WO2007128765A2 - Traitement d'infections bactériennes au moyen d'enzymes lytiques de bactériophages - Google Patents

Traitement d'infections bactériennes au moyen d'enzymes lytiques de bactériophages Download PDF

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Publication number
WO2007128765A2
WO2007128765A2 PCT/EP2007/054281 EP2007054281W WO2007128765A2 WO 2007128765 A2 WO2007128765 A2 WO 2007128765A2 EP 2007054281 W EP2007054281 W EP 2007054281W WO 2007128765 A2 WO2007128765 A2 WO 2007128765A2
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WIPO (PCT)
Prior art keywords
phage
organism
bacteria
lytic
composition
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Ceased
Application number
PCT/EP2007/054281
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English (en)
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WO2007128765A3 (fr
Inventor
Victoria Alday-Sanz
Iddya Karunasagar
Indrani Karunasagar
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Inve Technologies NV
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Inve Technologies NV
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Publication of WO2007128765A3 publication Critical patent/WO2007128765A3/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria

Definitions

  • the present invention relates to a method for the prophylactic or curative treatment of a bacterial infection of an organism, in particular of an aquaculture organism such as crustaceae, mollusca and fish.
  • the invention also relates to a composition for use in such a method and to a method for producing such a composition. More specifically, the invention relates to the prophylactic or curative treatment of vibriosis in aquaculture practices, in particular in shrimp (or prawn) cultures.
  • Bacterial diseases are a common problem in aquatic animal farming. Vibrio spp is the main genus involved and in particular V. harveyi is the species most consistently isolated related to disease outbreaks in the literature. Vibrio has a worldwide distribution and it is considered a primary pathogen for shrimp and fish larval stages and a secondary pathogen for juveniles and adults.
  • probiotics which are expected to compete and displace shrimp/fish pathogens. Often, results from probiotics are, however, not consistent. Therefore, the application of bacteriophages was screened as a possible therapy and prophilaxis against bacterial pathogens in aquaculture. Bacteriophages are widely distributed in the aquatic environment and they are considered to play an important role in maintaining the microbial balance in natural ecosystems.
  • Bacteriophages have been found to be specific for certain microbial species or even strains. Contrary to antibiotics, phage therapy achieves a very specific effect, working at species level without disturbing the normal flora of the shrimp or the environment. There are no residues after the treatment and phage levels decrease once the target population demises.
  • Karunasagar et al. (2005) described the successful use of live lytic phages against V. harveyi in aquaculture. However, for phage therapy it would be a disadvantage that a single bacteriophage may not be effective against all or at least against most of the strains of the pathogen.
  • a first drawback of the use of active phages is therefore that a typical phage is specific for very few bacterial strains even among the same bacterial species. Thus, a typical phage has a small host range and is only capable of killing a small subset of bacteria within a single genus of bacteria.
  • a further drawback is that the introduction of live phages in aquaculture applications, poses risks of transfer of genetic material between bacterial cells, such as pathogenic islands via bacteriophages. Also from a legal point of view, the introduction of live phages as a microbial additive in aquaculture practices requires additional impact assessment on the environment (EC1831/2003).
  • a further disadvantage is that the preservation and shelf life of the product containing live phages is limited to a few days or weeks as long as it is not frozen at -8O 0 C or freeze-dried, which has a high storage or production cost. lnstead of using the active phages it is also possible to use only the lytic phage enzymes. These lytic enzymes could be produced by sequencing the genome of the phage, determining the gene coding for the lytic enzyme, inserting it in a vector and over-expressing the gene in a host. This is however a time-consuming procedure which is moreover expensive and not always successful. Another procedure for producing the lytic enzyme is disclosed in US 2005/0136088 and in WO 2004/064732.
  • An object of the present invention is therefore to provide a new composition for the therapeutic or prophylactic treatment of a bacterial infection which contains a lytic phage enzyme but substantially no active bacteriophages and which is easier to produce on a larger scale.
  • the composition according to the invention which comprises at least a fraction of a phage lysate produced from bacteria which are infected with a lytic bacteriophage and containing at least one lytic enzyme having the ability to lyse the bacteria causing the bacterial infection, is characterised in that said bacterial lysate fraction further contains said bacteriophage which is inactivated by means of a chemical nucleic acid inactivating agent.
  • the method for producing the composition according to the invention comprises the additional step of inactivating the bacteriophages present in the phage lysate by means of a chemical nucleic acid inactivating agent.
