WO2016003307A1 - Souches de bactériophages, compositions et procédés associés - Google Patents

Souches de bactériophages, compositions et procédés associés Download PDF

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Publication number
WO2016003307A1
WO2016003307A1 PCT/RU2014/000483 RU2014000483W WO2016003307A1 WO 2016003307 A1 WO2016003307 A1 WO 2016003307A1 RU 2014000483 W RU2014000483 W RU 2014000483W WO 2016003307 A1 WO2016003307 A1 WO 2016003307A1
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bacteriophage
strain
composition
escherichia coli
seq
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PCT/RU2014/000483
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English (en)
Inventor
Andrey Vladimirovich ALESHKIN
Evgeny Olegovich RUBALSKY
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Limited Liability Company "Bphage"
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Priority to PCT/RU2014/000483 priority Critical patent/WO2016003307A1/fr
Publication of WO2016003307A1 publication Critical patent/WO2016003307A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C12N2795/00032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to bacteriophage strains, antibacterial compositions, methods for preparing these and their use in the food, pharmaceutical and healthcare industries.
  • the invention has particular application in the treatment and prophylaxis of diseases in humans and animals and for uses as processing aids and disinfectants.
  • Bacteriophages have been used for a wide range of antibacterial applications, including bio-control in the food industry and for the prophylaxis and treatment of diseases in humans and animals.
  • a problem with known antibacterial compositions containing bacteriophages is the necessity of inclusion in the composition of a wide variety of species and/or strains of bacteriophages in order to broaden the spectrum of their specific activity. This problem results from the absence of bacteriophages with lytic activity against a broad range of pathogenic bacterial strains that are clinically important for humans or that have lytic activity against several bacterial species (referred herein as polyvalent bacteriophages).
  • a further problem with known compositions is retaining suitable titer in storage.
  • bacteriophages with a lytic activity against several bacterial species polyvalent bacteriophages
  • polyvalent bacteriophages e.g. bacteriophage ⁇ , which lyses Escherichia coli, Klebsiella pneumonia and Aerobacter aerogenes
  • S. Welkos, M. Schreiber, H. Baer S. Welkos, M. Schreiber, H. Baer:
  • Korean patent filing KR 101101376 refers to a bacteriophage strain as an active component of a drug for prophylaxis and treatment of diseases caused by Escherichia coli or Shigella sonnei.
  • the strain is not sufficiently active against the wide range of pathogens (including Escherichia coli serotypes O104:H4, 0157:H7, as well as Shigella flexneri).
  • the disclosed compositions do not address the problem of stabilizing the bacteriophage titer in storage.
  • the present invention provides improved bacteriophage strains, compositions, methods of preparation and uses against bacteria which may be antibiotic resistant.
  • the invention provides an Escherichia coli bacteriophage strain which has been deposited at Leibniz-lnstitut DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstr. 7B, 38124 Braunschweig, Germany with deposit accession number DSM 28572 on 14 th March 2014 in accordance with the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (known hereinafter as bacteriophage strain no.
  • an Escherichia coli bacteriophage strain comprising a nucleic acid sequence at least 90%, 93%, 96%, 99% or 100% identical to SEQ ID NO:1 or SEQ ID NO:4.
  • the invention provides a nucleic acid sequence encoding the genome of a bacteriophage comprising a sequence at least 90%, 93%, 96%, 99% or 100 % identical to SEQ ID NO:1 or SEQ ID NO:4.
  • an antibacterial composition comprising at least one bacteriophage strain of the invention and one or more additives.
  • the antibacterial composition comprises bacteriophage strain BPhEd and bacteriophage strain BPhEc2.
  • the invention provides an endolysin protein obtainable from a strain of the invention comprising a sequence of amino acids at least 90 %, 93 %, 96%, 99% or 100 % identical to SEQ ID NO:2 or SEQ ID NO:5, and nucleic acids encoding these endolysin proteins.
  • the nucleic acid comprises a nucleotide sequence at least 90 %, 93 %, 96%, 99% or 100 % identical to SEQ ID NO:3 or SEQ ID NO: 6.
  • an antibacterial composition comprising at least one endolysin of the invention
  • the antibacterial compositions may further comprise one or more bacteriophages selected from the group: Staphylococcus bacteriophage, Salmonella enteritidis bacteriophage, Salmonella infantis bacteriophage, Salmonella typhimurium bacteriophage, Listeria
  • the composition may comprise one or more additives selected from the group: licorice root syrup, apple pectin, glycine, buffer solution, pharmaceutically acceptable carrier, excipient, adjuvant, or mixtures thereof.
  • the additives may be selected from the group licorice root syrup, apple pectin, glycine.
  • the composition may comprise a buffer solution acceptable for food industries or health-care facilities in the amount of 1 to 95 % of the composition volume.
  • the composition is suitable for prophylaxis and/or treatment of human or animal infectious diseases or for decontamination.
  • the composition may be used for decontamination of food raw materials or semi-finished or finished food products, or food storage containers, or equipment for food production, or work surfaces, or food production facilities or medical tools or equipment, or equipment of health facilities, or premises of health care facilities.
  • composition may comprise one or more additives selected from the group:
  • compositions suitable for prophylaxis and/or treatment of human or animal infectious diseases may be prepared in water or buffer solution or may be prepared in other forms, for example encapsulated in a capsule, such as a hard-shelled capsule, or in the form of a suppository.
  • an antibacterial composition comprising at least one bacteriophage strain and / or at least one endolysin of the invention for use as a medicament.
  • an antibacterial composition comprising at least one bacteriophage strain and / or at least one endolysin of the invention for the treatment or prophylaxis of a bacterial infection in humans or animals.
  • a pharmaceutical composition comprising at least one bacteriophage strain and / or at least one endolysin of the invention in a pharmaceutically acceptable carrier.
