness due to said bacteria and said treatment reduces the risk of bacterial infection or sepsis in said colonized person, and wherein said treatment of the colonized person reduces the level of colonization with bacteria susceptible to the bacteriophage by at least one log, wherein said composition is administered intravesicularly, topically, orally, rectally, ocularly, optically, nasally, or via inhalation.
  • said bacteria is E. coli.
  • the bacteriophage composition is an oral tablet, capsule or liquid, a nasal aerosol, a throat wash, a mouth wash or gargle, a toothpaste, and a topical ointment.
  • the colonized person is a person having a wound selected from the group consisting of an ulcer, a laceration, a deep penetrating wound and a surgical wound, and the bacteriophage produce lytic infections in bacteria capable of infecting these wounds.
  • a composition comprising at least one of the Deposited Bacteriophages ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML- 134 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • the composition further comprises a pharmaceutically acceptable carrier wherein the pharmaceutically acceptable carrier is an aerosol, a paste, a powder, or an injectable formulation.
  • a bacteriophage composition comprising at least one of the isolated bacteriophages ECTA-47, ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML-134 deposited under ATCC accession No. PTA- 8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E.
  • said contacting comprises spraying or misting the bacteriophage composition on the food product(s), by dipping or soaking the food product(s) in a solution containing a concentration of the bacteriophage composition sufficiently high to inhibit the growth of E. coli strains, or adding, injecting or inserting the bacteriophage composition into the food product(s).
  • Still another embodiment comprises the use of a bacteriophage composition
  • a bacteriophage composition comprising at least one of the isolated bacteriophages ECTA-47, ECML-83, ECML-117, ECML-119, ECML- 122, and/or ECML- 134 deposited under ATCC accession No. PTA- 8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E.
  • coli strains for the reduction of the incidence of food borne illnesses caused by E. coli strains comprising contacting a food product or products with a microbial growth inhibiting effective amount of said bacteriophage composition.
  • said contacting comprises spraying or misting the bacteriophage composition on the food product(s), by dipping or soaking the food product(s) in a solution containing a concentration of the bacteriophage composition sufficiently high to inhibit the growth of E. coli strains, or adding, injecting or inserting the bacteriophage composition into the food product(s).
  • Another embodiment comprises the use of a bacteriophage composition
  • a bacteriophage composition comprising at least one of the isolated bacteriophages ⁇ CML-83, ⁇ CML-117, ECML-119, ECML- 122, and/or ECML- 134 deposited under ATCC accession No. PTA-8348, PTA- 7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli O157:H7 strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E.
  • coli O157:H7 strains for the prevention of food borne illnesses caused by E. coli O157:H7 strains, comprising contacting a food product or products with a microbial growth inhibiting effective amount of said bacteriophage composition.
  • said contacting comprises spraying or misting the bacteriophage composition on the food product(s), by dipping or soaking the food product(s) in a solution containing a concentration of the bacteriophage composition sufficiently high to inhibit the growth of E. coli O157:H7 strains, or adding, injecting or inserting the bacteriophage composition into the food product(s).
  • a bacteriophage composition comprising at least one of the isolated bacteriophages ⁇ CML-83, ⁇ CML-117, ECML-119, ECML- 122, and/or ECML- 134 deposited under ATCC accession No. PTA-8348, PTA- 7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli O157:H7 strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli O157:H7 strains for the reduction of the incidence of food borne illnesses caused by E. coli O157:H7 strains comprising contacting a food product or products with a microbial growth inhibiting effective amount of said bacteriophage composition
  • said contacting comprises spraying or misting the bacteriophage composition on the food product(s), by dipping or soaking the food product(s) in a solution containing a concentration of the bacteriophage composition sufficiently high to inhibit the growth of E. coli O157:H7 strains, or adding, injecting or inserting the bacteriophage composition into the food product(s).
  • the compositions are pharmaceutical compositions.
  • compositions comprising at least one, two, three, four, five, or six of the Deposited Bacteriophages ⁇ CTA-47, ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML-134 deposited under ATCC accession No. PTA- 8347, PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E.
  • the Deposited Bacteriophages ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML-134 deposited under ATCC accession No. PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, have lytic activity against E. coli strains, wherein said E. coli strains are E. coli O157:H7 strains.
  • variants of the Deposited Bacteriophages ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML-134 deposited under ATCC accession No. PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively, retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli O157:H7 strains.
  • Figure 1 shows a Restriction Fragment Length Polymorphism (RFLP) profile of the ECTA-47, ECML-83, ECML-119, and ECML-122 bacteriophage in comparison to two other bacteriophages also specific for the Targeted Bacteria.
  • Reference RFLP profiles various bacteriophages specific for the Target Bacterium. Note the unique patterns of ECTA-47, ECML-83, ECML-119, and ECML-12 in comparison to the unrelated bacteriophage shown for comparison purposes. All phage DNA was digested with EcoRV.
  • Lane 1 ECML-85; Lane 2, ECML-83; Lane 3, ECML-13; Lane 4, ECML-47014-2; Lane 5, ECML-47014-1; Lane 6, ECML-47-2; Lane 7, ECML-47-1; Lane 8, ECML-I; Lane 9, ECML-6; Lane 10, ECML-10; Lane 11, ECTA-47; Lane 12, ECML-95; Lane 13, ECML- 122; Lane 14, ECML-119; Lanes M, DNA Ladder Mix (Fermentas).
  • Figure 2 shows a Restriction Fragment Length Polymorphism (RFLP) profile of the ECML-117 and ECML-134 bacteriophages in comparison to two other bacteriophages also specific for the Targeted Bacteria. Note the unique patterns of ECML-117 and ECML-134. The figure is of the indicated digests electrophoresed on an 0.8% agarose gel. Lane 1, DNA Ladder Mix (Fermentas); lane 2, ECML-134, EcoRV-digest; lane 3, ECML- 117, EcoRV-digest; lane 4, ECML-4, Afllll-digest.
  • RFLP Restriction Fragment Length Polymorphism
  • Table 1 shows the lytic specificity of the bacteriophages ECTA-47, ECML-83, ECML-119, and ECML-122 for E. coli ⁇ i.e., the Targeted Bacteria.) Lytic activity is indicated by a +.
  • Table 2 shows the lytic specificity of the bacteriophages ECML-117 and ECML- 134 for E. coli O157:H7 ⁇ i.e., the Targeted Bacteria.
  • Intralytix ID Original ID ECML-117 ECML-134
  • Intralytix ID Original ID ECML-117 ECML-134
  • Intralytix ID Original ID ECML-117 ECML-134
  • Intralytix ID Original ID ECML-117 ECML-134
  • Table 3 shows the lytic activity of ECML-117 and ECML-134 bacteriophages against non-0157:H7 strains of E. coli. TABLE 3
  • Table 4 shows the lytic specificity of the bacteriophages ECML- 117 and ECML- 134 for non-E. coli bacteria of other Gram-positive and Gram-negative bacterial species.
  • ECML-117 and ECML-134 phages did not lyse Staphylococcus aureus, Listeria monocytogenes, Salmonella, and Pseudomonas aeruginosa.
  • isolated refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment.
  • Isolated material may be, for example, foreign nucleic acid included in a vector system, foreign nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man.
  • Isolated material further encompasses bacteriophage specific for the Targeted Bacteria or particular Targeted Bacteria isolates, isolated and cultured separately from the environment in which it was located, where these isolates are present in purified compositions that do not contain any significant amount of other bacteriophage or bacterial strains, respectively.
  • significant refers broadly to an amount of a substance present in the total measured composition, wherein the substance is present in greater than 1% of the total volume or concentration of the composition.
  • Deposited Bacteriophage refers broadly to isolated bacteriophage ECTA-47, ECML-83, ECML-119, and ECML-122 deposited with the ATCC on April 19, 2007, and receiving ATCC Deposit Accession No. PTA-8347 for ECTA-47, PTA-8348 for ECML-83, PTA-8351 for ECML-119, and PTA-8352 for ECML-122.
