WO2015188230A1 - Procédé de production de bactériophage - Google Patents

Procédé de production de bactériophage Download PDF

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Publication number
WO2015188230A1
WO2015188230A1 PCT/AU2015/050320 AU2015050320W WO2015188230A1 WO 2015188230 A1 WO2015188230 A1 WO 2015188230A1 AU 2015050320 W AU2015050320 W AU 2015050320W WO 2015188230 A1 WO2015188230 A1 WO 2015188230A1
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bacteriophage
phage
bacterial
shrimp
host
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PCT/AU2015/050320
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English (en)
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Elizabeth Jane ELLIOT
James Edward RUSHBROOK
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Stock And Animal Products Pty Ltd
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Priority claimed from AU2014902215A external-priority patent/AU2014902215A0/en
Application filed by Stock And Animal Products Pty Ltd filed Critical Stock And Animal Products Pty Ltd
Publication of WO2015188230A1 publication Critical patent/WO2015188230A1/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
    • 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
    • C12N2795/00051—Methods of production or purification of viral material

Definitions

  • BACTERIOPHAGE PRODUCTION METHOD TECHNICAL FIELD relates to bacteriophage. More particularly, this invention relates to a method for large scale production of bacteriophage that may be used as antiseptic or antibacterial agents.
  • Bacteriophage are viruses which are capable of infecting and replicating in bacteria, ultimately leading to bacterial cell death. Bacteriophage were discovered in 1915 by Frederick Twort. It was fairly quickly realized that bacteriophage could be used as a powerful anti-bacterial therapy, as first pioneered by Felix d'Herelle in France in the 1920's and 1930's. Typically, bacteriophage therapy involves the targeted application of bacteriophages that, upon encounter with specific pathogenic bacteria, can infect and kill them. The phage then lyse the bacteria, releasing virion progeny that can continue the infection and replication cycle, including migrating to other sites of infection anywhere in the body.
  • Bacteriophage are unique among antibacterial agents in their ability to increase their numbers when in the presence of bacterial targets while only minimally impact non- target bacteria or body tissues.
  • bacteriophage are to become a more standard and widely-used antibacterial agent, industrial scale processes for their production need to be improved. Of particular concern is that bacteriophage produced on an industrial scale must be safe for administration to humans, while being produced at an economic cost that makes large scale bacteriophage production commercially viable.
  • the present invention is broadly directed to the selection and use of bacteria as optimal hosts for producing bacteriophage.
  • the bacterial hosts are non- pathogenic bacteria and allow production of bacteriophage that target pathogenic bacteria.
  • the bacterial hosts disclosed herein may facilitate efficient, large scale production of bacteriophage for a variety of subsequent antibacterial uses while minimizing the risk that the bacteriophage are contaminated with any pathogenic bacteria or components thereof.
  • the invention provides a method of producing a bacterial replication host for a bacteriophage, said method including the step of selecting a bacterial species, strain, serotype or isolate that is different to a pathogenic bacterium as host for replication of the bacteriophage.
  • the invention provides a method of producing bacteriophage including the step of propagating a bacteriophage that is capable of infecting a pathogenic bacterium in a host bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium under conditions that promote propagation of the bacteriophage. .
  • the invention provides bacteriophage produced by the method of the second aspect.
  • the invention provides a method of producing a bacteriophage composition including the steps of: preparing a bacteriophage that is capable of infecting a pathogenic bacterium by propagation in a different host bacterial species, strain, serotype or isolate under conditions that promote propagation of the bacteriophage; and forming a composition comprising the isolated bacteriophage.
  • the invention provides a bacteriophage composition produced according to the method of the aforementioned aspect.
  • a still further aspect provides use of the bacteriophage composition as an antiseptic or antibacterial agent.
  • the pathogenic bacterium is of the genus Vibrio, Aeromonas or Pseudomonas.
  • the pathogenic bacterium of the genus Vibrio is a pathogen of shrimp or prawns, such as Vibrio parahaemolyticus although without limitation thereto.
  • indefinite articles “a” and “an” are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers.
  • “a” may refer to one element, one or more elements or a plurality of elements.
  • FIG. 1 shows an example of desirable bacteriophage growth characteristics that include the ability to produce clear and distinct plaques (clearance zones) and an absence of "halo", where there are areas of partial clearing around the plaques.
  • FIG. 2 shows that there may be agglutination where the susceptible bacteria "group"' together following exposure to bacteriophage
  • FIG. 3 shows turbidity or cloudiness that can indicate the presence of undesired temperate phage.
  • FIG. 4 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1129 screened against 25, 50 and 100 ng/mL Mitomycin C
  • FIG. 5 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1128 screened against 25, 50 and 100 ng/mL Mitomycin C.
  • FIG. 6 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1333 screened against 25, 50 and lOOng/mL Mitomycin C.
  • FIG. 7 shows a Mitomycin C growth curve for Vibrio parahaemolyticus isolate # 1440 screened against 25, 50 and lOOng/mL Mitomycin C.
  • FIG. 8 shows PCR results for confirmation of EMS pathogen
  • M DNA molecular weight maker (IQ2000TM AHPNS/EMS specific sequence amplification kit).
  • FIG. 9 shows time-mortality relationship of V.parahaemolyticus challenging shrimp.
  • FIG.10 shows abnormal in colours of hepatopancrea of experimental shrimp.
  • FIG. 11 shows changes of HP tubule structure of EMS shrimp: hemocytic infiltration, lack of B,R, F cells in HP tubules and bacterial infection.
  • Biological systems including agricultural, aqua-culture, veterinary and human are prone to bacterial infection, infestation or contamination, as are synthetic systems such as formulated personal care, medications, water delivery, fluid circulation and the like, disease, dysfunction and spoilage.
  • antibiotics and antiseptics have been used to successfully address these infestations and contamination events however many bacterial strains are now resistant to such treatment regimes.
  • the invention disclosed herein provides bacteriophage isolated from contaminated or infected systems, where a pathogenic bacterium is present.
  • the pathogen bacterium while susceptible to the phage is not always the most appropriate replication host, for reasons of the need for complex growth media, difficulties in culture and the precautions required during handling of pathogenic bacteria.