  • a method of treatment of a bacterial infection in humans comprising administering a therapeutically effective amount of an anti-bacterial composition comprising at least one bacteriophage strain of the invention to a patient suffering from a bacterial infection.
  • a prophylactic method of treatment of humans comprising
  • an anti-bacterial composition comprising at least one bacteriophage strain of the invention.
  • the one or more bacteriophage strains each have a lytic activity of at least 10 6 pfu per ml of the
  • compositions against test strains such as Escherichia coli 0104:1-14 and K12 C600.
  • test strains such as Escherichia coli 0104:1-14 and K12 C600.
  • the one or more bacteriophage strains each have a lytic activity of at least 10 6 pfu per capsule or suppository against test strains.
  • a method for obtaining a bacteriophage comprising the steps of (i) inoculating a bacterial host strain at a titer of 10 8 - 10 9 CFU/ml and cultivating it up to log phase in a cultivation vessel with solid growth medium; (ii) inoculating the resulting lawn culture of the host strain with the bacteriophage strain at a titer of 10 5 -10 6 pfu/ml, sealing the cultivation vessel and cultivating the bacteriophage strain culture under a layer of air; (iii) then obtaining a phage lysate suspension by treating the culture with a physiological solution or a buffer solution with a pH of 6.3 - 7.5; (iv) mixing the phage lysate suspension with an organic solvent that is immiscible with water and subjecting the mixture to centrifugation and (v) filtering the resulting supernatant and subsequently passing the supernatant through a column
  • the solid growth medium has a thickness of 10-25mm and the layer of air has a thickness of 25-40mm over the surface of the solid growth medium.
  • the cultivation vessel may, for example be a glass mattress flask.
  • the supernatant is filtered through a filter with a pore size of 0.20 to 0.22 ⁇ .
  • One or more additional phage lysates may be mixed with the supernatant prior to filtration.
  • the host strain is not pathogenic for humans or animals and the bacteriophage strain has been previously passaged through a culture of a second host strain that is pathogenic for humans or animals.
  • the bacterial host strain may be inoculated with a bacteriophage strain according to the invention.
  • the bacterial host strain is Escherichia coli K12C600.
  • the bacteriophage strain BPhEd may have been previously passaged through a culture of
  • the bacteriophage strain BPhEc2 may have been previously passaged through a culture of Escherichia coli serotype 0157:1-17.
  • a particular advantage of the present invention is the simplification of the contents of phage-based antibacterial compositions while achieving the necessary spectrum of specific activity and selection of specific additives able to secure titer stability in storage, for example for 2 years in a liquid form and up to 5 years in a freeze-dried form.
  • compositions are provided by the inclusion in the antibacterial composition of an Escherichia coli bacteriophage strain BPhEd (deposit number DSM 28572) and / or Escherichia coli bacteriophage strain BPhEc2 (deposit number DSM 28789).
  • compositions comprise both BPhEd and BPhEc2 as the host range activities of each strain have been found to be complementary.
  • composition may further comprise one or more virulent bacteriophages selected from: a Staphylococcus
  • each of the bacteriophages have a lytic activity of at least 10 6 pfu per mL of the composition against susceptible test strains and human bacteria isolates.
  • compositions used for prophylaxis and treatment of infectious diseases in humans and animals may comprise one or more specific additives in the amount of 0.005 to 99.9% wt of the composition volume.
  • Additives may be selected from the group: licorice root syrup, apple pectin, glycine, buffer solution, pharmaceutically acceptable carrier, excipient, adjuvant, or mixtures thereof.
  • compositions in liquid form may comprise at least one bacteriophage of the invention and additives, such as licorice root syrup and /or apple pectin and /or glycine, in an amount that results in the composition having a stable lytic activity, for example for a period of around one year or more.
  • Licorice root syrup may be suitable for antispasmodic, enveloping properties and for improvement of organoleptic (taste) properties.
  • Licorice root syrup may be present in the range 0.01 to 50.0% wt of the composition, for example between 1 and 10% wt.
  • Apple pectin may be suitable as a toxin sorbent and viscosity regulator.
  • This additive may be present, for example in the range 0.005% to 6.0% wt of the composition, such as 0.005 to 2% wt or 0.005 to 0.015% wt.
  • Glycine may be suitable as a stabilizer of phage titer in storage and an antioxidant.
  • This additive may be present, for example, in the range 0.005 to 21 % wt, for example 0.005 to 20.2% wt, 0.005 to 10% wt, 0.005 to 2% wt or 0.005 to 0.015% wt of the composition.
  • compositions may comprise at least one bacteriophage of the invention and the additives licorice root syrup (1-10%wt), apple pectin (0.005 - 2%wt) and glycine (0.005 - 2% wt) in water or buffer solution.
  • Additives may be used to produce compositions suitable for use as a suppository.
  • additives may be selected from the group: glucose, solid fat, emulsifier, paraffin, and water.
  • Glucose may be present in the range 1-2% wt of the composition.
  • Solid fat, such as SolPro may be present in the range 60-80% wt of the composition.
  • Emulsifier, such as T-2 may be present in the range 5-7% of the composition.
  • Paraffin may be present in the range 5- 7% of the composition.
  • An antibacterial composition may comprise, for example, (%wt of the composition) 5-15% of a phage lysate filtrate comprising at least one bacteriophage of the invention, 1-2% of glucose, 60-80% of solid fat, 5-7% of emulsifier, 5-7% of paraffin and 7-10% of water.
  • Compositions may also comprise at least one bacteriophage of the invention in freeze- dried form.
  • the bacteriophage in freeze-dried form may comprise suitable additives, such as saccharose and / or gelatin. Additives may also be used to produce compositions suitable for encapsulation in a capsule.
  • additives may be selected from the group glucose, lactose and fumed silica.