  • this invention provides isolated bacteriophages ECML-117 deposited with the ATCC and receiving ATCC Deposit Accession No. PTA-7950, and ECML-134 receiving ATCC Deposit Accession No. PTA-7949.
  • All of the Deposited Bacteriophages described herein are lytic not lysogenic phages.
  • the Deposited Bacteriophages have lytic activity against E. coli strains.
  • the Deposited Bacteriophage ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML- 134 deposited under ATCC accession No. PTA- 8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949, respectively have lytic activity against E. coli O157:H7 strains.
  • “Targeted Bacteria” refers broadly to Escherichia coli bacteria strains, hi a more preferred embodiment, “Targeted Bacteria” refers broadly to Escherichia coli strain O157:H7.
  • progeny refers broadly to replicates of the Deposited bacteriophage, including descendants of the Deposited bacteriophage created by serial passage of the Deposited bacteriophage or by other means well known in the art, or bacteriophage whose RFLP profiles are substantially equivalent to the RFLP profile of the Deposited bacteriophage ⁇ See Figures 1 and 2).
  • substantially equivalent is used to describe variability between organisms in accordance with the standards advanced by Tenover et al. from the United States Centers for Disease Control and Prevention (Tenover, F.C. et al.
  • Tenover describes a system for interpreting chromosomal DNA Restriction Enzyme digest patterns ("RPLP”) using Pulsed-Field Gel Electrophoresis (PFGE). Tenover at page 2233. In particular, Tenover sets forth various categories of genetic and epidemiologic relatedness including those organisms that are "indistinguishable” from or “closely related” to each other. While Tenover provides a schematic (prophetic) example of PFGE patterns of genetically related bacteria, the same principles being applied for bacteria also apply to bacteriophage, because Tenover is analyzing genomic DNA.
  • RPLP chromosomal DNA Restriction Enzyme digest patterns
  • PFGE Pulsed-Field Gel Electrophoresis
  • variants refers broadly to bacteriophages that share the same phenotypic characteristics of the Deposited Bacteriophage and share the same lytic activity of the Deposited Bacteriophages against the Targeted Bacteria. Variants also include bacteriophages that are “substantially equivalent” to the Deposited Bacteriophages, or are “indistinguishable” from or “closely related” to the Deposited Bacteriophages as described in Tenover.
  • bacteriophages substantially equivalent to the Deposited Bacteriophages refer broadly to those bacteriophages that are “indistinguishable” from or “closely related” to the Deposited Bacteriophages as these terms are defined in Tenover.
  • Tenover describes that organisms are "genetically indistinguishable if their restriction patterns have the same numbers of bands and the corresponding bands are the same apparent size.”
  • Tenover at page 2235 Epidemiologically, these organisms are "all considered to represent the same strain; i.e., isolates demonstrating the common outbreak pattern represent the outbreak strain.”
  • Tenover at page 2235 Accordingly, under Tenover, a particular organism is "indistinguishable” from itself or its clone.
  • Tenover describes that an organism is "closely related” if its "PFGE pattern differs from the outbreak pattern by changes consistent with a single genetic event, i.e., a point mutation or an insertion or deletion of DNA. Such changes typically result in two to three band differences.”
  • Tenover states that such two to three band differences "have been observed in strains of some species when they are cultured repeatedly over time or isolated multiple times from the same patient.”
  • Tenover at page 2235 Accordingly, under Tenover, progeny of a organism (e.g., descendants of the organism created by serial passage of the organism), for example, are “closely related" to the parent organism.
  • recombinant bacteriophage refers broadly to all genetically modified versions of the Deposited Bacteriophage or its progeny, obtained by serial passaging (in vivo or in vitro) or genetic manipulations of the Deposited Bacteriophage or its progeny. Such manipulations include, but are not limited to, introducing genes or gene cassettes encoding alternative proteins or nonfunctional proteins, or noncoding nucleotide sequences into the genome of the Deposited Bacteriophage.
  • derivatives refers broadly to all substances that constitute subunits or expression products of the Deposited Bacteriophage or its progeny, including (but not limited to) phage nucleic acids, partial or complete phage genes, gene expression products, and structural components.
  • derivatives of the invention mean polyribonucleotide(s) and polydeoxyribonucleotide(s), including modified or unmodified bacteriophage DNA, cDNA, mRNA and synthetic polynucleotide sequences, as well as DNA/RNA hybrids.
  • Polynucleotides of the invention also encompass modified polynucleotides, such as for example phosphorylated DNAs.
  • substantially pure refers broadly to material essentially free of any similar macromolecules that would normally be found with it in nature.
  • a substantially pure bacteriophage is in a composition that contains no more than 1% of other bacteriophages.
  • bacteriophage composition refers broadly to a composition comprising, or alternatively consisting essentially of, or alternatively consisting of, the Deposited Bacteriophage.
  • a "bacteriophage composition” as used herein does not include the Deposited Bacteriophage as it exists in its natural environment prior to isolation and/or substantial purification.
  • a composition may comprise, consist of, or essentially consist of at least one of the Deposited Bacteriophages.
  • the compositions as described herein may comprise, consist of, or essentially consist of at least one, two, three, four, five, or all six of the Deposited Bacteriophages.
  • colonization refers broadly to the presence of Targeted Bacteria on foodstuff(s), or environmental surface(s), or in vivo such as in the gastrointestinal tract or skin of a mammalian organism without perceptible significant alteration other than the presence of bacteria.
  • colonization and “colonized” stand in contrast to the terms “infection” or “infected” which are commonly understood to require perceptible deleterious alteration as part of their definition.
  • Colonization and “colonized” may also refer to the presence of bacteria in or on a human or animal without perceptible damage, alteration, or disease.
  • ATCC will mean the American Type Culture Collection, located at 10801 University Boulevard, Manassas, Virginia, 20110-2209, USA.
  • ORF refers broadly to an Open Reading Frame which is an in- frame sequence of codons that (in view of the genetic code) correspond to or encode a protein or peptide sequence. Two ORFs correspond to each other if the sequences or their complementary sequences encode the same amino acid sequences.
  • An ORF sequence, operably associated with appropriate regulatory sequences, may be transcribed and translated into a polypeptide in vivo.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • the Deposited Bacteriophage has binding specificity for Targeted Bacteria, and is capable of lysing Targeted Bacteria (e.g., lytic bacteriophages).
  • the invention also contemplates progeny, variants, substantially equivalent bacteriophages, and bacteriophage derivative(s) of the Deposited Bacteriophage.
  • the invention further contemplates variants of the Deposited Bacteriophage, which are bacteriophage having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and phenotypic characteristics as the Deposited Bacteriophage.
  • Such variants are considered to be the Deposited Bacteriophage in accordance with the standards advanced by Tenover, et al. from the United States Centers for Disease Control and Prevention (Tenover, F. C. et al. (1995) Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. J. Clin. Microbiol. 33:2233-2239).
  • the progeny, variants, substantially equivalent bacteriophages, and bacteriophage derivative(s) of the Deposited Bacteriophage all retain the same target specificity ⁇ e.g., the Target Bacteria) and are lytic phages.
  • the invention as described herein pertains to the Deposited Bacteriophages.
  • the invention as described herein also pertains to progeny of the Deposited Bacteriophages and teaches RFLP methods for identifying progeny and other "substantially equivalent" bacteriophages.
  • RFLP analysis is a means of identifying closely related bacteriophages. See e.g., Schnabel and Jones (January 2001) "Isolation and Characterization of Five Erwinia anylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora.” Applied and Environmental Microbiology 67(1): 59-64 and Osawa, et al.
  • the Deposited Bacteriophage will able to isolated and identify variants of the Deposited Bacteriophages as described herein.