  • This invention disclosed herein provides a method where by bacteriophage are produced using a "replication host” bacterium that has been selected because of its "user friendly” nature.
  • the method disclosed herein includes selection of both replication host bacteria and bacteriophage that overcome a number of rate limiting and labour intensive steps typically associated with production and purification of bacteriophage.
  • the method is essentially a two phase, complementary process whereby replication host bacteria and bacteriophage can be pre-selected and thus optimised for subsequent use in the method. This enables rapid scale-up, resulting in high purity, high titer bacteriophage of >10 7 to >10 u pfu/mL that may be used to create safe and effective antibacterial and/or antiseptic compositions and formulations.
  • the invention provides a method of producing a bacterial replication host for a bacteriophage, said method including the step of selecting a bacterial species, strain, serotype or isolate that is different to a pathogenic bacterium as a host for replication of the bacteriophage.
  • the invention provides a method of producing bacteriophage including the step of propagating a bacteriophage that is capable of infecting a pathogenic bacterium in a host bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium under conditions that promote propagation of the bacteriophage.
  • the invention provides bacteriophage produced by the method of this aspect.
  • Bacteriophage includes and encompasses any virus that is capable of infecting and replicating in a bacterium.
  • Bacteriophage may have a DNA or RNA genome comprising single-stranded or double-stranded DNA or RNA. The genome is typically packaged or encapsulated by proteins encoded by the bacteriophage genome.
  • Bacteriophage may be enveloped (e.g encapsulated by bacterial host-derived lipids, glycolipids and/or lipoproteins) or may be non- enveloped.
  • Bacteriophage may exhibit a lytic cycle or a lysogenic cycle (e.g "temperate" phage) associated with replication in a bacterial host.
  • bacteriophage include Caudovirales such as Myoviridae, Siphoviridae and Podiviridae, Ligamenvirales such as Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae and Fuselloviridae and other families such as Globuloviridae, Inoviridae, Leviviridae, Microviridae, Plasmaviridae and Techtiviridae, although without limitation thereto.
  • Caudovirales such as Myoviridae, Siphoviridae and Podiviridae
  • Ligamenvirales such as Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Cystoviridae and Fuselloviridae and other families such as Globulovi
  • Cystoviridae are RNA phages as are Leviviridae which have a linear, positive-sense single-stranded RNA genome and infect Enterobacter and Pseudomonas bacteria;
  • Myoviridae are characterized by complex contractile tails and include, as examples, bacteriophage mu, PL P2, and T4 and the "T4-like" bacteriphage;
  • Podoviridae are characterized by short, non-contractile tails and include, as examples, bacteriophages N4, P22, T3, and T7;
  • Siphoviridae are characterized by long, non-contractile tails and include, as examples, hk022, ⁇ , T5, and BF23 bacteriophage,
  • Corticoviridae comprise icosahedral, lipid-containing, non- enveloped bacteriophages including bacteriophage PM2;
  • Inoviridae comprise rod- shaped or filamentous bacteriophage
  • the bacteriophage is capable of infecting and killing a pathogenic bacterium.
  • the bacteriophage is not a filamentous or temperate bacteriophage.
  • the bacteriophage is a lytic bacteriophage or at least displays a lytic cycle in a replication host.
  • Other preferred characteristics of the bacteriophage include that: the bacteriophage should generate clear and distinct plaques during in vitro propagation in bacteria that are substantially free from halo, aggregation or turbidity on solid media; and/or rapid growth characteristics.
  • the bacteriophage is typically isolated from an environmental or natural source wherever a susceptible bacterial host may be present.
  • environmental or natural sources include contaminated or infected water systems, aquaculture systems, industrial waste, sewage, mining waste, soil, medical waste, water and human and other animal materials such as sputum, wound fluid, urine, blood, faeces and throat swabs, although without limitation thereto.
  • the bacteriphage are capable of infecting a pathogenic bacterium and being propagated and/or replicated in a bacterium (referred to herein as a "replication host") that is different to the pathogenic bacterium.
  • bacteriophage produced according to the methods disclosed herein may be capable of infecting bacteria ( .e pathogenic bacteria and replication hosts) inclusive of gram positive and gram negative bacteria, rods, cocci, non-motile and motile bacteria, anaerobes, facultative anaerobes, aerobes and photosynthetic bacteria, although without limitation thereto.
  • the bacteria may be of a genus or other taxonomic group such as Aeromonas, Altermonas, Cytophaga, Flavobacterium, Lactococcus, Mycoplasma, Photobacterium, Proteobacteria, Vagococcus, Achromobacter, Actinomyces, Staphylococcus, Bacillus, Yersinia, Hemophilus, Helicobacter, Alphaproteobacteria, Mycobacterium, Streptococcus, Neisseria, Klebsiella, Brucella, Rickettsia, Bordatella, Clostridium, Listeria, Legionella, Vibrio, Enlerobacter, Proteus, Pasteurella, Bacteroides, Campylobacter, Morganella, Edwardsiella, Lactococcus, Diplococcus, Pseudomonas, Borrelia, Citrobacter, Corynebacterium, Moraxella, Neisseria, Escherichi
  • pathogenic bacteria or bacteria that otherwise cause or are associated with one or more diseases of humans, animals or plants include Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, Achromobacter anitratum Actinobacillus lignieresi, Aeromonas hydrophila, Aeromonas salmonicida, Alcaligenes faecalis, Bordatella bronchiseptica, Brucella ovis, Bacillus anthracis, Bordatella pertussis, Borrelia burgdorferi, Campylobacter jejuni, Campylobacter novyi, Campylobacter fetus, Chlamydia psittaci ovis, Citrobacter freundii, Clostridium chauvoei, Clostridium colinum, Clostridium hemolyticum, Clostridium perfringens, Clostridium septicum, Coryne
  • Paratuberculosis Mycobacterium chelonei, Mycobacterium tuberculosis, Mycobacterium, leprae, Mycobacterium, asiaticwn, Mycobacterium intracellulare, Mycoplasma pneumoniae, Mycoplasma hominis, Neisseria meningitidis, Neisseria gonorrhoeae, Nocardia asteroides, Rickettsia rickettsii, Brucella, abortis, Brucella can is, Brucella suis, Legionella pneuophila, Klebsiella pneumoniae, , Propionibacterium acnes, Paenibacillus larvae, Pasteurella multocida, Photobacterium damselae subsp.