  • Glucose may be present in the range 2-5% wt of the composition.
  • Lactose may be present in the range 35-62.5% wt of the composition.
  • Fumed silica such as Aerosil A300, may be present in the range 0.5-1.0%.
  • An antibacterial composition may comprise, for example, (%wt of the composition) 35-50% of freeze-dried bacteriophage, 2-5% of glucose, 0.5-1.0% of fumed silica, and the remainder (35-62.5%) of lactose.
  • compositions used for decontamination of food raw materials or semi-finished or finished food products, or containers for storage, equipment for food production, or work surfaces, or food production facilities may include a buffer solution acceptable for food industries in the amount of 1 to 95% of the composition volume.
  • Compositions used for the decontamination of medical tools, equipment or facilities may include a buffer solution acceptable for healthcare facilities in the amount of 1 to 95% of the composition volume.
  • Antibacterial compositions may include various buffer solutions for maintaining the pH at 6.8 - 7.8 and acceptable in specific applications, such as for the treatment and prevention of human and animal diseases and as disinfectants.
  • the Escherichia coli bacteriophage strain BPhEd was isolated from faeces of broiler chickens in a bacterial culture of the Escherichia coli strain O104:H4 N°112027 and has been deposited at DSMZ with deposit accession number DSM 28572 on March 14, 2014.
  • the 5 Escherichia coli bacteriophage strain BPhEd can be characterized by the following
  • nucleotide sequence SEQ ID NO:1 The nucleotide sequence SEQ ID NO:1 and passed bioinformatic analysis. It was ascertained that it is free of the genes encoding known toxins (stxl , stx2, eaeA, hlyA) and the genes responsible for the lysogenic type of the phage infection (integrases, repressor proteins etc.). During storage or subculture, including in deposit collections, the genome of Escherichia coli
  • the present invention includes the genome of an Escherichia coli bacteriophage strain having a nucleotide sequence from about 90 % to about 100 % identical to SEQ ID NO:1 , for example a sequence at least 90%, 93%, 96%, 99% or 100% identical to SEQ ID NO:1.
  • the Escherichia coli bacteriophage strain BPhEc2 was isolated from cowshed effluent and has been deposited at DSMZ with deposit accession number DSM 28789 on May 9 th , 2014.
  • the Escherichia coli bacteriophage strain BPhEc2 can be characterized by the following properties:
  • the genome of Escherichia coli phage strain BPhEc2 may undergo natural mutagenesis, which will not alter the encoded proteins or ribonucleic acids functionally or structurally. Accordingly the present invention includes the genome of an Escherichia coli bacteriophage strain having a nucleotide sequence from about 90 % to about 100 % identical to SEQ ID NO:4 , for example a sequence at least 90%, 93%, 96%, 99% or 100% identical to SEQ ID NO:4.
  • the nucleic acids which encode the genome of the virulent bacteriophages and which are a nucleotide sequence from 90 % to 100 % identical to SEQ ID NO:1 or SEQ ID NO:4, for example a sequence at least 90%, 93%, 96%, 99% or 100% identical to SEQ ID No.1 or SEQ ID NO:4, can be transformed into a sensitive bacterial cell (for example, into Escherichia coli strain K12C600) with a subsequent generation of a bacteriophage.
  • transformation means the process of cellular uptake of free DNA molecules from the environment.
  • Escherichia coli bacteriophage strains BPhEd and BPhEc2 produce the protein endolysin.
  • the term "endolysin” is a generic term common for bacteriophage enzymes that destroy cell membranes and peptidoglycan bacteria. An endolysin of Escherichia coli
  • bacteriophage strain BPhEd is encoded by a nucleic acid comprising a nucleotide sequence from 90 % - 100 % identical to SEQ ID NO:3, for example a nucleotide sequence at least 90%, 93%, 96%, 99% or 100% identical to SEQ ID No:3.
  • An endolysin protein obtainable from the Escherichia coli bacteriophage strain BPhEd comprises a sequence of amino acids at least 90%, 93%, 96%, 99% or 100 % identical to SEQ ID NO:2.
  • An endolysin of Escherichia coli bacteriophage strain BPhEc2 is encoded by a nucleic acid comprising a nucleotide sequence from 90 % - 100 % identical to SEQ ID NO:6, for example a nucleotide sequence at least 90%, 93%, 96%, 99% or 100% identical to SEQ ID No. 6.
  • An endolysin protein obtainable from the Escherichia coli bacteriophage strain BPhEc2 comprises a sequence of amino acids at least 90%, 93%, 96%, 99% or 100 % identical to SEQ ID NO:5.
  • the present invention encompasses other endolysins of Escherichia coli
  • compositions for anti-bacterial use may comprise at least one endolysin of the Escherichia coli bacteriophage strain BPhEd and / or BPhEc2 and / or comprising a sequence of amino acids at least 90%, 93%, 96%, 99% or 100 % identical to SEQ ID NO:2 or SEQ ID NO:5.
  • the percentage identity between two nucleotide sequences or between two amino acid sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps which need to be introduced for optimal alignment and the length of each gap.
  • Various computer programs e.g. BioEdit, Geneious
  • mathematical algorithms e.g. ClustalW, MUSCLE, MAFFT
  • Anti-bacterial compositions of the invention may further comprise a staphylococcus bacteriophage, salmonellosis bacteriophage or listeriosis bacteriophage.
  • the composition may also include any bacteriophages with their respective specific activities in the form of phage lysates (phage lysate filtrates), suitable for making preparations for prophylaxis and treatment of human or animal infectious diseases or for decontamination of food raw materials or semi-finished or finished food products, or food storage containers, equipment for food production, or work surfaces, or food production facilities or medical tools or equipment, or equipment of health facilities, or premises of health care facilities (for example as described in: Antibacterial, antiviral and serum preparations for treatment and prophylaxis. Allergens.