  • the variants of the Deposited Bacteriophage having minor variation(s) in the genomic sequence and polypeptides encoded thereby while retaining the same general genotypic and/or phenotypic characteristics as the Deposited Bacteriophage.
  • Such variants are considered to be the Deposited Bacteriophage in accordance with the standards advanced by Tenover.
  • these variants may be the result of successive passaging of the Deposited Bacteriophage where the variants accumulate silent mutations, conservative mutations, minor deletions, and/or minor replications of genetic material.
  • the variants described herein of the Deposited Bacteriophage retain the phenotypic characteristics of the Deposited Bacteriophage, in a preferred embodiment, the variants have lytic activity against the Target Bacteria.
  • bacteriophages substantially equivalent to the Deposited Bacteriophages are those bacteriophages that are "indistinguishable” from or "closely related” to the Deposited Bacteriophages. See Tenover at page 2235. Accordingly, under Tenover, progeny of a organism (e.g., descendants of the organism created by serial passage of the organism), for example, are "closely related" to the parent organism.
  • the Deposited Bacteriophages can be used to isolate derivatives, in particular all substances that constitute subunits or expression products of the Deposited bacteriophage or its progeny, including (but not limited to) phage nucleic acids, partial or complete phage genes, gene expression products, and structural components.
  • derivatives of the invention mean polyribonucleotide(s) and polydeoxyribonucleotide(s), including modified or unmodified bacteriophage DNA, cDNA, mRNA and synthetic polynucleotide sequences, as well as DNA/RNA hybrids.
  • Polynucleotides of the invention also encompass modified polynucleotides, such as for example phosphorylated DNAs.
  • the nucleic acid can be either DNA or RNA but not both and it can exist in various forms. Further, the nucleic acids of phages often contain unusual or modified bases. These modified bases protect phage nucleic acid from nucleases that break down host nucleic acids during phage infection. The size of the nucleic acid varies depending upon the phage. The phages can have only enough nucleic acid to code for 3-5 average size gene products while the some phages may code for over 100 gene products. [0074] Additionally, the Deposited Bacteriophage comprises an isolated bacteriophage ECTA-47, ECML-83, ECML-119, or ECML-122 deposited under ATCC accession No.
  • PTA-8347, PTA-8348, PTA8351, or PTA-8352 said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • the Deposited Bacteriophage also comprises isolated progeny of bacteriophage ECTA-47, ECML-83, ECML-119, or ECML-122 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA8351, or PTA-8352, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • the Deposited Bacteriophage comprises an isolated bacteriophage substantially equivalent to the bacteriophage ECTA-47, ECML-83, ECML-119, or ECML- 122 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA8351, or PTA-8352, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • the Deposited Bacteriophage also comprises isolated progeny of bacteriophage substantially equivalent to the bacteriophage ECTA-47, ECML-83, ECML-119, or ECML- 122 deposited under ATCC accession No. PTA-8347, PTA-8348, PTA8351, or PTA-8352, said bacteriophage having lytic activity against E. coli strains, and variants thereof, wherein said variants retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli strains.
  • PTA- 7950 and PTA-7949 retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli O157:H7 strains.
  • the Deposited Bacteriophage also encompasses progeny of the Deposited Bacteriophages ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML- 134 deposited under ATCC accession No.
  • PTA-8348, PTA-7950, PTA8351, PTA-8352, and PTA-7949 respectively, and variants thereof which retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli O157:H7 strains.
  • the Deposited Bacteriophages also comprise bacteriophages substantially equivalent to the bacteriophage ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML- 134 deposited under ATCC accession No. PTA-8348, PTA-7950, PTA8351, PTA- 8352, and PTA-7949, respectively, have lytic activity against E. coli strains, wherein said E. coli strains are E. coli O157:H7 strains and variants of the Deposited Bacteriophages ECML-117 and ECML-134 deposited under ATCC accession No.
  • PTA-7950 and PTA- 7949 retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli O157:H7 strains.
  • the Deposited Bacteriophage also encompasses progeny substantially equivalent to the Deposited Bacteriophages ECML-83, ECML-117, ECML-119, ECML-122, and/or ECML-134 deposited under ATCC accession No.
  • PTA-8348, PTA-7950, PTA8351, PTA- 8352, and PTA-7949 respectively, and variants thereof which retain the phenotypic characteristics of said bacteriophage and wherein said bacteriophage and variants thereof have lytic activity against E. coli O157:H7 strains.
  • the Deposited Bacteriophage are formulated in compositions containing the bacteriophage and a carrier, and can be stored as a concentrated aqueous solution or lyophilized powder preparation.
  • Bacteriophage may be formulated by resuspending purified phage preparation in aqueous medium, such as deionized water, buffer solution (e.g., Tris-HCl pH 7.4), mineral water, 5% sucrose solution, glycerol, dextran, polyethylene glycol, sorbitol, or other formulations that maintain phage viability, and are non-toxic to humans.
  • aqueous medium such as deionized water, buffer solution (e.g., Tris-HCl pH 7.4), mineral water, 5% sucrose solution, glycerol, dextran, polyethylene glycol, sorbitol, or other formulations that maintain phage viability, and are non-toxic to humans.
  • Suitable formulations, wherein the carrier is a liquid, for administration e
  • a spray comprising a composition of the present invention can be produced by forcing a suspension or solution of a compound disclosed herein through a nozzle under pressure.
  • the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
  • An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
  • the Deposited Bacteriophage are preferably formulated in pharmaceutical compositions containing the bacteriophage and a pharmaceutically acceptable carrier, and can be stored as a concentrated aqueous solution or lyophilized powder preparation.
  • Bacteriophage may be formulated for oral administration by resuspending purified phage preparation in aqueous medium, such as deionized water, mineral water, 5% sucrose solution, glycerol, dextran, polyethylene glycol, sorbitol, or such other formulations that maintain phage viability, and are non-toxic to humans.
  • the pharmaceutical composition can further comprise an adjuvant.
  • the pharmaceutical composition may contain other components so long as the other components do not reduce the effectiveness (ineffectivity) of the bacteriophage so much that the therapy is negated.
  • Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA., 1985).
  • the pharmaceutical compositions containing Deposited Bacteriophage may be administered by parenteral (subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally), topical, oral, rectal, inhalation, ocular, optic, or nasal route, as necessitated by choice of drug and disease.
  • parenteral subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally
  • topical topical
  • oral rectal, inhalation, ocular, optic, or nasal route, as necessitated by choice of drug and disease.
  • the invention provides a pharmaceutical composition comprising at least one of the Deposited Bacteriophages, progeny, and/or variants thereof and a pharmaceutical carrier.
  • the Deposited Bacteriophage(s) of the invention may be administered in a powdered form in combination with additional components.
  • the additional components can include stabilizing agents, such as salts, preservatives and antibiotics.
  • the additional components can include nutritive components, such as those used to make a nutrient broth as described herein, or other useful components as determined by one skilled in the art.
  • a pharmaceutical composition includes at least one Deposited Bacteriophage in combination with a pharmaceutically acceptable carrier. Examples of acceptable carriers include a solid, gelled or liquid diluent or an ingestible capsule.
  • One or more of the bacteriophages of the invention, or a mixture thereof, may be administered orally in the form of a pharmaceutical unit dosage form comprising the bacteriophage in combination with a pharmaceutically acceptable carrier.
  • a unit dosage of the bacteriophage may also be administered without a carrier material.
  • compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels.
  • An oral dosage form may be formulated such that the bacteriophage(s) of the invention are released into the intestine after passing through the stomach.
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • the bacteriophages according to the invention may also be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the bacteriophage(s) of the invention may be in powder form, obtained by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile saline, before use.
  • the bacteriophage(s) may be formulated as ointments, creams or lotions.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • compositions suitable for topical administration in the mouth include unit dosage forms such as lozenges comprising a bacteriophage(s) of the invention in a flavored base, usually sucrose and acadia or tragacanth. Pastilles comprising one or more bacteriophages in an inert base such as gelatin and glycerin or sucrose and acacia are also provided. Mucoadherent gels and mouthwashes comprising a bacteriophage(s) of the invention in a suitable liquid carrier are additionally provided.