  • the replication host bacteria are suitably selected according to one or more criteria that include: they do not harbour a pro-phage; are capable of supporting bacteriophage replication; have prolific and reliable growth characteristics in vitro; do not produce toxins, or produce minimal toxins; are non-pathogenic; and/or have a requirement for inexpensive and/or minimal growth media. Non-pathogenicity and an ability to grow on inexpensive and/or minimal media are particularly desirable.
  • Vibrio spp As will be described in more detail in the Examples, a variety of different replication host bacteria have been identified according to the invention, including Vibrio spp, Aeromonas spp and Pseudomonas spp, although without limitation thereto.
  • bacteriophage are selected and grown under advantageous conditions that improve or enhance plaque selection and in vitro growth. Furthermore, the bacteriophage are harvested in "low complexity" media that reduce the need for complex, downstream purification.
  • the media preferably comprise one or more salts that include at least one monovalent cation. These may include sodium, potassium, rubidium and/or caesium.
  • a preferred concentration is between 0.001% and 15% (w/v) or any ranges therebetween such as 0.005% and 10% (w/v); 0.02% and 5% (w/v); 0.05% and 2% (w/v); and 0.01% and 1%) (w/v), although without limitation thereto.
  • the media preferably further comprise one or more salts that include at least one divalent cation.
  • the divalent cation is preferably a metal cation that is water-soluble.
  • metals are selected from magnesium, calcium, strontium, iron, manganese, cobalt, nickel, copper, zinc and silver.
  • a preferred concentration is between 0.003 to 3% (w/v) or ranges therebetween such as 0.005% and 1% (w/v); 0.01% and 0.5% (w/v); 0.02% and 0.2% (w/v); and 0.05% and 0.1 % (w/v), although without limitation thereto.
  • Preferred harvest media typically comprise water, sodium, magnesium and/or calcium salts.
  • a particularly preferred solution will comprise purified water and 5.8g/L sodium chloride plus 1.5g/L magnesium sulfate with the pH adjusted to the range 7.2 to 7.5.
  • the method disclosed herein provides an efficient means of producing bacteriophage in relatively large quantities for use in any of a variety of different applications. More particularly, the bacteriophage produced by the method may be substantially free of pathogenic bacteria-derived molecules and by-products that could prevent subsequent use of the bacteriophage.
  • the invention also provides a bacteriophage composition or formulation that may be useful for any of a variety of different applications.
  • the invention provides a method of producing a bacteriophage composition including the steps of: preparing a bacteriophage that is capable of infecting a pathogenic bacterium by propagation in a different bacterium under conditions that promote propagation of the bacteriophage; and forming a composition comprising the isolated bacteriophage.
  • the invention provides a bacteriophage composition produced or formulated according to the method of the aforementioned aspect.
  • the bacteriophage composition is suitable for use as an antibacterial agent or an antiseptic agent.
  • agents may be used prophylactically, remedially or therapeutically to inhibit, prevent, remove, remediate or treat bacterial infection and/or contamination.
  • the composition may comprise a plurality of different bacteriophage that are respectively capable of infecting and killing different bacterial pathogens.
  • bacteriophage compositions include agriculture, aqua- culture, medications for veterinary and human health, personal care products, water supply, decontamination of foods, food utensils, food preparation surfaces and food storage containers, decontamination of medical and veterinary equipment and workspaces and the like.
  • Particular applications include aquaculture uses such as in fish farming to target Aeromonas spp and prawn farming to target pathogenic bacteria such as Vibrio spp, medical and veterinary treatment infections caused by antibiotic- resistant bacteria such as MRSA and Pseudomonas spp, although without limitation thereto.
  • veterinary applications include Early Mortality Syndrome (EMS) or Acute Hepatopancreas Necrosis Disease Syndrome (AHPNS), bovine respiratory disease (BRD) in feedlot cattle, mastitis in dairy cattle, Streptococcus, Staphylococcus and Salmonella in feed mills, laying hens and other intensively reared animals.
  • EMS Early Mortality Syndrome
  • AHPNS Acute Hepatopancreas Necrosis Disease Syndrome
  • BTD bovine respiratory disease
  • compositions and formulations disclosed herein may comprise one or more other components such as suitable carriers, diluents or excipients. These may include one or more of water, saline, buffers, binders, fillers, lubricants, alcohols and/or polyols, stabilizers, sugars, sugar alcohols and other carbohydrates, lipids, preservatives, peptides and proteins and/or any other substances that facilitate formulation, storage, preservation and/or delivery of the bacteriophage composition.
  • suitable carriers such as suitable carriers, diluents or excipients. These may include one or more of water, saline, buffers, binders, fillers, lubricants, alcohols and/or polyols, stabilizers, sugars, sugar alcohols and other carbohydrates, lipids, preservatives, peptides and proteins and/or any other substances that facilitate formulation, storage, preservation and/or delivery of the bacteriophage composition.
  • compositions and formulations may be suitable for delivery by spraying or aerosolization (e.g as a dilutable concentrate), topical application as a cream, lotion or paint, as a bandage, dressing or swab impregnated with the bacteriphage, by enteral or parenteral application such as by a capsule or tablet or by delivery as a solid powder or granule, although without limitation thereto.
  • spraying or aerosolization e.g as a dilutable concentrate
  • topical application as a cream, lotion or paint
  • a bandage dressing or swab impregnated with the bacteriphage
  • enteral or parenteral application such as by a capsule or tablet or by delivery as a solid powder or granule, although without limitation thereto.
  • Phage therapies based on viral infection of a bacterial pathogen are one means of addressing this problem.
  • One of the advantage of this approach is that bacteriophage are highly bacterial host- specific, although as a consequence, systems for rapidly growing and supplying high purity phage are lacking because the bacterial host might not be the optimal host for large scale production of the bacteriophage.
  • the method disclosed herein describes how to find the optimal phage for use in remedial regimes by strategically eliminating undesirable traits from both the phage and replication host. As a consequence, scale up methods that produce high titre results and high purity are provided.