  • the Staphylococcus bacteriophage of the composition may be represented by a bacteriophage virulent to clinical strains of Staphylococcus aureus, including MRSA strains (methycillin-resistant strains of Staphylococcus aureus).
  • the salmonellosis bacteriophage of the composition of the present invention may be represented by bacteriophages active against Salmonella enteritidis (including the strains SE6, SE19 and/or SE40), against Salmonella typhimurium and/or against Salmonella infantis.
  • the listeriosis bacteriophage of the composition may be represented by a bacteriophage able to lyse Listeria monocytogenes (for example, strains 2, 6, 660, 944, 4908, 4909, 4910, 4913, 4944, 7973, 10522, 766GISC, 4IP, 12IP, 16IP, 20IP, 46IP, 53IP, 61 IP, 766IP, Aorig., C20/10357, C-52, C-212, EGD, EGDe, Gim03, M-5, M-6).
  • Listeria monocytogenes for example, strains 2, 6, 660, 944, 4908, 4909, 4910, 4913, 4944, 7973, 10522, 766GISC, 4IP, 12IP, 16IP, 20IP, 46IP, 53IP, 61 IP, 766IP, Aorig., C20/10357, C-52, C-212, EGD, EGDe, Gim03, M-5, M-6).
  • typhimurium strain ⁇ 1 Salmonella infantis strain 1271 ; Listeria monocytogenes strains EGDe, Gim03, 7973, C20/10357, C-52, Aorig., EGD, M-6, 12IP, 20IP, 4908, 4944, 944, C- 212, 6, M-3, 2, 16IP, 660, 4909, 4913, 766 GISC, 61 IP, 53IP, M-5, 766IP, 46IP, 10522, 4IP, 4910, WSLC 1001.
  • compositions comprising at least one bacteriophage strain of the invention may be used for the treatment or prophylaxis of bacterial infections, such as an infection caused by Escherichia coli, for example Escherichia coli serotype 0104:1-14.
  • the treatment of a patient with a bacterial infection is carried out for a period between 7 and 30 days. More preferably, the treatment period is between 14 and 21 days. Typically, if no effect is observed after 10 days then the treatment with the composition is terminated. If an improvement is observed then the maximum dosage period is typically 30 days.
  • the treatment may then be suspended, for example for a period of 14 days, and the treatment course repeated.
  • the composition is typically administered for a period between 2 and 7 days. During the treatment period the composition may be administered, for example, between 2 and 3 times a day.
  • a patient may be administered with an antibacterial composition with a volume of between 30 and 50ml_ wherein the at least one bacteriophage of the invention are each present in the composition at a lytic activity of at least 10 6 pfu per mL of the composition, tested against bacterial test strains such as Escherichia coli 0104:1-14 and K12 C600.
  • the volume of the composition may be varied depending on the lytic activity of the bacteriophage within the composition.
  • the anti-bacterial composition may also be orally dosed in a freeze dried form, for example in a capsule, such as a hard-shelled capsule.
  • each of the at least one bacteriophage of the invention has a lytic activity of at least 10 6 pfu per capsule.
  • the anti-bacterial composition may also used in the form of a suppository.
  • each of the at least one bacteriophage of the invention has a lytic activity of at least 10 s pfu per suppository.
  • the present invention provides a method for producing bacteriophages which is suitable for producing various types of bacteriophages, such as BPhEd and BPhEc2.
  • This method of phage lysate preparation enables effective cultivation of bacteriophages and a stable high titer of 10 11 -10 13 pfu/ml to be obtained, making it easier to produce antibacterial compositions with a consistent titer of the bacteriophages included therein.
  • the phage lysate is purified of endotoxins.
  • Bacteriophages of the invention may be produced as described hereinafter.
  • the bacteriophages may be prepared using a susceptible bacterial host strain that is not pathogenic for humans or animals, for example for BPhEd and BPhEc2 Escherichia coli K12 C600 may be used.
  • the host strain is inoculated at a titer of 10 8 - 10 9 CFU (colony- forming units) and cultivated up to log phase in a cultivation vessel with solid growth medium, such as a slant solid growth medium.
  • the host strain is inoculated on a growth medium with a thickness of 10 - 25 mm.
  • the cultivation may be carried out in a suitable vessel.
  • cultivation is carried out in a glass mattress flask due to its optical properties and thermal conductivity.
  • the host strain is cultivated at an optimal temperature for the strain, for example 37°C for Escherichia coli K12 C600, up to log phase.
  • the host strain is cultivated, for example, for a period of 3 - 3.5 hours.
  • the resulting lawn culture of the host strain is inoculated with the bacteriophage strain at a titer of 10 5 - 10 6 pfu/mL.
  • the bacteriophage strain may have previously been passaged through a culture of a host strain that is pathogenic for humans or animals.
  • the Escherichia coli bacteriophage strain BPhEd the bacteriophage may have previously been passaged through an Escherichia coli serotype O104:H4 culture.
  • the bacteriophage may have been previously passaged through an Escherichia coli serotype 0157:1-17 culture.
  • the cultivation vessel is then hermetically sealed, and the bacteriophage strain culture is cultivated under a layer of air.
  • the layer of air is 25-
  • the culture is cultivated at an optimal temperature for growing a bacteriophage strain culture, for example at a temperature of 37°C. Preferably, the culture is cultivated for a period of 13 -15 hours.
  • a phage lysate suspension is obtained by treating the culture with a physiological solution or a buffer solution with a pH of 6.3 to 7.5, for example between pH 7.0 - 7.2.
  • the buffer solution is in the quantity 0.04 - 0.09 ml per 1 cm 2 of the medium surface, such as 0.040 - 0.045 ml.
  • the phage lysate is then transferred into a sterile container and a water-immiscible organic solvent added.