  • the lytic enzymes (most probably lysin and/or holin enzymes) present in the composition according to the invention allow a significant reduction of Vibrio bacterial numbers, especially of Vibrio harveyi, Vibrio campbelli and other relevant shrimp bacterial pathogens, without requiring the presence of the active bacteriophages. It has been found quite by surprise that the antibacterial effect could be maintained during the chemical inactivation process of the phage, notwithstanding the fact that during this inactivation process they are also subjected to inactivation compounds that react not only with nucleic acids but also with proteins.
  • bacteriophages which are inactivated by means of a chemical nucleic acid inactivating agent instead of by means of heat remain active against bacterial infections and are even more active than the non-inactivated bacteriophages.
  • the nucleic acid inactivating agent i.e. the RNA or DNA inactivating agent by means of which the bacteriophage is inactivated, comprises preferably an alkylating agent, in particular at least one alkylating agent selected from the group consisting of alkyl halides, dialkyl sulfates, alkyl sulfonates, alkyl leads, ⁇ -propiolactone (BPL), butyrolactone, alkyl iodides, alkyl bromides, alkyle ⁇ imsnes, including substituted alkylenimines, the alkylating agent being preferably selected from the group consisting of ⁇ -propiolactone (BPL), butyrolactone, methyl iodide, ethyl iodide, propyl iodide, methyl bromide, ethyl bromide, propyl bromide, dimethyl sulphate, diethyl
  • Alkylating agents appeared to be effective for inactivating vibrio phages.
  • Such alkylating agents are mutagenic chemicals that react with nucleic acid and modify it by adding alkyl groups. They enable to inactivate viruses, i.e. to modify viruses in such a manner that they are no longer able to reproduce themselves.
  • the inactivation of viruses by alkylation in primarily the result of reaction with the nucleic acid (RNA or DNA) than with the proteins of the virus (Herriot 1948), the alkylating agents also react with proteins / enzymes thus affecting their enzymatic activity (Herriot 1946).
  • BPL Broo et al. (2001) have found that BPL would even react with viral capsid proteins. Quite surprisingly, it has thus been found that the lytic enzymes present in the phage lysate were not affected by the alkylating agent used to inactivate the phage, in particular not by BPL.
  • the bacteriophage is a lytic Vibrio phage, in particular a lytic WMo harveyi or a Vibrio campbelli phage.
  • the phage lysate is substantially free of bacteria and bacterial debris.
  • Such a lysate can easily be obtained by filtering and/or especially by centrifuging the phage lysate, more particularly at relatively low centrifugal accelerations lower than 25 000 g, and preferably lower than 15 00O g. Due to the absence of bacteria, there is no risk on infecting the organism with possibly pathogenic bacteria.
  • said fraction of the phage lysate is freeze- dried. In this way, it can be stored easily.
  • composition according to the invention is further preferably in the form of a feed for the organism.
  • said fraction of the phage lysate can be mixed, either in liquid or in solid form (f.e. freeze-dried form) with other feed components which are normally used for feeding the organism.
  • the invention can be brought in inclusion in liposomes in an oil emulsion, so that they for instance could be delivered to filter feeding organisms like life food organisms such as rotifers or Artemia or to bivalve organisms.
  • the invention can also be encapsulated in a fatty solution to protect the active enzymes from interference with other enzymes or from physical or chemical aggression during the feed process. Said incapsulation could be useful for incorporation in feeds such as pellets.
  • the freeze-dried form is useful to incorporate the invention in solid feeds, preferably as a coating, mixed or not with other temperature unstable additives like for instance vitamins, micro-organisms.
  • an advantage of this embodiment wherein the composition is in the form of a feed is that more of the composition will actually be taken in by the organism so that a smaller amount is required to treat the bacterial infection.
  • the present invention also relates to the first medical use wherein the composition described hereabove is used for the manufacture of a medicament for the therapeutic or prophylactic treatment of a bacterial infection of an organism, in particular of an aquaculture organism such as crustaceae, mollusca and fish, and more particularly for the manufacture of a medicament for the therapeutic or prophylactic treatment of a Vibrio infection, in particular of a Vibrio harveyi or a Vibrio campbelli infection of an aquaculture organism such as shrimp.
  • the invention further also relates to a method for producing the composition described hereabove, which method comprises the steps of culturing bacteria infected with said lytic bacteriophage, lysing said bacteria to produce a phage lysate, and inactivating the bacteriophages present in said phage lysate by means of a chemical nucleic acid inactivating agent, in particular by means of the alkylating agents as described hereabove.
  • the bacteria used to produce the phage lysate are preferably Vibrio bacteria, and are more particularly Vibrio harveyi or Vibrio campbelli bacteria.
  • Lysis of the bacteria can be achieved by means of the lytic enzymes produced by the bacteriophages themselves and/or can be achieved by the addition of lytic additives.
  • any remaining bacteria and bacterial debris are preferably removed from the phage lysate, in particular by centrifuging the lysate, preferably at a centrifugal acceleration smaller than 25 000 g and preferably smaller than 15 000 g.
  • This separation step can be performed after the inactivation step, in which case a first preliminary separation step is preferably carried out before the inactivation step to remove some of the bacteria and bacterial debris. In this way, the inactivating agent can be used more efficiently.