  • This method is used to isolate and identify both bacteria and phage that overcome a number of rate limiting and labour intensive steps typically associated with production and purification of biological agents such as bacteriophage.
  • the method is essentially a two phase, complementary process whereby bacteria and phage can be pre-selected and thus optimised for subsequent synergistic use. This enables rapid scale-up, resulting in high purity, high titer phage of >10 7 to >10 n pfu/mL.
  • Bacteriophage can be isolated from contaminated or infected systems, wherever a susceptible bacterial host is present. Phage to be isolated via this method are intended to be specific for particular bacteria, and thus are only suitable for particular antibacterial applications.
  • Filamentous phage typically produce toxins and as a consequence, are typically unsuitable for therapeutic or remedial uses without significant downstream processing. Similarly, temperate phage cycle between lytic and lysogenic cycles, and as a consequence cycle between deleterious and recovery relationships with their host, which is an undesirable trait in therapeutic and remedial phage.
  • the first step in this method is to ensure that neither filamentous nor temperate phage are selected.
  • Critical to this method is the selection of phage that are virulent (i.e obligate lytic phage), as these phage do not integrate their DNA into the host cell DNA and kill their bacterial host by lysis. This is a preferred phage property well suited for antibacterial remediation.
  • a reliable way of reducing the likelihood of isolation of non-virulent phage is to source potential candidates only from 'environmental' sources and not directly from their bacterial hosts. Collection of 'environmental samples' from sources such as water, sputum, wounds, sewerage and animals, preferentially selects phage that adopt virulent reproduction methods.
  • swabs are collected, swabs dilution or dispersion into a sterile fluid may be required followed by filtration (0.45 um, 0.2 um) such that viable bacteria are retained and smaller, viral particles are passed through. Such filtrates are collected and screened.
  • more dilute samples are collected, for example water samples; such samples may be added in equal volume of double strength microbial nutrient media, such that the final volume is at lx concentration.
  • the resultant solution is then allowed to incubate nominally for 24 to 48 hours such that the resident bacteria can replicate (as well as any phage present in the sample).
  • the solution is then filtered, with bacteria retained and phage passed through for further screening.
  • phage can be further selected for "scale-up" performance by ensuring that their in vitro performance on solid growth media is optimal.
  • This optimisation has a dual purpose: it enables identification and rejection of phage that may have temperate characteristics; and also identifies phage that may be more rapid or powerfully infective of a host bacterium.
  • Desirable characteristics include the ability to produce clear and distinct plaques (clearance zones) and an absence of "halo", where there are areas of partial clearing around the plaques, as shown in FIG. 1. As shown in FIG. 2, there also may be agglutination where the susceptible bacteria "group”' together following exposure to phage. As shown in FIG. 3, turbidity or cloudiness can indicate the presence of temperate phage or in any other circumstance where other than clear and distinct clearance is observed. Rapid plaque development is a desirable characteristic that is preferentially selected for. Acceptable plates examined will typically have 95% bacterial cell clearance.
  • Optimal in vitro characteristics screening can also be used help to differentiate between various phage contained within an environmental sample. These include the following:
  • the third step relates to purification regimes that should include careful selection and serial dilution of individually selected high performing plaques.
  • the plaques continue to be examined for optimal in vitro performance on solid media as the purification takes place.
  • a single plaque is selected using a sterile instrument such as a sterile pipette tip; the 'plug' is then dispensed into a 1.5 mL vial and suspended in 500uL low nutrient aqueous solution, typically comprising water, sodium, magnesium and/or calcium salts.
  • a sterile instrument such as a sterile pipette tip
  • 500uL low nutrient aqueous solution typically comprising water, sodium, magnesium and/or calcium salts.
  • a preferred solution will comprise purified water and 5.8g/L sodium chloride plus 1.5g/L magnesium sulfate, with the pH adjusted to the range 7.2 to 7.5. (referred to as PD(Neb))
  • Some other buffers may also make a suitable solution, with certain exceptions; namely some phosphate buffers, which result in the precipitation of insoluble magnesium phosphate or calcium phosphate, depending on the cations selected.
  • Tris (hydroxymethyl)aminomethane) is generally avoided as it contains a primary amine, which in the presence of other amine groups can result in the formation of nitrosamines, which are carcinogenic. Good's Buffers are typically appropriate.
  • Solutions and media used will be essentially free from phosphates and amines and will contain salts added such as: at least one monovalent cation is present in the solution including Sodium, Potassium, Rubidium and/or Caesium at a concentration of between 0.001 and 15% w/v); and/or at least one divalent cation selected from the metals group is available in solution; especially favourable will include one or more selected from magnesium, calcium, strontium, iron, manganese, cobalt, nickel, copper, zinc, silver at a concentration of between 0.003 to 3% (w/v).
  • the plaque and solution is then mixed and allowed to stand (or incubate) typically overnight, during which time the phage migrate from the plaque into the solution.
  • the vial may then be centrifuged and/or the supernatant passed through a sub-micron filter (0.45, 0.2 um), and the filtrate collected.
  • the filtrate is then subject to serial dilution and the process repeated twice over, so that on each occasion a single plaque is selected and its in vitro performance and purity verified.
  • a particularly useful method of doing this is a modified Miles and Misra plate regime such as hereinbefore described.
  • Phage that have been selected by this method can then be assessed for their potential against a number of bacterial isolates (host range analysis), and subjected to more specific and discrete verification regimes such as TEM before validation and assessment of suitability for remedial or therapeutic use via the production of high titre phage.
  • phage To enter and thus infect a host cell, phage must attach to a specific receptor or region of the bacterial cell. Phage are obligate cellular parasites and rely on random encounters as well as affinity with their host in order to achieve infection. Host growth conditions as well as the presence or absence of phage receptors will influence the phage's ability to be infective, and thus be useful in this technology. This part of the process selects for phage that have a wide range of susceptible hosts, and as a consequence are more attractive as remedial or therapeutic agents.