  • Pharmaceutical compositions suitable for rectal administration are most preferably presented as unit dose suppositories. Suitable carriers include saline solution, nutrient broths, and other materials commonly used in the art.
  • Pharmaceutical compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays that contain a carrier in addition to a bacteriophage. Such carriers are well known in the art.
  • the bacteriophage(s) according to the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the bacteriophage(s) of the invention may take the form of a dry powder composition, for example, a powder mix of the bacteriophage(s) and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • the bacteriophage(s) of the invention may be administered via a liquid spray, such as via a plastic bottle atomizer.
  • the bacteriophage(s) according to the invention can be administered as drops and gels.
  • compositions of the invention may also contain other adjuvants such as flavorings, colorings, anti-microbial agents, or preservatives.
  • the invention also provides kits containing packaging and a bacteriophage(s) of the invention.
  • Dose and duration of therapy will depend on a variety of factors, including the patient age, patient weight, and tolerance of the phage.
  • Bacteriophage may be administered to patients in need of the therapy provided by this invention by oral administration. Based on previous human experience in Europe, a dose of phage between 10 7 and 10 11 PFU will be suitable in most instances.
  • the phage may be administered orally in, for example, mineral water, optionally with 2.0 grams of sodium bicarbonate added to reduce stomach acidity.
  • sodium bicarbonate may be administered separately to the patient just prior to dosing with the phage.
  • Phages also may be incorporated in a tablet or capsule which will enable transfer of phages through the stomach with no reduction of phage viability due to gastric acidity, and release of fully active phages in the small intestine.
  • the frequency of dosing will vary depending on how well the phage is tolerated by the patient and how effective a single versus multiple dose is at reducing bacterial (e.g. , E. col ⁇ ) gastrointestinal colonization.
  • the dose of Deposited Bacteriophage and duration of therapy for a particular patient can be determined by the skilled clinician using standard pharmacological approaches in view of the above factors.
  • the response to treatment may be monitored by, analysis of blood or body fluid levels of E. coli, or E. coli levels in relevant tissues or monitoring disease state in the patient.
  • the skilled clinician will adjust the dose and duration of therapy based on the response to treatment revealed by these measurements.
  • One of the major concerns about the use of phages in clinical settings is the possible development of bacterial resistance against them. However, as with antimicrobial resistance, the development of resistance to phages takes time.
  • phage preparations may be constructed by mixing several separately grown and well-characterized lytic monophages, in order to (i) achieve the desired, broad target activity of the phage preparation, (ii) ensure that the preparation has stable lytic properties, and (iii) minimize the development of resistance against the preparation.
  • phages having the same spectrum of activity may be administered at different times during the course of therapy.
  • therapeutic phages may be genetically engineered which will have a broad lytic range and/or be less immunogenic in humans and animals.
  • the amount of the present bacteriophages, required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage.
  • the invention contemplates a method for the prevention of food borne illnesses caused by the Targeted Bacteria, comprising contacting a food product or products with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the Deposited Bacteriophage.
  • the modes of contact include, but are not limited to, spraying or misting the Deposited Bacteriophage composition on the food product(s), or by dipping or soaking the food product(s) in a solution containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria, or adding, injecting or inserting the Deposited Bacteriophage into the food product(s).
  • the invention contemplates a method for the reduction in the incidence of food borne illnesses caused by the Targeted Bacteria, comprising contacting a food product or products with a microbial growth inhibiting effective amount of a bacteriophage composition comprising the Deposited Bacteriophage.
  • the modes of contact include, but are not limited to, spraying or misting the Deposited Bacteriophage composition on the food product(s), or by dipping or soaking the food product(s) in a solution containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria, or adding, injecting or inserting the Deposited Bacteriophage into the food product(s).
  • the invention contemplates the application of the Deposited Bacteriophage composition to equipment associated with the processing of food product(s), such as cutting instruments, conveyor belts, and any other implements utilized in the mass production of food products, including the preparation, storage and packaging steps of food processing.
  • the Deposited Bacteriophage can additionally be introduced into packaging materials used to contain food product(s), prior to or following transfer of the food product(s) to the packaging materials.
  • the Deposited Bacteriophage can be useful in the local processing of food products located, for example, in the home or in a restaurant kitchen, using the same modes of contact as described supra.
  • the Deposited Bacteriophage is added as a component of paper products, either during processing or after completion of processing of the paper products.
  • Paper products to which the Deposited Bacteriophage may be added include, but are not limited to, paper towels, toilet paper, moist paper wipes.
  • the Deposited Bacteriophage is added as a component of cleansing wipes.
  • the Deposited Bacteriophage may be added in an aqueous state to a liquid-saturated paper product, or alternatively may be added in powder form, such as lyophilized, to dry paper products, or any combination thereof.
  • the Deposited Bacteriophage may be incorporated into films such as those used for packaging foods, such as by impregnating or coating the film.
  • the methods of the invention further contemplate the application of the Deposited Bacteriophage to the floors, walls, ceilings, drains, or other environmental surfaces in structures such as the industrial food processing, military, or home environments.
  • the Deposited Bacteriophage is applied to refrigerated devices used to store or transport food or food products, including but not limited to, home and industrial refrigerators, deli-meat and cheese counters, refrigerated trucks, and mobile food-service vehicles.
  • the Deposited Bacteriophage of the invention is useful in preventing the colonization of, or inhibiting the growth of, Targeted Bacteria on processed or unprocessed food products by infecting, lysing or inactivating Targeted Bacteria present on said food product.
  • Processed or unprocessed food products in which the Deposited Bacteriophage is particularly useful in preventing the growth or colonization of Targeted Bacteria include, but are not limited to beef (particularly ground beef), food products made with ground beef such as hamburgers, sloppy joes, lasagna, stews, and other ground beef preparations, fresh vegetables exposed to Targeted Bacteria presumably via animal waste, such as lettuce, spinach, green onions, and other fresh vegetables commonly grown out of doors in fields, drinking water, and foodstuffs secondarily contaminated with Targeted Bacteria through contact with contaminated foods, sewage, or animal feces.
  • beef particularly ground beef
  • food products made with ground beef such as hamburgers, sloppy joes, lasagna, stews, and other ground beef preparations
  • fresh vegetables exposed to Targeted Bacteria presumably via animal waste, such as lettuce, spinach, green onions, and other fresh vegetables commonly grown out of doors in fields, drinking water, and foodstuffs secondarily contaminated with Targeted Bact
  • the Deposited Bacteriophage can also be administered to potable and non- potable water sources to reduce or eliminate the presence of Targeted Bacteria.
  • Bacteriophage compositions of the invention may be provided in aqueous or nonaqueous embodiments for the preservation of food.
  • Aqueous embodiments of the Deposited Bacteriophage include aqueous compositions comprising, or alternatively consisting of, the Deposited Bacteriophage alone or in combination with other bacteriophage or bacteriophages.
  • Aqueous embodiments of the Deposited Bacteriophage are available in solutions that include, but are not limited to, phosphate buffered saline, Luria-Bertani Broth or chlorine-free water.
  • Non-aqueous embodiments of the Deposited Bacteriophage include, but are not limited to, lyophilized compositions or spray-dried compositions comprising, or alternatively consisting of, the Deposited Bacteriophage alone or in combination with other bacteriophage(s). Freeze-dried and spray-dried compositions may also include soluble and/or insoluble carrier materials as, for example, processing aids.
  • the Deposited Bacteriophage can be administered at a concentration effective to prevent the initial colonization of foods with Targeted Bacteria, or to inhibit the growth or colonization of food or food products, as well as the equipment used to process or store food.