  • the invention thus provides a simple and cheap method to treat the phage lysate, containing the active phage and the products encoded by its genome, without any risk of transfer of (virulent) genetic material, without the need of additional assessment impact on the environment and which can be used to treat the animals infected with the target bacteria or to prevent infection of said bacteria.
  • the present invention finally also relates to a method for the prophylactic or curative treatment of a bacterial infection of an organism, in particular of an aquaculture organism such as crustaceae, mollusca and fish, which method comprises the step of administering an effective amount of the above described composition to the organism, in particular to shrimp.
  • the composition contains a lytic enzyme of a lytic Vibrio phage and is used for the prophylactic or curative treatement of vibriosis, in particular of luminous vibriosis caused by Vibrio harveyi or vibriosis caused by Vibrio campbelli.
  • Isolation of the WMo bacteriophages Samples of water from shrimp farm, hatcheries and seawater were taken for phage isolation. 25 ml of water sample was centrifuged at 10 000 g for 15 min. 1 ml of the supernatant was added to an 8 h old V. harveyi culture grown in 5 ml Tryptic soya broth with 1 % NaCI (TSBS) and further incubated at room temperature for 8-12 h. The mixture was centrifuged at 10 00Og for 15 min at 4 0 C. The supernatant was filtered through 0.45 ⁇ m filters and collected in a sterile container.
  • TSBS Tryptic soya broth with 1 % NaCI
  • phage was tested by inoculating 25 ⁇ l of the filtrate on lawns of V. harveyi in tryptic soya agar. Areas of clearing were cut out to inoculate fresh V. harveyi culture in TSBS for enrichment of phages. Bacteria and debris were separated by centrifugation at 10 000 g for 15 min at 4 0 C. The supernatant was filtered through 0.45 ⁇ m filters and inoculated on lawns of V. harveyi to confirm the presence of bacteriophages.
  • the host-phage mixture was incubated in a shaker incubator at 28 0 C for 3 h and the degree of lysis was observed by reduction in A 6 oo to about 0.2.
  • the bacterial debris was pelleted by high speed centrifugation at 10 000 g for 30 min at 4 0 C, and the supernatant containing phage was filtered through 0.22 ⁇ m membrane filter (Millipore Corporation, USA) and the filtrate stored at -8O 0 C for further studies.
  • the suspension with the inactivated phage was added to a bacterial culture (V. harveyi) in a liguid growth medium. A 1 :10 dilution of this preparation was also tested in the bacterial culture.
  • Figure 1 shows the Vibrio counts after 24 hours and 48 hours when no inactivated phage preparation is added (control), compared to the bacterial growth when the inactivated phage suspension was added directly (1 ml) and in a dilution of 1 :10 (0.1 ml).
  • V. harveyi and 4 lytic V. harveyi phages were used.
  • the bacterial strains wild type: strains W1 to strain W9 were exposed to the live phages and resistant colonies (resistant to all four phages) were found to grow in the clearing zones (resistant variants: strain R1 to strain R9).
  • resistant variants were picked up with a loop and cultured in a microbiological medium. Then, the isolates were tested against four inactivated phage preparations.
  • the inactivated phage preparations were produced according to the method described above.
  • the inactivated phage preparations (designated Vihai , Viha2, Viha3 and Viha4) produced from live phages with a titre of 10 9 pfu / ml and tenfold dilutions (10 " ⁇ 10 ⁇ 2 , 10 ⁇ 3 and 10 ⁇ 4 ) of the inactivated phage preparations were applied on a lawn of the isolated culture. Growth inhibition was recorded for the highest dilution of the inactivated phage suspension.
  • Table 1 Highest dilution of inactivated phage preparation (Vihai , Viha2, Viha3 and Viha4) at which growth inhibition of 9 resistant strains of V. Harveyi (Strain R1 to R9) was recorded.
  • the growth of all wild types was affected by the live phages and the four inactivated phage preparations in a dilution till 10 ⁇ 4 .
  • resistant variants of these strains cannot be controlled by the live phages. Nevertheless, when these resistant variants were brought in contact with the inactivated phage preparations, the growth of these variants was inhibited, sometimes even when a dilution of 10 ⁇ 2 or 10 ⁇ 3 of the inactivated phage preparation was used.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Farming Of Fish And Shellfish (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé destiné au traitement prophylactique ou curatif d'une infection bactérienne dans des organismes d'aquaculture, tel que les crustacés, les mollusques et les poissons, au moyen d'une nouvelle composition qui contient une enzyme phagique lytique, ne contient pratiquement pas de bactériophages actifs et peut être produite à grande échelle. Plus spécifiquement, l'invention concerne le traitement prophylactique ou curatif de la vibriose dans les méthodes d'aquaculture, en particulier dans les cultures de crevettes. L'invention concerne également une composition destinée à être utilisée dans ledit procédé et un procédé destiné à produire ladite composition dans laquelle les bactéries infectées avec un bactériophage lytique sont cultivées, puis sont lysées et les bactériophages présents dans le lysat phagique sont inactivés au moyen d'un agent chimique d'inactivation des acides nucléiques.
PCT/EP2007/054281 2006-05-09 2007-05-03 Traitement d'infections bactériennes au moyen d'enzymes lytiques de bactériophages Ceased WO2007128765A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EPPCT/EP2006/062176 2006-05-09
PCT/EP2006/062176 WO2007128348A1 (fr) 2006-05-09 2006-05-09 COMPOSITIONS COMPRENANT DES ENZYMES LYTIQUES DE BACTÉRIOPHAGES permettant LE TRAITEMENT D'INFECTIONS BACTÉRIENNES

