DESC:
F O R M 2

THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]

1. TITLE OF THE INVENTION: BACTERIOPHAGE THERAPY OF INFECTIOUS DISEASES IN AQUACULTURE

2. APPLICANT (A) NAME: ARISTOGENE BIOSCIENCES PVT LTD

(B) ADDRESS: A-67(A), FIRST CROSS, RAJAJI NAGAR INDUSTRIAL ESTATE BANGALORE, KARNATAKA, INDIA, 560010

3. NATIONALITY (C) INDIA

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE NATURE OF THIS
INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

[001] TECHNICAL FIELD OF THE INVENTION
[002] The present invention is in the technical field of bacteriophage compositions to treat infectious diseases, more particularly, methods for production of bacteriophage compositions, their storage, preservation, and methods for prophylactic or curative treatment of bacterial infection of an organism in aquaculture.
[003] BACKGROUND OF THE INVENTION
[004] Bacteriophages (phages) are the viruses that infect bacteria. They are distinguished from animal and plant viruses. Bacteriophages can have either lytic or lysogenic life cycle.
[005] Phages in a lytic cycle cause lysis of the host bacterial cell. They are also known as virulent phages. Phages that replicate in lysogenic life cycle are called temperate phages. They have the possibility of an alternative life cycle where their genomic DNA is integrated into the host bacterial chromosome, called “prophage”. Prophage is propagated passively through the bacterial chromosome's replication apparatus. Although largely inert, prophages can, under some circumstances excise from the host genome, enter lytic mode, produce numerous progeny that leads to lysis of the host bacterium. Phages that lack the capability to enter the alternative lysogenic life cycle, are the most suitable type of phage to employ therapy.
[006] Bacterial diseases are a common problem in aquatic animal farming. Vibrio species is the main genus involved and in particular V. harveyi and V.parahemolyticus are the most consistently isolated related to disease outbreaks in the literature. Vibrio has a worldwide distribution, and it is considered as a primary pathogen for shrimp and fish larval stages and a secondary pathogen for juveniles and adults.
[007] Vibrio-related infections frequently occur in hatcheries. Vibrio species are part of the natural microflora of wild and cultured shrimps and become opportunistic pathogens when natural defense mechanisms are suppressed (Brock and Lightner, 1990). They are usually associated with multiple etiological agents. However, some Vibrio species, or strains of certain species, have been identified as primary pathogens. Pathogenic strains of V. harveyi, V. vulnificus and V. parahaemolyticus have caused massive epidemics in Thailand and the Philippines Luminescent V. harveyi appears to release exotoxins and may cause 80-100% mortality in P. monodon hatcheries.
[008] Antibiotics are currently used for control of vibriosis. The indiscriminate use of antibiotics in aquaculture has led to the emergence and spread of antibiotic-resistant bacteria. In a study of a luminous vibriosis outbreak in a shrimp hatchery, isolates of V. harveyi were resistant to the antibiotics used; thus, they were ineffective in controlling the disease among shrimp larvae.
[009] At present, there is severe antibiotic-therapy crisis, which is the result of three major factors: a dramatic rise in antibiotic resistance among bacteria, shortage of novel classes of antibiotics, and the withdrawal of the pharmaceutical industry from the discovery and development of new antibiotics.
[010] In addition, other concerns associated with the use of antibiotics is the problem of residues, which has resulted in rejection by seafood importing countries of shrimp containing traces of antibiotics.
[011] Therefore, antibiotics are no longer the preferred treatment against vibriosis in shrimp culture and alternative methods are being sought. Hence, there is a need to eradicate vibriosis in aquaculture without the use of antibiotic.
[012] Phages are viruses that only infect bacteria and are used as therapeutic for bacterial infections in humans dating back roughly a century, but with the introduction of antibiotics in the mid-1900s, phages fell out of favor in most parts of the world. Now, with the growing threat of antibiotic-resistant bacterial infections, phage therapy returns to the spotlight. The world may be heading for a post-antibiotic age.
[013] In summary, there is an urgent need in the art to develop a highly specific cost-effective bacteriophage therapy for aquaculture to curb antibiotic overuse and misuse, in humans and animals.
[014] SUMMARY OF THE INVENTION
[015] The primary objective of the present invention is to provide bacteriophage compositions to treat infectious diseases, more particularly, methods for production of bacteriophage compositions, their storage, preservation and methods for prophylactic or curative treatment of bacterial infection of an organism in aquaculture. Furthermore, the invention also provides methods for detecting the presence of the targeted bacteria in various environments including processed or unprocessed food products, and in equipment used to process or manufacture the food products.