  • the Deposited Bacteriophages typically administered at a growth inhibiting effective amount of a concentration of about 10 7 to about 10 11 Plaque Forming Units (PFU)/ml.
  • PFU Plaque Forming Units
  • One of skill in the art is capable of ascertaining bacteriophage concentrations using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).
  • the Deposited Bacteriophage at such concentrations may be applied at, for example, about 1 ml/500 cm 2 of food surface.
  • the Deposited Bacteriophage is administered to environments to control the growth or viability of Targeted Bacteria.
  • Environments in which the Deposited Bacteriophage is useful to control the growth or viability of Targeted Bacteria include, but are not limited to, abattoirs, meat processing facilities, feedlots, vegetable processing facilities, medical facilities (including hospitals, out-patient clinics, school and/or university infirmaries, and doctors offices), military facilities, veterinary offices, animal husbandry facilities, public and private restrooms, and nursing and nursing home facilities.
  • the invention further contemplates the use of the Deposited Bacteriophage for the battlefield decontamination of food stuffs, the environment, and personnel and equipment, both military and non-military.
  • the Deposited Bacteriophage is additionally useful alone or in combination with other bacteriophage(s) and/or other compounds, for preventing the formation of biofilms, or controlling the growth of biof ⁇ lms, in various environments.
  • Aqueous embodiments of the Deposited Bacteriophage are available in solutions that include, but are not limited to, phosphate buffered saline, Luria-Bertani Broth or chlorine-free water.
  • the Deposited Bacteriophage is used to control biofilm formation and growth in municipal water systems, industrial water systems, and personal water systems, as well as biof ⁇ lms present in refrigerated environments.
  • the modes of administration include, but are not limited to, spraying, hosing, and any other reasonable means of dispersing aqueous or non-aqueous Bacteriophage compositions, in an amount sufficiently high to inhibit the growth or viability of Targeted Bacteria.
  • the Deposited Bacteriophage is useful in preventing the growth or viability of Targeted Bacteria by infecting, lysing or inactivating Targeted Bacteria present in said environment.
  • Administration of the Deposited Bacteriophage composition includes application to the floors, walls, counter-tops, ceilings, drains or any other environmental surface.
  • Bacteriophage compositions of the invention are available in aqueous or nonaqueous embodiments discussed earlier for Food Preservation applications.
  • the Deposited Bacteriophage is added as a component of paper products, either during processing or after completion of processing of the paper products.
  • Paper products to which the Deposited Bacteriophage may be added include, but are not limited to, paper towels, toilet paper, and moist paper wipes.
  • the Deposited Bacteriophage is added as a component of cleansing wipes; it may be added in an aqueous state to a liquid-saturated paper product, or alternatively may be added in powder form such as a lyophilized preparation, to dry paper products, or any combination thereof.
  • the Deposited Bacteriophage can be administered at a concentration effective to inhibit the growth or viability of Targeted Bacteria in a particular environment.
  • the Deposited Bacteriophage is administered at a concentration of about 10 7 to 10 ⁇ PFU/ml.
  • One of skill in the art is capable of ascertaining bacteriophage concentrations using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.).
  • the invention contemplates a method for the prevention or treatment of illnesses caused by the Targeted Bacteria, comprising contacting a microbial growth inhibiting effective amount of a bacteriophage composition comprising the Deposited Bacteriophage with a site or sites of infection of a host mammal infected with Targeted Bacteria.
  • the infected mammalian host may be a human host or animal host.
  • the host may be a bovine, poultry, or porcine host.
  • Prevention of the infection by Targeted Bacteria, or treatment of infected persons or animals, is particularly preferred in immunocompromised persons, pregnant females, and newborns and infants, who maybe at an elevated risk of infection by Targeted Bacteria.
  • the modes of contact include, but are not limited to, spraying or misting the bacteriophage composition on the infected mammalian host, by injecting at a site or sites of infection a pharmaceutically acceptable composition containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria, or by ingesting a solution containing a concentration of the Deposited Bacteriophage sufficiently high to inhibit the growth of Targeted Bacteria.
  • Additional routes of administration include but are not limited to oral, rectal, topical, ophthalmic, buccal, intravenous, optic, nasal, vaginal, inhalation, and intrapleural.
  • the Deposited Bacteriophage is useful for preparing bacterial vaccines or bacterins that eliminate or reduce colonization of the Targeted Bacteria in, and/or their being shed by, various agriculturally-important animals.
  • One example of a practical application for that type of vaccine is in the cattle-raising industry, where its administration may significantly reduce colonization of cattle with the Targeted Bacteria; thus, improving public safety by reducing contamination of beef with the Targeted Bacteria.
  • Bacteriophage compositions of the invention are available in aqueous or nonaqueous embodiments discussed earlier for Food Preservation applications.
  • the Deposited Bacteriophage can be administered at a concentration effective to inhibit the growth or viability of Targeted Bacteria in the infected host.
  • the Deposited Bacteriophage is administered at a concentration of about 10 7 to 10 1 1 PFU/ml.
  • One of skill in the art is capable of ascertaining bacteriophage concentrations using widely known bacteriophage assay techniques (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.)
  • the Deposited Bacteriophage can be administered to animals (including humans) (i) orally, in tablet or liquid formulation (10 5 - 10 1 1 PFU/dose), (ii) rectally, (iii) locally (skin, eye, ear, nasal mucosa, etc.), in tampons, rinses and creams, (iv) as aerosols or intrapleunal injections and (v) intravenously.
  • the invention provides homologous recombination techniques are used to introduce sequences encoding alternative proteins, non-functional proteins, or non-coding sequences into the bacteriophage DNA sequence. Such techniques are useful to "knock-out" undesired traits of the Deposited Bacteriophage, or alternatively to introduce different traits.
  • homologous recombination is used to "knock-out" ORFs encoding proteins that maybe involved in a lysogenic cycle of the bacteriophage.
  • homologous recombination is used to introduce or knock-out genes involved in burst size.
  • homologous recombination is used to introduce alternative bacteriophage genes which delay the burst event or increase the phage burst size.
  • References disclosing alternative bacteriophage genes involved in the timing of the burst event or the size of the phage burst include, but are not limited to (Johnson-Boaz, et al. (1994) "A dominant mutation in the bacteriophage lambda S gene causes premature lysis and an absolute defective plating phenotype.” MoI Microbiol 13(3): 495-504; Wang, et al. (2000) "Holins: the protein clocks of bacteriophage infections.” Annu Rev Microbiol 54: 799-825, each of which is hereby incorporated by reference in their entirety.
  • recombinant bacteriophage harboring reporter system(s) is generated for various practical applications.
  • One example of possible application of such system is species identification/confirmation of Targeted Bacteria as bacterial diagnostics.
  • Another possible application is the detection of the presence of viable cells of Targeted Bacteria to which the Deposited Bacteriophage have specificity.
  • Loessner et al. for example, one of skill in the art can generate recombinant reporter bacteriophage. See Loessner, et al.
  • the Vibrio harveyi luxAB gene may be introduced into the bacteriophage DNA sequence using techniques such as homologous recombination.
  • An ideal target for the introduction of the luxAB gene is immediately downstream and in frame with an ORF encoding bacteriophage capsid protein, thereby creating a sequence encoding a fusion protein.
  • the preferable location of introduction of the luxAB gene sequence is particularly before any sequence encoding a transcriptional terminator downstream of the ORF encoding a capsid protein.
  • Other bacteriophage ORF sequences which may function as useful sources of luxAB gene-fusions include gene sequences encoding tail-sheath proteins, or any other late gene region sequences encoding phage head or tail proteins.
  • the resulting recombinant bacteriophage may be used with methods of the invention to detect the presence of viable cells of Targeted Bacteria.
  • reporter genes which are useful for the generation of reporter bacteriophage include, but are not limited to, the firefly luciferase gene.
  • the invention further contemplates the introduction of one or more of the above- described recombinant events.