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WO2007128765A3 WO2007128765A3 (fr) 2008-01-03

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PCT/EP2006/062176 Ceased WO2007128348A1 (fr) 2006-05-09 2006-05-09 COMPOSITIONS COMPRENANT DES ENZYMES LYTIQUES DE BACTÉRIOPHAGES permettant LE TRAITEMENT D'INFECTIONS BACTÉRIENNES
PCT/EP2007/054281 Ceased WO2007128765A2 (fr) 2006-05-09 2007-05-03 Traitement d'infections bactériennes au moyen d'enzymes lytiques de bactériophages

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CN108192842A (zh) * 2018-01-17 2018-06-22 厦门昶科生物工程有限公司 一种哈维弧菌噬菌体微生态制剂及其制备方法和应用
CN110184222B (zh) * 2019-06-06 2021-06-08 厦门惠盈动物科技有限公司 一种噬菌蛭弧菌冻干粉制剂及其应用
CN112391355B (zh) * 2019-08-14 2023-12-01 宁波大学 哈维氏弧菌高效裂解性噬菌体vB_VhaS-yong3及其应用
CN112063595B (zh) * 2020-09-24 2021-11-26 瑞科盟(青岛)生物工程有限公司 一株裂解性哈维弧菌噬菌体rdp-vp-19012及其应用

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WO2004064732A2 (fr) * 2003-01-16 2004-08-05 New Horizons Diagnostics Corporation Utilisation de proteines lytiques associees a des bacteriophages pour la prevention et le traitement d'infections bacteriennes chez l'etre humain, les animaux et les volailles
ES2135355B1 (es) * 1998-04-27 2000-07-01 Univ Santiago Compostela Vacuna anti-vibrio anguillarum (gava-3) para la prevencion de la enfermedad vibriosis del rodaballo y peces salmonidos, y procedimiento de obtencion.
US6913757B1 (en) * 2000-07-26 2005-07-05 The Board Of Governors For Higher Education, State Of Rhode Island And Providence Plantations Live, avirulent strain of V. anguillarum that protects fish against infection by virulent V. anguillarum and method of making the same
ATE421249T1 (de) * 2002-02-13 2009-02-15 Immunology Lab Inc Zusammensetzungen und verfahren für die behandlung von mikroorganismen-infektionen
EP1677834B1 (fr) * 2003-10-06 2014-01-08 Gangagen, Inc. Phage therapeutique dose de maniere definie

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