  • Bacterial hosts are actively source and maintained as a library as described in
  • a selection of host bacteria are used to determine the range of hosts that may be susceptible to the isolated phage. Phage are identified as being especially active against a wide array of hosts and/or especially active against specifically desirable hosts (for example antibiotic- resistant bacteria or specific strains or isolates of known bacterial pathogens)
  • the opportunity for host infection can be preferentially influenced, especially in aquaculture systems through the use of attractants.
  • Crude attractants attractants such as squid meal or oil, egg, worm meal and molasses can interfer with the shelf life and vitality of the phage where as highly purified, discrete attractants such as those described in Chemical Communication in Crustaceans ISBN 978-0-387-77100- 7 Thomas Breithaupt 1 Martin Thiel Editors have attractant capability without diluting or adversely effecting phage vitality and/or shelf life.
  • metabolites such as isophorone and 6-methyl-5-hepten-2- one, Chlorodesmin, Pachydictyol A, Dictyol E, Pteroenone, 'hair crab ceramide', Isatin, Tyrosol, attractin pheromone, also free amino acids especially ASP, GLU, ASN, SER, THR, GLN, HIS, GLY, ARG, 0-ALA, TAUR, TYR, A ABA, VAL.
  • metabolites such as isophorone and 6-methyl-5-hepten-2- one, Chlorodesmin, Pachydictyol A, Dictyol E, Pteroenone, 'hair crab ceramide', Isatin, Tyrosol, attractin pheromone, also free amino acids especially ASP, GLU, ASN, SER, THR, GLN, HIS, GLY, ARG, 0-ALA, TAUR, TYR, A ABA, VAL.
  • TRP TRP, PHE, ILE, LEU, ORN and LYS
  • ⁇ ImM low concentrations
  • small peptides including Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones nucleotides
  • volatile info chemicals such as diemthyl sulfide can provide chemically mediated trophic effect.
  • a pathogenic bacterium When a pathogenic bacterium is susceptible to a particular phage, it might not necessarily be the most appropriate replication host. This may be for reasons of the requirement for complex growth media, that it is difficult to culture the bacterial pathogen and/or the precautions required during handling the bacterial pathogen.
  • This method disclosed herein whereby virulent phage are produced using a "replication host" bacterium provides a much more "user friendly" system for producing bacteriophage.
  • Suitable 'replication host' characteristics will ideally include
  • bacterial pathogens that cause known diseases are identified. Non-limiting examples will be described in more detail.
  • Vibrio parahaemolyticus is a known causative agent for EMS when characterised as being 'PCR positive'. There are however a number of strains that are not considered pathogens (or environmental risks) that are more ideally suited a role as a replication host. Vibrio species including parahemalytica isolates have been obtained from commercial and wild sources (for example crustaceans and their commercial pond environments) as well as commercially available specimens such as those available via ATCC (such as ATCC 17802)
  • Aeromonas species are responsible for gastroenteritis and wound infections. Antibiotic resistance poses a potential problem in antimicrobial therapy of these infections. While most strains are susceptible to chloramphenicol, ciprofloxacin, co-trimoxazole and the aminoglycosides, the activity of amoxycillin/clavulanate and the acylureidopenicillins is inconsistent. These organisms are ubiquitous in fresh water environments and over growth in same can also be a cause of disease in aquiculture environments. Aeromonas isolates have been collected from a number of "wild" environments including hatcheries, dams, septic systems, waterways, as well as wounds of animals and humans. In addition to this, characterised cultures have been screened.
  • Pseudomonas are also opportunistic pathogens and potentially suitable isolates have been identified in samples obtained from crayfish, dogs lungs, horse uterus, human wounds and aquatic environments.
  • Staphylococcus the causative agent in mastitis and other dermal infections
  • the purpose of the isolation was to obtain Pseudomonas isolates, and as a consequence on samples 3, 4, 5, 7 and 8 were progressed to identification phase.
  • Biolog GEN III system analysis indicated that the isolates were most likely (respectively) Pseudomonas aeruginosa, Pseudomonas fluorescense Biotype G, Pseudomonas citronellolis and Pseudomonas Nitroreducens/azelaica.
  • the Pseudomonas citronellolis (#7 isolate) was excluded from immediate consideration as it is an unlikely human pathogen. The remaining isolates were progressed for additional analysis.
  • prophage The presence of a prophage is an unacceptable trait in isolates selected for production of high purity, high titre phage, and thus hosts of such phage must be eliminated.
  • the antibiotic substance Mitomycin C stresses bacterial hosts to the extent that prophage production can be visualised by measuring the optical density of growth media and observing particular trends in the data over ⁇ 8 hours.
  • a prophage can be identified as a 'dip' and recovery in the optical density, indicating that the population begins to reduce (as a consequence of lysis brought on by the prophage) and then recovers as the prophage exits this cycle and reincorporates into the host DNA.
  • Antibiotic-resistant strains are driving the need for alternative therapies to be developed and as a consequence phage that are especially effective against antibiotic stains are especially valuable. For this reason it is important to have access to antibiotic resistant strains that can be used to select especially valuable phage.
  • antimicrobial resistance screening is undertaken. Most typically, Staphylococcus isolates will be screened for resistance using challenges with Ampicillin (lOug), Cefoxitin 30ug, Oxacillin lug, Penicillin lOug, Ciproxin 5ug, Amibacin 30ug and Erythromycin 15ug as well as, potentially Cephalosporins at suitable dilutions.
  • Bacterial isolates are typically subject to strategic biochemical screening using both commercially available (such as MicroSys V36, Biogen III Microplate) and specifically designed biochemical-screening regimes ⁇ i.e. media variations), with the objective of maximising growth and minimising media complexity.
  • biochemical screening is undertaken for species identification purposes, however on this occasion the results are also used to identify isolates that have discrete and clearly identifiable nutritional needs.
  • V.parahemalytic strains that had previously been identified as having optimally low nutrition requirements (stored on beads at -80°C) were resuscitated in Nutrient Broth + 2% NaCl, and then incubated at 30°C for 48 hours; there after aliquots were transferred to 'PVSS' broth (comprising 5g/L bacteriological peptone, 1 g/L yeast extract and 33g/L synthetic sea salts) and allowed to grow to early exponential phase. The quality and quantity of growth was recorded for each bacterial isolate.