  • a recombinant bacteriophage of the invention may harbor one or more reporter gene(s) as well as lack one or more genes associated with certain undesirable biological functions of the bacteriophage.
  • Derivatives such as polypeptides, including but not limited to bacteriophage lytic enzymes, encoded by the bacteriophage or the bacteriophage progeny are used for applications designed to prevent the growth of Targeted Bacteria through cell wall lysis.
  • lytic polypeptides are useful for the prevention of the growth of Targeted Bacteria on processed and unprocessed food products, as well as equipment used for the processing of said food products.
  • bacteriophage derivatives are useful for the treatment of one or more infections in a mammal, including humans, by administering their therapeutically effective amounts to the patient.
  • This method is useful for the treatment of infections of the gastrointestinal system.
  • this method is useful in a prophylactic setting for the prevention of infection by Targeted Bacteria in pregnant mammals, including humans.
  • This method of treatment is further useful for the prevention or other disorders or infections caused by Targeted Bacteria, such as acute bloody or non-bloody diarrhea, sometimes associated with hemolytic-uremic syndrome.
  • bacteriophage derivatives such as lysins will be useful for preparing bacterial vaccines or bacterins that eliminate or reduce colonization of the Targeted Bacteria in, and/or their being shed by, various agriculturally-important animals.
  • lysins bacteriophage derivatives
  • One example of a practical application for that type of vaccine is in the cattle-raising industry, where administration of such vaccines/bacterins may significantly reduce colonization of cattle with the Targeted Bacteria; thus, improving public safety by reducing contamination of beef with the Targeted Bacteria.
  • the Deposited Bacteriophage, its progeny, recombinant bacteriophage, or derivatives of the above are useful in methods of screening environmental samples (including food products and food processing equipment) and clinical specimens for the presence of viable cells of Targeted Bacteria.
  • recombinant bacteriophage containing a reporter system such as, for example, a luciferase reporter system is applied to the sample and analyzed at some time point in the future for the activation of the reporter molecule. The activation of the reporter molecule is indicative of the presence of viable cells of Targeted Bacteria.
  • the Deposited Bacteriophage, its progeny, recombinant bacteriophage, or derivatives such as lytic enzymes are useful in methods of screening environmental samples including food products and food processing equipment and clinical specimens for the presence of viable cells of Targeted Bacteria, by monitoring and measuring bacterial metabolism products such as bacterial adenosine kinase (AK) or adenosine triphosphate (ATP) released as a result of specific lysis of Targeted Bacteria.
  • AK bacterial adenosine kinase
  • ATP adenosine triphosphate
  • the original Deposited Bacteriophage, its progeny, recombinant bacteriophage, or derivatives such as lytic enzymes will specifically lyse Targeted Bacteria without affecting any other prokaryotic or eukaryotic cells that may be present in the sample, thus providing means for accurately and specifically identifying and detecting Targeted Bacteria.
  • the Deposited Bacteriophage, and/or its progeny and derivatives may be further useful as a tool for the epidemiological typing of Targeted Bacteria.
  • one of skill in the art can use the Deposited Bacteriophage of the invention to screen a panel of Targeted Bacteria isolates to aid in the taxonomic identification of the Targeted Bacteria, by determining which isolates yield a positive lytic reaction to the Deposited Bacteriophage. See e.g., van der Mee-Marquet, et al.
  • the Deposited Bacteriophage, and/or its progeny and derivatives, also may be valuable for preparing bacterial lysates to be used as vaccines or bacterins.
  • the immunogenicity of such vaccines or bacterins may be superior to that of so-called dead cell vaccines because phage-mediated lysis is a more effective and gentler approach for exposing protective antigens of bacteria than are approaches used to prepare the latter vaccines.
  • methods commonly used to inactivate bacterial pathogens for dead- cell vaccines including but not limited to heat treatment, UV-irradiation, and chemical treatment, may deleteriously affect a vaccine's effectiveness by reducing the antigenicity of relevant immunological epitopes (Holt, et al.
  • Example 1 - ECTA-47, ECML-83, ECML-119, and ECML-122 Bacteriophage Isolation
  • the ECTA-47 and ECML-119 bacteriophages were isolated from Baltimore Inner Harbor waters using lysis of the Targeted Bacteria to form plaques in bacterial lawns as a means of detecting the presence of bacteriophage having lytic specificity for the Targeted Bacteria.
  • ECML-83 bacteriophage was isolated from pooled environmental waters from the Baltimore, Maryland region using lysis of the Targeted Bacteria to form plaques in bacterial lawns as a means of detecting the presence of bacteriophage having lytic specificity for the Targeted Bacteria.
  • ECML- 122 bacteriophage was isolated from pond effluent from a pond near chicken farms in Minneapolis, MD, using lysis of the Targeted Bacteria to form plaques in bacterial lawns as a means of detecting the presence of bacteriophage having lytic specificity for the Targeted Bacteria. Plaques were harvested, diluted, and re-plated on bacterial lawns through a process of serial enrichment until a single bacteriophage species, or monophage, was obtained as determined by a stable restriction fragment length profile of the bacteriophage DNA. The isolates obtained using the technique recited supra may be cultured using the techniques as set forth herein. The bacteriophage was deposited with the ATCC.
  • Concentration of the Deposited Bacteriophage may be determined using techniques known in the art (Adams, M. H. (1959). Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519.). When a single phage particle encounters a permissive bacterium it will lyse it with the concomitant release of newly formed phage particles. When phages are mixed with host cells and poured in a layer of soft agar on the surface of a nutrient agar plate supporting bacterial growth, the cells will resume growth. In areas where no phages are present the bacteria will grow to stationary phase, forming a smooth opaque layer or lawn in the overlay.
  • phage progeny from each infected bacterium will infect neighboring bacteria, resulting in a growing zone of lysis full of liberated phage which eventually becomes visible to the naked eye as a plaque in the otherwise smooth bacterial lawn. These plaques can be counted, and their number is widely used for expressing phage titer in plaque- forming units or PFU.
  • concentration of the Deposited Bacteriophage may be determined. Briefly: (1) Various dilutions of the Deposited Bacteriophage preparation are prepared; for example, by mixing 0.1 ml of phage sample with 9.9 ml of sterile LB broth. The samples are gently but thoroughly mixed.
  • 0.5 ml of this mixture (which is a 10 '2 dilution of the original sample) is mixed with 4.5 ml of sterile LB broth (10 ⁇ 3 dilution).
  • Several 10-fold dilutions are prepared in a similar fashion; (2) the contents of the tubes (1 ml of various dilutions) are transferred into sterile 10 ml culture tubes and 0.1 ml of host bacterial culture are added. The sample is mixed gently before proceeding immediately to the next step; (3) 3 - 5 ml of warm (45-50°C) 0.7% LB agar (top agar) are added. The sample is mixed quickly and very gently.
  • the Deposited Bacteriophage is produced using a culture system. More specifically, strain of the host Targeted Bacteria or other closely-related bacterial species on which the bacteriophage can propagate is cultured in batch culture, followed by inoculation of the bacteriophage at the pre-determined multiplicity of infection (MOI). Following incubation and bacterial lysis, the bacteriophage is harvested and purified and/or concentrated to yield phage progeny suitable for the uses enumerated herein. Purification and concentration procedures included variously processing through filtration system(s), centrifugation (including continuous-flow centrifugation) or other well known bacteriophage purification and concentration techniques (Adams, M. H. (1959).
  • the invention provides compositions comprising active viral particles of the bacteriophage capable of lysing strains of Targeted Bacteria.
  • concentration of bacteriophage is determined using phage titration protocols.
  • the final concentration of the bacteriophage is adjusted by concentration, if a more concentrated phage composition is desired, via filtration, centrifugation, or other means, or by dilution, if a less concentrated phage composition is desired, with water or buffer to yield a phage titer of 10 6 to 10 12 PFU/ml, preferably 10 10 to 10 1 1 PFU/ml.