  • the organic solvent may be chloroform.
  • the mixture may then be incubated, for example with continuous shaking for a period of about 30- 45 minutes, and then subjected to centrifugation, for example for 30 - 45 minutes at 5000 - 6000 rpm.
  • the resulting supernatant is then filtered, for example to remove any bacteria, including phage-resistant bacteria.
  • the filter has a pore size of 0.20 to 0.22 ⁇ which enables efficient filtration of the phage lysate.
  • the supernatant may be initially be passed through a filter with a pore size of 0.40 to 0.50 ⁇ , for example 0.45 ⁇ , and then passed through a filter with a pore size of 0.20 to 0.22 pm.
  • the filtrate is then passed through a column containing an agent with an affinity to one or more endotoxins.
  • the refined phage lysate should have an acceptable level of endotoxins, for example less than 50 units of endotoxin per ml of refined phage lysate (EU/mL).
  • the supernatants may be mixed with one or more additional phage lysates prior to filtration, or may be mixed after filtration.
  • the present invention provides the use of virulent bacteriophages lytically active against a broad range of pathogenic bacterial strains that are clinically important for humans.
  • the invention enables simplification of the formulation of anti-bacterial compositions.
  • the composition may also include additional monovalent virulent bacteriophages to achieve the desired range of specific activity for a given purpose. Additives may be used to stabilize the bacteriophages' lytic activity.
  • the present invention will be described in more detail and with reference to non-limiting examples.
  • the examples demonstrate the possibility of simplification of the formulation of the antibacterial compositions based on bacteriophages, while retaining the ability to achieve the desired range of specific activity by inclusion as an active ingredient at least one
  • compositions contain a number of
  • bacteriophage strains in order to provide a broad spectrum of activity against food borne infections (which is important, for example, in clinical situations when the cause of an infection is not immediately identifiable).
  • the examples include application of the composition to bacterial infections caused by E.coli strains specifically lysed by the bacteriophage strains of the invention and also the treatment or prophylaxis of other bacterial infections showing the broad utility of the described compositions.
  • the examples also include preparation of an Escherichia coli phage lysate containing a Escherichia coli bacteriophage strain, assessment of the range of antibacterial action, assessment of the effect of bacteriophages of the antibacterial composition on industrial strains of lactobacilli, bifidobacteria and normal intestinal microflora, and further specific applications of the claimed invention.
  • Example 1 Obtaining a phage lysate of Escherichia coli containing a polyvalent Escherichia coli bacteriophage strain BPhEd.
  • One milliliter of night broth culture of Escherichia coli K12 C600 was mixed with 4 ml LB broth (10 g of BactoTM tryptone, 5 g of BactoTM yeast extract and 10 g of sodium chloride per 1 litre of the medium) and 10 ⁇ suspension of Escherichia coli bacteriophage strain BPhEd at 10 9 pfu/ml.
  • the mixture was incubated in a shake flask at 37°C until the bacterial culture acquired a lighter shade, then 0.5 ml chloroform was added and the mixture was stirred vigorously for 20 minutes.
  • Bacterial cell fragments were then removed by low speed centrifugation (10 000 g, 15 min) and the phage lysate was titered using the double-layered agar technique (agar overlay method). The concentration of phage lysate was measured in plaque forming units (pfu) per 1 ml of phage lysate. The resulting phage lysate had a bacteriophage concentration of 10 9 pfu/ml.
  • a phage lysate of Escherichia coli bacteriophage strain BPhEc2 was also obtained using the same method as described above.
  • the resulting phage lysate had a bacteriophage concentration of 10 10 pfu/ml.
  • Example 2 Testing the spectrum of antibacterial activity of the bacteriophage strains BPhEd and BPhEc2 (host range activity).
  • the spectrum of antibacterial activity of the Escherichia coli bacteriophage strains BPhEd and BPhEc2 were tested using the spot test method.
  • 0.5 ml doses of night broth bacterial culture, as indicated in Table 1 were mixed with 4 ml of semisolid agar (LB broth containing 0.7% of agar) at 47°C and distributed on the surface of nutrient agar (e.g. Nutrient Agar, HIMEDIA) in a Petri dish.
  • a drop of a bacteriophage strain (10 7 pfu/ml) obtained according to the method of Example 1 was applied on the surface and incubated at 37°C during the night.
  • Example 3 Stability testing of a liquid antibacterial composition.
  • An antibacterial composition which included the following: a Escherichia coli phage lysate filtrate containing Escherichia coli bacteriophage BPhEd ; a Escherichia coli phage lysate filtrate containing Escherichia coli bacteriophage BPhEc2; a Staphylococcus phage lysate filtrate containing a Staphylococcus bacteriophage that is a virulent
  • bacteriophage a Salmonella enteritidis phage lysate filtrate containing a Salmonella bacteriophage that is a virulent bacteriophage lytically active against Salmonella enteritidis; a Salmonella infantis phage lysate filtrate containing a Salmonella bacteriophage that is a virulent bacteriophage lytically active against Salmonella infantis; a Salmonella typhimurium phage lysate filtrate containing a Salmonella bacteriophage that is a virulent bacteriophage lytically active against Salmonella typhimurium; a Listeria monocytogenes phage lysate filtrate containing a Listeria bacteriophage that is a virulent bacteriophage.
  • the mixed phage lysate filtrates were diluted with phosphate buffered saline (PBS) maintaining the lytic activity of each phage in the composition to a level at least 10 6 pfu per mL of the composition against test strains, as well as human bacterial isolates.