  • Vibrio isolate is identified as having optimal growth capacity coupled with minimal or reduced nutrition requirements it is sequestered for further investigation for its ability to act as a growth substrate (replication/ surrogate host) for virulent phage.
  • purified phage were then introduced to the potential replication hosts. These co-cultures then each contained a bacterial isolates with known minimal nutritional needs and a phage known to be effective against a bacterial pathogen. The cultures were allowed to progress for a defined period and thereafter the degree of clearance for these cultures was observed and measured, with a score of 3+ being maximum clearance (that is high level phage production) down to 0 (phage appeared to be produced or effective against that host).
  • Aeromonas replication hosts can be identified in a similar way, for future remedial use in aquatic, terrestrial and industrial systems.
  • the potential presence of lytic prophage in forecast replication hosts need to be determined. This can be achieved by growing the isolates in Tryptone soy broth and then screening of isolates against Mitomycin C, which acts to induce prophage production (an undesirable trait). Cultures that do not survive the Mitomycin C challenge are then progressed to further screening.
  • Phage-resistant colonies can then be determined via screening on solid media, such as Tryptone soy agar, using growth of the cultures to a clear lawn density and then application of various dilution of phage. Phage resistant colonies are of particular interest as they represent a specific challenge to be satisfied in phage library development and optimisation.
  • kits can be obtained from HIMEDIA or Pliva-Lachema Diagnostika (for example ENTEROtest 24 - kit for identification of Gram- negative fermentative rods). Again on this occasion the purpose of using the biochemical screening kit was not to identify the host, but was rather to determine the absolute requirements for media composition.
  • phage can now be grown "on demand” and supplied as needed in responses to specific infection and/or contamination events. These might include for example fmgerliiig hatcheries, food manufacturing and other water rich environments.
  • parahaemolyticus 1 x 10 9 pfu/ml parahaemolyticus (PCR Positive for (PCR Negative) EMS)
  • Aeromonas (aqua- 1 x io 9 pfu7mi through Multiple Yes
  • Bacteriophage are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals.
  • one of the densest natural sources for bacteriophage and other viruses is sea water, where up to 9> ⁇ 10 8 virions per/ml have been found in microbial mats at the surface.
  • Recent investigations have revealed that bacteriophage are much more abundant in the water column of both freshwater and marine habitats than previously thought. Due to the density of phage found in these locations, water sources are seen as prime habitats for phage localisation, discovery and amplification.
  • Water samples used for phage amplification should be obtained from the environment in which the bacteria are most likely to be found, e.g - freshwater, marine water, sewerage, etc.
  • the host strains to be used for phage amplification should be cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture on an agar plate in a way that distinct colonies will be obtained. Isolates retrieved from cryopreservation must be cultivated twice after retrieval to ensure optimal results.
  • phage filtrates are spotted (20 ⁇ 1) onto a suitable agar medium and left to incubate overnight under optimal growth conditions to test for bacterial contamination. Should bacterial contamination occur, the phage is to be filtered again through a 0.45 ⁇ filter. B - In some cases, phage filtrates may need to be treated with 0.5% chloroform and then centrifuged at 5000g, 10°C for 15mins before filtering.
  • the Miles and Misra Method is a technique used in to determine the number of colony forming units in a bacterial suspension or homogenate.
  • the technique was first described in 1938 by Miles, Misra and Irwin at the London School of Hygiene and Tropical Medicine.
  • the Miles and Misra method has been shown to be precise.
  • a modified procedure has been developed by the inventors based on this method. Its purpose is to measure plaque forming units/ml of a phage filtrate/cocktail against a bacterial isolate. This procedure has been termed a modified Miles and Misra technique.
  • the organisms to be used in the trial are cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture on an agar plate in a way that distinct colonies will be obtained. Isolates retrieved from cryopreservation are cultivated twice after retrieval to ensure optimal results.
  • Each phage filtrate is designated one standard petri dish.
  • the plate is first divided into eight separate sections of approximately the same size.
  • a prepared template is available to assist with this but it is also possible to do so using a ruler and a permanent marker.
  • phage titre can be calculated as plaque forming units per milliliter (pfu/ml)
  • bacteriophages To enter a host cell, bacteriophages must attach to specific receptors on the surface of bacteria, including lipopolysaccharides, teichoic acids, proteins, or even flagella. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn determines the phage's host range. Host growth conditions also influence the ability of the phage to attach and invade them. As phage virions do not move independently, they must rely on random encounters with the right receptors when in solution (blood, lymphatic circulation, irrigation, soil water, etc). Adsorption is a key stage in virus recognition of a sensitive host cell, i.e. specificity of phage infection is defined at this moment. Since bacteriophage, like any other virus, are obligate intracellular parasites, successful penetration into the bacterial cell is an essential condition for continuation of their life cycle.
  • Bacterial cultivation of trial isolates • The organisms to be used in the trial should be cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture on an agar plate in a way that distinct colonies will be obtained. NB - Isolates retrieved from cryopreservation must be cultivated twice after retrieval to ensure optimal results.
  • Each of the trial isolates is designated one standard petri dish.
  • the plate is allocated areas for each of the phage filtrates to be tested. These areas can be marked using a permanent marker on the underside of the petri dish.
  • Bacterial isolates were identified by an assay using the Biolog Gen III Microplate identification system. Test data are separated into a colour coded spreadsheet for the purpose of pattern observation in which we can group similar isolates and discern different ones.
  • Tetrazolium redox dyes are used to colourimetrically indicate utilisation of the carbon sources. All of the wells start out colourless when inoculated. During incubation there is increased respiration in the wells where cells can utilize a carbon source and/or grow. Increased respiration causes reduction of the tetrazolium redox dye, forming a purple colour. Negative wells remain colourless, as does the negative control well with no carbon source. There is also a positive control well used as a reference. After incubation, the phenotypic fingerprint of purple wells is compared to an extensive species library. If a match is found, a species identification of the isolate is made.
  • a prophage is a phage (viral) genome inserted and integrated into the circular bacterial DNA chromosome or existing as an extrachromosomal plasmid. This is a latent form of a bacteriophage, in which the viral genes are present in the bacterium without causing disruption of the bacterial cell.