  • the resulting bacteriophage compositions are generally stored at 4° C; alternatively, preparations can be freeze or spray-dried for storage, or can be encapsulated and stabilized with protein, lipid, polysaccharide, or mixtures thereof.
  • the phage titer can be verified using phage titration protocols and host bacteria.
  • One of skill in the art is capable of determining bacteriophage titers using widely known bacteriophage assay techniques. Adams (1959) Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519, hereby incorporated by reference in its entirety.
  • the bacteriophage produced using the methods of the present invention may be dispersed in an appropriate aqueous solution or lyophilized or freeze-dried powder and applied to the surface of food products.
  • the bacteriophage may be included with a cheese culture or other microbially active foodstuff prior to or during processing.
  • Example 5 Isolation of the Bacteriophage DNA (ECTA-47, ECML-83, ECML-119, ECML-122)
  • Bacteriophage DNA a derivative of the bacteriophage, can be used for various applications such as for preparing DNA-based vaccines, and also for analytical purposes, for identifying the bacteriophage such as RFLP profile determination and comparison.
  • Phage DNA can be isolated using a suitable commercial kit such as the Lambda Mini Kit (Qiagen, Inc.; Valencia, CA) or the standard phenol extraction technique. For example, 0.75 ml of phage in phosphate-buffered saline solution at a titer of 10 8 -10 u PFU/ml is collected.
  • the supernatant (approximately 600 ⁇ l) is carefully removed and transferred to a clean eppendorf tube. 0.6 ml of chloroform is added to the supernatant, mixed well, and centrifuged at 13,000 RPM for five (5) min. The supernatant is then carefully extracted (approximately 500 ⁇ l). Next, 0.1 volumes of 3M sodium acetate (40 ml) is added to the solution, followed by 2.5 volumes of cold 95% ethanol (1 ml) to precipitate the bacteriophage DNA. The solution is allowed to incubate at -2O 0 C for 1 hour, followed by centrifugation at 13,000 RPM for thirty (30) min.
  • RFLP can be used to identify the Deposited Bacteriophage or its progeny.
  • the progeny will have a substantially equivalent RFLP DNA profile as the RFLP DNA profile of the original bacteriophage.
  • a reference RFLP profile of the Deposited Bacteriophage is shown in Figure 1. DNA was isolated from the bacteriophage using Qiagen Plasmid
  • DNA was quantitated by measuring absorbance at 260 nm. Approximately 0.5 - 1 ⁇ g of
  • DNA was digested with an appropriate restriction enzyme (Figure 1), and RFLP profile was determined on 1% agarose gel after staining with ethidium bromide.
  • Example 7 Lytic Specificity of the Bacteriophage (ECML-117 and ECML- 134)
  • ECML-117 and ECML- 134) Two hundred twenty-six E. coli strains were screened for their susceptibility to the bacteriophage by the drop-on-lawn method, also known as the "spot test" method. Strains were streaked onto LB agar plates and incubated at 37°C overnight. One colony of each strain was inoculated into a separate well of a 96-well microtiter plate containing LB broth and incubated at 37°C until the OD600 reached 0.2-0.3. One hundred microliters of each strain were mixed with LB soft agar and poured onto an LB agar plate.
  • the ECML-117 bacteriophage was isolated from Baltimore Inner Harbor waters using lysis of the Targeted Bacteria to form plaques in bacterial lawns as a means of detecting the presence of bacteriophage having lytic specificity for the Targeted Bacteria. Plaques were harvested, diluted, and re-plated on bacterial lawns through a process of serial enrichment until a single bacteriophage species, or monophage, was obtained as determined by a stable restriction fragment length profile of the bacteriophage DNA. The isolates obtained using the technique recited supra may be cultured using the techniques as set forth herein. The bacteriophage was deposited with the ATCC.
  • Concentration of the bacteriophage may be determined using techniques known in the art. Adams (1959) Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519, hereby incorporated by reference in its entirety. When a single phage particle encounters a permissive bacterium it will lyse it with the concomitant release of newly formed phage particles. When phages are mixed with host cells and poured in a layer of soft agar on the surface of a nutrient agar plate supporting bacterial growth, the cells will resume growth. In areas where no phages are present the bacteria will grow to stationary phase, forming a smooth opaque layer or lawn in the overlay.
  • phage progeny from each infected bacterium will infect neighboring bacteria, resulting in a growing zone of lysis full of liberated phage which eventually becomes visible to the naked eye as a plaque in the otherwise smooth bacterial lawn.
  • These plaques can be counted, and their number is widely used for expressing phage titer in plaque-forming units or PFU.
  • concentrations of the ECML-117 or ECML-134 bacteriophages may be determined.
  • the sample was mixed gently before proceeding immediately to the next step; (3) 3 - 5 ml of warm (45-50°C) 0.7% LB agar (top agar) were added. The sample was mixed quickly and very gently. Then, the entire contents of the culture tube were poured onto a plate containing solidified LB agar (bottom agar).
  • the plates were slid in circles a few times on the bench top immediately after pouring; (4) after sitting at room temperature for 10 min to allow the top agar to harden, the plates were inverted and placed into a 37°C incubator and incubated overnight; (5) the next morning, the number of plaques on the plate with 30-300 individual well spaced plaques were counted and the titer calculated and expressed as PFU/ml of the starting sample.
  • the bacteriophage was produced using a culture system. More specifically, strain of the host Targeted Bacteria or other closely-related bacterial species on which the bacteriophage can propagate was cultured in batch culture, followed by inoculation of the bacteriophage at the pre-determined multiplicity of infection (MOI). Following incubation and bacterial lysis, the bacteriophage was harvested and purified and/or concentrated to yield phage progeny suitable for the uses enumerated herein. Purification and concentration procedures included variously processing through filtration system(s), centrifugation (including continuous-flow centrifugation) or other well known bacteriophage purification and concentration techniques. Adams (1959) Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519, hereby incorporated by reference in its entirety.
  • the invention provides compositions comprising active viral particles of the bacteriophage capable of lysing strains of Targeted Bacteria.
  • concentration of bacteriophage was determined using phage titration protocols.
  • the final concentration of the bacteriophage was adjusted by concentration, if a more concentrated phage composition is desired, via filtration, centrifugation, or other means, or by dilution, if a less concentrated phage composition is desired, with water or buffer to yield a phage titer of 10 6 to 10 12 PFU/ml, preferably 10 10 to 10 11 PFU/ml.
  • the resulting bacteriophage compositions were generally stored at 4° C; alternatively, preparations can be freeze or spray-dried for storage, or can be encapsulated and stabilized with protein, lipid, polysaccharide, or mixtures thereof.
  • the phage titer can be verified using phage titration protocols and host bacteria.
  • One of skill in the art is capable of determining bacteriophage titers using widely known bacteriophage assay techniques. Adams (1959) Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd.: 443-519, hereby incorporated by reference in its entirety.
  • Example 10 Application of the Bacteriophage for Preservation of Food Products
  • the bacteriophage produced using the methods of the present invention may be dispersed in an appropriate aqueous solution or lyophilized or freeze-dried powder and applied to the surface of food products.
  • the bacteriophage may be included with a cheese culture or other microbially active foodstuff prior to or during processing.
  • Bacteriophage DNA a derivative of the bacteriophage, can be used for various applications such as for preparing DNA-based vaccines, and also for analytical purposes, for identifying the bacteriophage such as RFLP profile determination and comparison.
  • Phage DNA can be isolated using a suitable commercial kit such as the Lambda Mini Kit (Qiagen, Inc.; Valencia, CA) or the standard phenol extraction technique. For example, 0.75 ml of phage in phosphate-buffered saline solution at a titer of 10 8 -10 u PFU/ml is collected.