  • the composition also included the additives (%wt of the composition) licorice root syrup (4.0%), apple pectin (0.01%) and glycine (0.01%). Samples of the composition were studied with regard to stability of the lytic activity of the Escherichia coli bacteriophage strains BPhEd and BPhEc2 for two years of storage, as well as lysogeny tests of all of the bacteriophages of the composition. Lytic activity of the polyvalent bacteriophage strain of Escherichia coli BPhEd against the susceptible
  • Lytic activity of the bacteriophage strain of Escherichia coli BPhEc2 against the susceptible Escherichia coli strains serotypes 0157:H7, 0157:H " and K12 C600 was at least 10 6 pfu/mL. Lytic activity of other bacteriophage strains of the composition was also at least
  • Example 4 Stability testing of a freeze-dried antibacterial composition.
  • An anti-bacterial liquid composition was prepared which contained the same mixture of bacteriophage strains as included in the composition of Example 3, but with additives suitable for producing a freeze dried composition.
  • the composition was arranged to have a lytic activity of each phage of at least 10 11 pfu per mL of the composition against test strains as well as human bacterial isolates.
  • the composition also included the additives (%wt of the
  • composition saccharose (10.0%) and gelatin (1.5%).
  • the antibacterial composition was freeze-dried.
  • Samples of the composition were studied with regard to stability of polyvalent activity of the bacteriophage strain of Escherichia coli BPhEd for five years of storage, as well as lysogeny tests of all of the bacteriophages of the composition. After storage, the samples were made up with water to the same volume as in the original composition.
  • Lytic activity of the bacteriophage strain of Escherichia coli BPhEc2 against the susceptible Escherichia coli strains serotypes 0157:H7, 0157:1-1 " and K12 C600 was at least 10 11 pfu/mL Lytic activity of each of the other bacteriophage strains of the composition was also at least 10 11 pfu/ml. Lysogeny tests were all negative.
  • An anti-bacterial composition was prepared which contained the same mixture of bacteriophage strains as included in the composition of Example 3.
  • the antibacterial composition was obtained with a lytic activity of each phage of at least 10 6 pfu per mL of the composition against test strains as well as human bacterial isolates.
  • the composition also included the additives (%wt of the composition) licorice root syrup (4.0%), apple pectin (0.01%), glycine (0.01%), distilled water (95.88% wt).
  • An anti-bacterial composition was prepared which contained the same mixture of bacteriophage strains as included in the composition of Example 3, but with additives suitable for producing a suppository.
  • the antibacterial composition was obtained with a lytic activity of each phage of at least 10 6 pfu per suppository of the composition against test strains as well as human bacterial isolates.
  • the composition included 0.132ml of the phage lysate filtrates diluted with buffer per suppository.
  • the composition also included the additives per suppository: 0.0236g of glucose, 1.043 g of solid fat "SolPro", 0.088 g of emulsifier T-2, 0.088 g of paraffin P-2, 0.125 ml of purified water.
  • Samples of the composition were studied with regard to stability of lytic activities of the bacteriophage strains of Escherichia coli BPhEd and BPhEc2 for five years of storage, as well as lysogeny tests of all of the bacteriophages of the composition.
  • Escherichia coli BPhEc2 against the susceptible Escherichia coli strains serotypes 0157:H7, 0157: H " and K12 C600 was at least 10 6 pfu per suppository. Lytic activity of each of the other bacteriophage strains of the composition was also at least 10 6 pfu per suppository. Lysogeny tests were all negative.
  • Example 7 Stability testing of an antibacterial composition in the form of capsule.
  • a freeze dried anti-bacterial composition was prepared according to the method of Example 4 and then formed into a capsule.
  • the freeze dried mixture was provided a lytic activity of each phage of at least 10 6 pfu per capsule of the composition against test strains as well as human bacterial isolates.
  • the composition included 0.176g of the freeze-dried bacteriophages per capsule.
  • the composition also included the additives per capsule: 0.0236g of glucose, 0.295 g of lactose, 0.005 g of fumed silica (Aerosil A 300).
  • Lytic activity of the bacteriophage strain of Escherichia coli BPhEc2 against the susceptible Escherichia coli strains serotypes 0157:1-17, 0157:1-1 " and K12 C600 was at least 10 6 pfu per 1 capsule.
  • Lytic activity of each of the other bacteriophage strains of the composition was also at least 10 6 pfu per 1 capsule. Lysogeny tests were all negative.
  • Example 8 Assessment of the effect of the bacteriophages of the antibacterial composition on industrial strains of lactobacilli and bifidobacteria.
  • One of the tubes was used as a control tube (with only an industrial strain introduced into the culture).
  • Two other test tubes were treated with 1 ml doses of the antibacterial composition as described in Example 3.
  • the fourth tube for comparison was treated with a 1 ml dose of Intesti-bacteriophage (Scientific Development and Production Center "Microgen" of Ministry of Health Care and Social Development of Russia).
  • the contents of the tubes were mixed by pipetting, and incubated for 17 hours at 38°C. Evaluation of growth of the industrial strains was carried out by visual comparison of the optical densities of the contents of the tubes. Uncertain cases were tested by titering. Optical densities of each row of four tubes were identical.
  • Example 10 Assessment of effect of a liquid antibacterial composition - enterocolitis.
  • a microbiological test of a stool sample from a patient with enterocolitis showed presence of Escherichia coli serotype O104:H4.
  • the patient was subsequently treated orally in 40 ml doses 3 times a day for 7 consecutive days with an antibacterial composition containing the phage strains described in Example 3 in phosphate buffered saline (with a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates), and additives (licorice root syrup, apple pectin and glycine) in a total amount of 1.0 % wt of the composition.
  • clinical symptoms of enterocolitis were resolved and no indication of presence of E. coli serotype O104:H4 was found using microbiological methods.
  • Microbiological testing of a stool sample of a patient with salmonellosis showed presence of Salmonella typhi murium.
  • the patient was subsequently treated orally with 30 ml doses 3 times a day for 7 consecutive days of an antibacterial composition containing the phage strains described in Example 3 (with a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates), and additives in the amount of 45.0 % wt of the composition.