  • the prophage Upon detection of host cell damage, such as UV light or certain chemicals, the prophage is excised from the bacterial chromosome in a process called prophage induction. After induction, viral replication begins via the lytic cycle. To determine if isolated bacteria were hosts for temperate bacteriophage, bacteria are stressed using mitomycin C with the hope of inducing the phage to enter the lytic cycle.
  • Mitomycin C dilutions must be made as follows - lOOng/ml, 200ng/ml and 400 ng/ml.
  • mitomycin C is currently purchased in vials containing 2mg of dried mitomycin C. Begin by adding 5ml of filtered dH 2 0 to the vial to make a stock 400ug/ml solution. This must be kept in a lightproof vial at 4°C to reduce deterioration. Lower dilutions should not be kept for storage purposes.
  • the organisms to be used in the trial should be cultivated on a suitable agar medium under optimal incubation conditions to obtain a fresh overnight grown culture. Streak out the pure culture onto an agar plate in a way that distinct colonies will be obtained.
  • the objective of this study is to establish the capability of selected isolates of Vibrio parahaemolyticus to infect Pacific white shrimp Litopenaeus vannamei with EMS/AHPND in a challenge trial.
  • the average numbers of feed particles that the shrimp ate in the morning and afternoon were 24.7 and 19.2 and the number of particles likely to be eaten by 91.6% and 90.2% of shrimp were 12 in the morning and 10 in the afternoon. Those numbers was used as doses to treat experimental shrimp on the first day of challenge. After the first day, the shrimp were fed with normal feed (not coated with bacteria). The experiment was carried out with 4 treatments:
  • parahaemolyticus isolates during challenge trial. They were treated in the same way as the challenge groups.
  • parahaemolyticus isolate 27-20 in the concentration of approximately 10 4 cfu/feed pellet.
  • Shrimp are fed twice daily with a high quality, pelleted shrimp feed (Tomboy, No. 2, 38% minimum Crude Protein). Feeding rate is to be regulated to minimise the quantity uneaten feed but as consistently as possible across the experiment (see 2. Challenge treatment).
  • Uneaten feed and faeces are removed from the tanks using a fine scoop net 2 hours after feeding to help maintain water quality. Nets are labeled for each tank (or treatment) and after use, they will be dipped into BKC solution and then clean water before being left to dry inside the wet lab. The nets are not to be removed from the room.
  • a routine of maintaining tanks in order of increased bio-security risk is adopted.
  • the sequence of maintenance is Treatments Tl, then T2, then T3 and then T4.
  • shrimp are checked every 2 hours during the day and as frequently as practical during the night. Moribund, freshly dead and clearly dead shrimp are removed from the tanks and placed in labelled specimen jars and fixed according to standard techniques, for histological examination. Details including treatment, tank number, date and time of removal and condition of the shrimp (moribund, freshly dead, dead) are included on the label. These shrimp are examined, using histology when necessary, to determine, as precisely as possible, the cause of death. As a planning number, a minimum of 10 dead shrimp, if there are that many, are to be examined using histology and PCR to determine cause of death or presence of EMS/AHPNS. At the end of the experiment, samples of surviving shrimp from each treatment are taken for PCR and histological examination for signs of EMS/AHPNS.
  • V.parahaemolyticus from some of the hepatopancrea of tested shrimp will be isolated and cultured on ChromoAgar to obtain a sample of the virulent/pathogenic strain of the bacteria.
  • Shrimp were transferred to the experiment tank and during the first two days of the adaptation period, the shrimp ate about 8.1 and 12.0 feed particles for morning and afternoon meal. 8 shrimp died at this stage and were replaced by new 8 shrimp from the same batch. In next three days, the amount of consumed food increased to an average of 24.7 and 19.3 particles for morning and afternoon meal. This indicated that the shrimp adapted well to experimental condition. During this period, the death of 7 shrimp was observed, most of them were dead after moulting. 10 more shrimp jumped out of the tanks and died when a lab worker tried to remove shrimp faeces and uneaten food after 2 hours of feeding. Experience in caring for experimental shrimp was gained and this phenomenon did not happen during the challenge period. Details of shrimp health status and number of consumed feed particles for each meal during adaptation period are summarized in Table 3.
  • Table 4 The consumption of experimental feed on the first day of challenge
  • the objective of this study will be to determine the effectiveness of a phage preparation to reduce the incidence of EMS/AHPND in Pacific white shrimp Litopenaeus vannamei that have been challenged with one or more strains or isolates of Vibrio parahaemolyticus, such as that in Example 2, which cause cause significant mortality in shrimp through EMS/AHPND.
  • Isolate Pl/1 was identified as an AHPNS isolate suitable for subsequent challenge experiments due to its susceptibility to bacteriophage.
  • Isolate 27-20 identified in Example 2 was also shown to be an AHPNS isolate susceptible to bacteriophage that may be useful for challenge experiments.
  • Propagation of bacteriophage will be performed using non-pathogenic Vibrio replication hosts. Isolation of phage from the replication host will also be performed essentially as described in Example 1.
  • Control (C). These shrimp will not be exposed to any phage or V. parahaemolyticus during challenge trial. They will be treated in the same way as the treatment and challenge groups. Other controls may include exposure to non- AHPND bacteria as a negative control.
  • phage will be added to the tanks to give an initial concentration in the tank water of approximately 10 8 pfu/mL.
  • the phage will be added at the same dosage once each day at about the same time for a total of five days.
  • the tanks will be filled with filtered seawater at a salinity of about 20 ppt that has been prepared from previously concentrated and sterilised seawater. Evaporative loss of water from tanks will be made up using de-ionised water or if de-ionised water is not available, with chlorine- free drinking water. This strategy may reduce the necessity for more detailed monitoring of salinity.
  • the tanks will not have a continuous flow through of water but will be considered "static".
  • the tanks will be aerated with a single air-stone to maintain dissolved oxygen levels above 5 ppm.
  • the temperature in the tanks will be at about 28°C which will be maintained by the ambient air temperature using the temperature control of the air conditioner in the laboratory.
  • the tank water salinity, pH and ammonia levels of treated water source will be monitored at the beginning. If these parameters are not acceptable, they should be improved accordingly.