  • the supernatant (approximately 600 ⁇ l) is carefully removed and transferred to a clean eppendorf tube. 0.6 ml of chloroform is added to the supernatant, mixed well, and centrifuged at 13,000 RPM for five (5) min. The supernatant is then carefully extracted (approximately 500 ⁇ l). Next, 0.1 volumes of 3M sodium acetate (40 ml) is added to the solution, followed by 2.5 volumes of cold 95% ethanol (1 ml) to precipitate the bacteriophage DNA. The solution is allowed to incubate at -2O 0 C for 1 hour, followed by centrifugation at 13,000 RPM for thirty (30) min.
  • E. coli O157:H7 strains were screened for their susceptibility to the bacteriophage by the drop-on-lawn method, also known as the "spot test" method. Strains were streaked onto LB agar plates and incubated at 37°C overnight. One colony of each strain was inoculated into a separate well of a 96-well microtiter plate containing LB broth and incubated at 37°C until the OD600 reached 0.2-0.3. One hundred microliters of each strain were mixed with LB soft agar and poured onto an LB agar plate.
  • the bacteriophage or its derivative, such as lytic enzyme, produced using the methods of the present invention is used to specifically lyse Targeted Bacteria without affecting any other prokaryotic or eukaryotic cells that may be present in the sample; thus, specifically eliciting their release of measurable bacterial products such as AK or ATP.
  • Example 14 Preparing Vaccines and Bacterins (ECML-117 and ECML-134)
  • lytic phages ECML-117 or ECML-134 to lyse specific strains of the Targeted Bacteria, which will yield bacterial lysates containing minimally-affected immunological epitopes of the bacteria.
  • the phage may be removed from the final vaccine/bacterin preparation. Alternatively, it may be retained unaltered in the preparation because its lytic activity against Targeted Bacteria that may be present in the mammalian organism being vaccinated may increase the preparation's efficacy.
  • the most prevalent, problematic strains of the Targeted Bacteria are chosen so that the vaccine/bacterin contains the immunological epitopes that are most relevant for protecting against the infection, and (ii) the bacteriophage is kept unaltered in the final vaccine/bacterin, at levels ranging from 10 6 -10 10 PFU/ml.
  • Bacteriophage-based vaccines and bacterins also may be prepared by using derivatives of bacteriophages ECML- 117 or ECML-134 to lyse the Targeted Bacteria.
  • An example of the general methodology can be briefly outlined from a recent study (Panthel, K., W. Jechlinger, et al. (2003). "Helicobacter pylori ghosts by PhiX protein E-mediated inactivation and their evaluation as vaccine candidates.” Infect Immun 71(1): 109-16.) of an Helicobacter pylori bacterin. The authors used E. coli—H.
  • the authors triggered e gene-expression in order to elicit bacterial lysis, and they found that the lysate protected BALB/c mice against H. pylori infection.
  • the bacteriophage or its derivative, such as lytic enzyme, produced using the methods of the present invention is used to specifically lyse Targeted Bacteria without affecting any other prokaryotic or eukaryotic cells that may be present in the sample; thus, specifically eliciting their release of measurable bacterial products such as AK or ATP.
  • Targeted Bacteria cells it may be possible to establish a correlation between the luminometer readings and the number of Targeted Bacteria cells lysed (in general, the average amount of ATP per bacterial cell is 0.5-1.0 fg; precise correlation between the luminometer readings and the number of Targeted Bacteria cells should be experimentally established). If Targeted Bacteria cells are not present in the food samples analyzed, bacterial lysis and ATP -release will not occur.
  • bacteriophages to prepare vaccines and bacterins is to use the lytic Deposited Bacteriophage to lyse specific strains of the Targeted Bacteria, which will yield bacterial lysates containing minimally-affected immunological epitopes of the bacteria.
  • the phage may be removed from the final vaccine/bacterin preparation. Alternatively, it may be retained unaltered in the preparation because its lytic activity against Targeted Bacteria that may be present in the mammalian organism being vaccinated may increase the preparation's efficacy.
  • the most prevalent, problematic strains of the Targeted Bacteria are chosen so that the vaccine/bacterin contains the immunological epitopes that are most relevant for protecting against the infection, and (ii) the bacteriophage is kept unaltered in the final vaccine/bacterin, at levels ranging from 10 6 -10 10 PFU/ml.
  • Bacteriophage-based vaccines and bacterins also may be prepared by using derivatives of the Deposited Bacteriophage to lyse the Targeted Bacteria.
  • An example of the general methodology can be briefly outlined from a recent study — Panthel, et al. (2003) "Helicobacter pylori ghosts by PhiX protein E-mediated inactivation and their evaluation as vaccine candidates.” Infect Immun 71(1): 109-16, herein incorporated by reference in its entiritey, of an Helicobacter pylori bacterin. The authors used E. coli-H. pylori shuttle plasmid pHel2 and lysis gene e of bacteriophage ⁇ X174 to construct H.
  • pylori lysis plasmid pHPC38 which they introduced into H. pylori strain P79.
  • the authors triggered e gene-expression in order to elicit bacterial lysis, and they found that the lysate protected BALB/c mice against H. pylori infection.

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Abstract

La présente invention porte sur un bactériophage isolé ayant une forte activité lytique contre des souches d'E. coli comprenant, mais sans y être limité, les souches d'E. coli O157:H7 et sur des procédés d'utilisation de ce bactériophage, et/ou de la descendance ou de dérivés de celui-ci, pour contrôler la croissance d'E. coli dans divers environnements.
PCT/US2008/013659 2007-12-12 2008-12-12 Nouveaux bactériophages d'e. coli et leurs utilisations WO2009075884A1 (fr)

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US11/955,176 2007-12-12
US11/955,145 2007-12-12
US11/955,145 US7625556B2 (en) 2007-12-12 2007-12-12 E. coli bacteriophage and uses thereof
US11/955,176 US7625741B2 (en) 2007-12-12 2007-12-12 E. coli O157:H7 bacteriophage and uses thereof

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WO2015112693A1 (fr) * 2014-01-24 2015-07-30 The Procter & Gamble Company Articles absorbants jetables comprenant une ou plusieurs compositions médicales pour la peau et procédés associés
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EP2893933A1 (fr) * 2014-01-10 2015-07-15 Pherecydes Pharma Thérapie bactériophage des infections d'E. coli
WO2015104388A1 (fr) * 2014-01-10 2015-07-16 Pherecydes Pharma Phagothérapie des infections à e. coli
AU2015205512B2 (en) * 2014-01-10 2018-11-08 Erytech Pharma Phage therapy of E coli infections
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US10918680B2 (en) 2014-01-10 2021-02-16 Pherecydes Pharma Phage therapy of E coli infections
EP3586855A1 (fr) * 2014-01-10 2020-01-01 Pherecydes Pharma Therapie par bacteriophage des infections par e.coli
WO2015112697A1 (fr) * 2014-01-24 2015-07-30 The Procter & Gamble Company Lingettes de soin de la peau comprenant des agents antibactériens naturels ajoutés
WO2015112693A1 (fr) * 2014-01-24 2015-07-30 The Procter & Gamble Company Articles absorbants jetables comprenant une ou plusieurs compositions médicales pour la peau et procédés associés
US9937087B2 (en) 2014-01-24 2018-04-10 The Procter & Gamble Company Disposable absorbent articles comprising skin health composition(s) and related methods
US10898530B2 (en) 2014-11-07 2021-01-26 Pherecydes Pharma Phage therapy
US11779617B2 (en) 2014-11-07 2023-10-10 Pherecydes Pharma Phage therapy
US11058131B2 (en) 2015-04-16 2021-07-13 Kennesaw State University Research And Service Foundation, Inc. Escherichia coli O157:H7 bacteriophage Φ241
WO2019211635A1 (fr) * 2018-05-02 2019-11-07 Jsc "Biochimpharm" Composition antimicrobienne et son utilisation
WO2019211634A1 (fr) * 2018-05-02 2019-11-07 Jsc "Biochimpharm" Composition antimicrobienne contre les infections gastro-intestinales et son utilisation

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