  • an antibacterial composition containing the phage strains described in Example 3 (with a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates), and additives in the amount of 45.0 % wt of the composition.
  • Microbiological testing of stool sample of a patient with enterocolitis showed presence of Escherichia coli serotype 0157:1-17.
  • the patient was subsequently treated orally with 40 ml doses 3 times a day for 14 consecutive days of an antibacterial composition containing the phage strains described in Example 3 (with a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates), and additives in an amount of 95.0 % wt of composition.
  • Clinical symptoms of enterocolitis were resolved and no indication of presence of E. coli serotype 0157:H7 was found using microbiological methods.
  • Example 13 Assessment of prophylactic effect - nosocomial infection caused by Salmonella typhimurium.
  • Example 14 Assessment of prophylactic effect - infection by Salmonella enteritidis Cases of an infection caused by Salmonella enteritidis were confirmed within a population. A group of 100 people were treated orally 3 times a day for 6 consecutive days with 40 ml doses of antibacterial composition containing the phage strains described in Example 3 (with a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates) and additives in an amount of 55.0 % wt of the composition. Among the group of people treated with the antibacterial composition no signs of infection caused by Salmonella enteritidis were demonstrated. Amongst a group of people not treated with the composition there were cases of infection.
  • Example 15 Assessment of prophylactic effect - food infection by Staphyloccus aureus An infection caused by Staphyloccus aureus was discovered amongst the clients of a catering facility. A group of the clients of the facility were treated orally 3 times a day for 5 consecutive days with 40 ml doses of antibacterial composition containing the phage strains described in Example 3 (at a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates), and additives in an amount of 95.0 % wt of the composition. Within the treatment group, there were no signs of toxic food infection. Among clients not treated with antibacterial composition, there were cases of toxic food infection, caused by Staphylococcus aureus.
  • the catering facility was shut for 5 days and during this period the catering staff were treated 3 times a day for 5 consecutive days with 40 ml doses of the antibacterial composition. After the treatment, cases of toxic food infection caused by Staphylococcus aureus subsided among the facility's clients.
  • Example 16 Using antibacterial composition as a processing aid
  • Escherichia coli serotype 0157:H7 In order to prevent further cases of contamination beef samples were pulverized with an antibacterial composition before vacuum sealing, the composition including the phage strains described in Example 3 (at a concentration of each phage of at least 10 6 pfu per mL of the composition against test strains, as well as human bacterial isolates), and additives in an amount of 95.0 % wt of the composition. Within the expiration period in sealed package, stated by the manufacturer, Escherichia coli serotype 0157:H7 was not found.
  • Example 17 Using antibacterial composition as a disinfectant
  • Staphylococcus aureus all of the surfaces of the facility and medical equipment were treated for 1 week with an antibacterial composition, which included the phage strains described in Example 3 (at a concentration of each phage of at least 10 6 pfu per ml of the composition against test strains, as well as human bacterial isolates), and additives in an amount 45.0 % wt of the composition. After the disinfection, cases of nosocomial infection declined.
  • a microbiological test of a stool sample from a patient with enterocolitis showed presence of Escherichia coli serotype 0104:H4.
  • the patient was subsequently treated orally with one capsule containing an antibacterial composition, as described in Example 7, twice a day for 14 consecutive days.
  • clinical symptoms of enterocolitis were resolved and no indication of presence of E. coli serotype O104:H4 was found using microbiological methods.
  • Example 19 Assessment of effect of antibacterial composition in the form of a suppository - enterocolitis.
  • Microbiological testing of stool sample of a patient with enterocolitis showed presence of Escherichia coli serotype 0157:H7.
  • the patient was subsequently treated rectally 2 times a day for 14 consecutive days with one suppository of an antibacterial composition as described in Example 6.
  • clinical symptoms of enterocolitis were resolved and no indication of presence of E. coli serotype 0157:1-17 was found using microbiological methods.
  • Example 20 Methods of obtaining bacteriophage active against disease-causing bacteria - salmonella
  • Typhimurium bacterial culture of the host strain - Salmonella entehca serovar Typhimurium - with a titer of 10 8 CFU/ml was inoculated in a cultivation vessel on a slant solid growth medium 10 - 25 mm in thickness.
  • the host strain was cultivated for 3.5 hours at an optimal temperature (+37°C), then the resulting bacterial lawn of Salmonella entehca serovar Typhimuhum was inoculated with a stock bacteriophage with a titer of 10 5 pfu/ml, the vessel was then hermetically sealed (glass mattress flask) and the bacteriophage was cultivated for 13 hours at an optimal temperature (+37°C) for growing a bacteriophage strain culture with a layer of air 25 - 40 mm in thickness over the surface of the solid growth medium.
  • Phage lysate was obtained by suspending the bacteriophage from the surface of the solid growth medium with saline in a proportion of 0.04 ml per 1 cm 2 of the surface of the solid growth medium.
  • the obtained refined phage lysate in the form of an eluate contained bacteriophages with a titer of 10 13 pfu/ml, free from bacteria or ingredients of the growth medium that had been used and with a concentration of endotoxin less than 50 units of endotoxin per 1 ml of refined phage lysate (EU/ml).
  • Example 21 Methods of obtaining cocktail of bacteriophages
  • bacterial culture of the non-pathogenic host strain - Escherichia coli K12 C600 - with a titer of 10 9 CFU /ml was inoculated in a cultivation vessel on a slant solid growth medium 10 - 25 mm in thickness.