  • salinity, pH and ammonia levels will be monitored at a representative sample of randomly selected tanks within the laboratory every two days. If there are any indications that the pH and ammonia levels are moving towards unacceptable levels, more detailed sampling will be carried out and water exchanges will be made to restore water quality.
  • V.parahaemolyticus from some of the hepatopancrea of tested shrimp to be isolated and cultured on ChromoAgar to obtain a sample of the virulent/pathogenic strain of the bacteria.
  • the bacteria used to compete for nutrient against the AHPNS isolate Pl/1 were Vibrio parahaemolyticus which had been previously identified not to cause death/AHPNS in prawns.
  • Six (6) non-AHPNS causing V. parahaemolyticus isolates were tested against the phage cocktail to identify those which were not susceptible and those which were.
  • Two isolates which were not susceptible to the phage (MC and VplO) were used in a subsequent challenge trial along with one bacterial isolate to which the phage was effective (Vp5).
  • Including the non-AHPNS causing isolate Vp5, which was susceptible to the phage cocktail was an attempt at giving the phage an additional, non-pathogenic replication host in which to replicate to maintain high concentrations throughout the experiment.

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Abstract

L'invention concerne un procédé de production d'un hôte de réplication bactérien pour un bactériophage, comprenant la sélection d'une espèce bactérienne, d'une souche bactérienne, d'un sérotype bactérien ou d'un isolat bactérien, qui est différent d'une bactérie pathogène, en tant qu'hôte pour la réplication du bactériophage. L'hôte de réplication bactérien constitue un véhicule non toxique, efficace et sans danger pour la propagation d'un bactériophage qui est capable d'infecter une bactérie pathogène qui est d'une espèce bactérienne, d'une souche bactérienne, d'un sérotype bactérien ou d'un isolat bactérien différent de l'hôte de réplication. La bactérie pathogène peut être du genre Vibrio, Aeromonas ou Pseudomonas. Des bactéries Vibrio particulières, telles que Vibrio parahaemolyticus, sont des agents pathogènes pour les crevettes et les crevettes bouquets. L'invention peut par conséquent avoir une applicabilité particulière en aquaculture.
PCT/AU2015/050320 2014-06-11 2015-06-11 Procédé de production de bactériophage WO2015188230A1 (fr)

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CN107686832A (zh) * 2016-08-05 2018-02-13 菲吉乐科(南京)生物科技有限公司 新的副溶血性弧菌噬菌体及其组合物、制备方法和应用
WO2019030257A1 (fr) * 2017-08-08 2019-02-14 Snipr Technologies Limited Cellules de propagateur et procédés de propagation de phages, en particulier pour l'administration de composants crispr-cas par l'intermédiaire d'organismes probiotiques
CN112646785A (zh) * 2020-12-30 2021-04-13 瑞科盟(青岛)生物工程有限公司 一株耐高温烈性变形杆菌噬菌体rdp-sa-20018及其应用
CN112725287A (zh) * 2021-01-15 2021-04-30 瑞科盟(青岛)生物工程有限公司 一株强裂解性金黄色葡萄球菌噬菌体rdp-sr-20001及其应用
CN113201501A (zh) * 2020-08-06 2021-08-03 青岛诺安百特生物技术有限公司 一株具有跨种裂解能力的弧菌噬菌体及其应用
CN113512539A (zh) * 2021-04-25 2021-10-19 中国海洋大学 一种噬菌体及其应用
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Cited By (13)

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Publication number Priority date Publication date Assignee Title
US10357522B2 (en) 2016-06-22 2019-07-23 The United States Of America As Represented By The Secretary Of The Navy Bacteriophage compositions and methods of selection of components against specific bacteria
EP3474872A4 (fr) * 2016-06-22 2019-11-20 The United States Of America As Represented By The Secretary Of The Navy Compositions à base de bactériophages et procédés de sélection de constituants dirigés contre des bactéries spécifiques
WO2017223101A1 (fr) * 2016-06-22 2017-12-28 The United States Of America As Represented By The Secretary Of The Navy Compositions à base de bactériophages et procédés de sélection de constituants dirigés contre des bactéries spécifiques
CN107686832A (zh) * 2016-08-05 2018-02-13 菲吉乐科(南京)生物科技有限公司 新的副溶血性弧菌噬菌体及其组合物、制备方法和应用
CN107686832B (zh) * 2016-08-05 2020-08-14 菲吉乐科(南京)生物科技有限公司 新的副溶血性弧菌噬菌体及其组合物、制备方法和应用
WO2019030257A1 (fr) * 2017-08-08 2019-02-14 Snipr Technologies Limited Cellules de propagateur et procédés de propagation de phages, en particulier pour l'administration de composants crispr-cas par l'intermédiaire d'organismes probiotiques
CN113201501A (zh) * 2020-08-06 2021-08-03 青岛诺安百特生物技术有限公司 一株具有跨种裂解能力的弧菌噬菌体及其应用
IT202000022519A1 (it) * 2020-09-24 2022-03-24 Craniomed Group Srl "metodica innovativa per il rilevamento dei virus che possono essere dei fagi o avere un meccanismo fagico, incluso sars cov-2 e congeneri, e che interessano la specie umana, il regno l’animale, il regno vegetale, e gli altri regni".
CN112646785A (zh) * 2020-12-30 2021-04-13 瑞科盟(青岛)生物工程有限公司 一株耐高温烈性变形杆菌噬菌体rdp-sa-20018及其应用
CN112725287B (zh) * 2021-01-15 2022-03-22 瑞科盟(青岛)生物工程有限公司 一株强裂解性金黄色葡萄球菌噬菌体rdp-sr-20001及其应用
CN112725287A (zh) * 2021-01-15 2021-04-30 瑞科盟(青岛)生物工程有限公司 一株强裂解性金黄色葡萄球菌噬菌体rdp-sr-20001及其应用
CN113512539A (zh) * 2021-04-25 2021-10-19 中国海洋大学 一种噬菌体及其应用
CN113512539B (zh) * 2021-04-25 2023-06-27 中国海洋大学 一种噬菌体及其应用

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