  • the host strain was cultivated for 3.2 hours at an optimal temperature (+37°C), then to the resulting bacterial lawn of Escherichia coli K12 C600 a stock bacteriophage was inoculated with a titer of 10 6 pfu/ml, the vessel (glass mattress flask) was then hermetically sealed and the bacteriophage was cultivated for 14 hours at an optimal temperature (+37°C) for growing a bacteriophage strain culture, with a layer of air 25 - 40 mm in thickness over the surface of the solid growth medium. Phage lysate was obtained by suspending the
  • bacteriophage from the surface of the solid growth medium with buffer at pH 7.0 in a proportion of 0.042 ml per 1 cm 2 of the surface of the solid growth medium.
  • the phage lysate was then pumped into a sterile vessel and mixed with chloroform and matured for 40 minutes with continuous shuttling, then centrifuged for 40 minutes at 5500 rpm.
  • a bacterial culture of a non-pathogenic host strain - Listeria innocua - with a titer of 10 9 CFU/ml was inoculated in a cultivation vessel on a slant solid growth medium 10 - 25 mm in thickness.
  • the host strain was cultivated for 3 hours at an optimal temperature (+37°C), then the resulting bacterial lawn of Listeria innocua was inoculated with a stock bacteriophage at a titer of
  • the cultivation vessel (glass mattress flask) was then hermetically sealed and the bacteriophage was cultivated for 15 hours at an optimal temperature (+22°C) for growing a bacteriophage strain culture, with a layer of air 25 - 40 mm in thickness over the surface of the solid growth medium.
  • Phage lysate was obtained by suspending the bacteriophage from the surface of the solid growth medium with buffer at pH 7.2 in a proportion of 0.045 ml per 1 cm 2 of the surface of the solid growth medium. The phage lysate was then pumped into a sterile vessel and mixed with chloroform and matured for 45 minutes with continuous shuttling, then centrifuged for 45 minutes at 5000 rpm.
  • the obtained refined phage lysate mix in the form of an eluate contained an coliphage with a titer of 10 12 pfu/ml, and a Listeria bacteriophage with a titer of 10 11 pfu/ml, free from bacteria or ingredients of the used growth medium and with a concentration of endotoxin less than 50 (EU/ml).
  • a bacterial culture of the host strain - Salmonella enterica serovar Typhimurium - with a titer of 10 8 CFU/ml was inoculated in a cultivation vessel on a slant solid growth medium 10 - 25 mm in thickness.
  • the host strain was cultivated for 3.5 hours at an optimal temperature (+37°C), then to the resulting bacterial lawn of Salmonella enterica serovar Typhimurium a stock bacteriophage was inoculated with a titer of 10 5 pfu/ml, the vessel (glass mattress flask) was then hermetically sealed and the bacteriophage was cultivated for 13 hours at an optimal temperature (+37 °C) for growing a bacteriophage strain culture with a layer of air 25 - 40 mm in thickness over the surface of the solid growth medium.
  • Phage lysate was obtained by suspending the bacteriophage from the surface of the solid growth medium with saline with pH 7.1 in a proportion of 0.042 ml per 1 cm 2 of the surface of the solid growth medium. The phage lysate was then pumped into a sterile vessel and mixed with chloroform and matured for 32 minutes with continuous shuttling, then centrifuged for 32 minutes at 6000 rpm.
  • a bacterial culture of non-pathogenic host strain - Listeria innocua - with a titer of 10 9 CFU /ml was inoculated in a cultivation vessel on a slant solid growth medium 10 - 25 mm in thickness.
  • the host strain was cultivated for 3 hours at an optimal temperature (+37°C), then to the resulting bacterial lawn of Listeria innocua a stock bacteriophage was inoculated with a titer of 10 5 pfu/ml, the cultivation vessel (glass mattress flask) was then hermetically sealed and the bacteriophage was cultivated for 15 hours at an optimal temperature (+24°C) for growing a bacteriophage strain culture, with a layer of air 25 - 40 mm in thickness over the surface of the solid growth medium.
  • Phage lysate was obtained by suspending the bacteriophage from the surface of the solid growth medium with saline at pH 7.2 in a proportion of 0.045 ml per 1 cm 2 of the surface of the solid growth medium. The phage lysate was then pumped into a sterile vessel and mixed with chloroform and matured for 45 minutes with continuous shuttling, then centrifuged for 45 minutes at 5000 rpm. Supernatants of the two resulting phage lysates were then mixed. The supernatant mix was then sterilized by filtration through a filter with a pore diameter of 0.22 pm; then passed through a column containing an agent with affinity to endotoxins.
  • the obtained refined phage lysate mix (cocktail) in the form of an eluate contained a salmonellosis bacteriophage with a titer of 10 12 pfu/ml, and a listeria bacteriophage with a titer of 10 11 pfu/ml, free from bacteria or ingredients of the used growth medium and with a concentration of endotoxin less than 50 (EU/ml).

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Abstract

L'invention concerne des souches de bactériophages Escherichia coli ainsi que des compositions anti-bactériennes et des procédés de production de bactériophages. Les bactériophages selon l'invention sont lytiquement actifs contre une large gamme de souches bactériennes pathogènes qui sont cliniquement importantes pour les humains. Les bactériophages et compositions selon l'invention ont par conséquent une utilité particulière dans le traitement et la prophylaxie de maladies chez l'homme et l'animal et pour des utilisations en tant qu'auxiliaires de traitement et désinfectants.
PCT/RU2014/000483 2014-07-02 2014-07-02 Souches de bactériophages, compositions et procédés associés WO2016003307A1 (fr)

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CN115851617A (zh) * 2022-10-17 2023-03-28 青岛农业大学 一种大肠杆菌噬菌体lhe83及其应用
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CN113980914B (zh) * 2021-12-06 2024-05-10 青岛新万福食品有限公司 一种宽裂解谱噬菌体及其在肉类冷冻保鲜中的应用
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CN115851617A (zh) * 2022-10-17 2023-03-28 青岛农业大学 一种大肠杆菌噬菌体lhe83及其应用

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