[016] According to a further exemplary aspect of the present invention, it provides a method for producing bacteriophage composition comprising: a) providing stabilized bacteriophage, phage components, or a combination thereof; and b) encapsulating the stabilized bacteriophage, phage components, or a combination thereof, to produce the bacteriophage composition.
[017] According to a further exemplary aspect of the present invention, the present invention is directed to isolate bacteriophages having specificity and lytic activity against Vibrio species, methods of using the bacteriophages, progeny and derivatives derived therefrom, to control the growth of vibrio species in various settings.
[018] According to a further aspect of the present invention, the invention describes phage compositions comprising alone or in any combination of VH1a, VH3, VH17, VH20, VH28, VP13 and MH1 bacteriophages having lytic specificity for the targeted bacteria.
[019] According to yet another aspect of the present invention, the invention provides an antibacterial composition that includes at least one phage strain selected from among the 7 strains VH1a, VH3, VH17, VH20, VH28, VP13 and MH1 or any combination of them, to be applied as prophylaxis, control and/or treatment of the infection caused by Vibrio in all types of crops of fish, mollusks and crustaceans susceptible to this pathogenic bacterium.
[020] In one of the embodiment, the present invention provides isolation, characterization and testing of new native bacteriophages, and their different mixes, originating from places related to aquaculture or marine environments that are capable of infecting and destroying Vibrio, so as to control the infection of this pathogen in species that are important to aquaculture.
[021] In one of the embodiments, the present invention provides seven strains of new native isolated bacteriophages, specifically against bacteria belonging to the Vibrio genus, particularly V. harveyi, V. parahemolyticus, V.campbelli, V.alginolyticus species that is effective for the prophylaxis, control and/or treatment of the infection caused by these vibrios and other members of Vibrio genre in all types of species (fish, mollusks, crustaceans) that are important for aquacultures susceptible to this bacterium. The strains were isolated from different marine sources and characterized for storage under different temperatures, salinity, sensitivity to chloroform, morphological structure, RAPD, SDS-PAGE determining the range of hosts and frequency of phage-resistant bacteria.
[022] In one of the embodiments, the present invention provides a method for the prophylactic or curative treatment of bacterial infection of an organism, in particular of an aquaculture organism such as crustaceae, mollusca and fish.
[023] In one particular embodiment, invention also describes antibacterial compositions based on the strains of bacteriophages, and optionally excipients and formulations of the antibacterial compositions to be used in the prophylaxis, control and/or treatment of the infection caused by Vibrio in all types of fish, mollusk and crustacean species of importance to the aquaculture industry susceptible to this bacterium. The compositions and formulations of this invention are added directly to the water in which the species to be treated are being cultivated, or rather, they are used to associate them to controlled liberation matrixes or to include them in the food.
[024] In yet another embodiment, these phages alone or in combination are provided to control the growth of the targeted bacteria, which may reduce their ability to contaminate and colonize various environments, including but not limited to (i) raw, unprocessed food products, (ii) equipment used to process or manufacture various food products, (iii) various food products processed or manufactured with equipment contaminated with the targeted bacteria, (iv) animals contaminated with the targeted bacteria, (v) animal environments contaminated with the targeted bacteria, and (vi) various processed food products for humans or animals containing ingredients contaminated with the targeted bacteria.
[025] According to a further exemplary aspect of the present invention, it provides a method for detecting the presence of the targeted bacteria in various environments including processed or unprocessed food products, and in equipment used to process or manufacture the food products.
[026] As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages.
[027] The advantages of phage therapy are;
[028] Phages are self-replicating therapeutic.
[029] Self-limiting i.e. when the bacteria gets eliminated phage also does not multiply.
[030] One phage/bacterium is sufficient.
[031] Highly host-specific.
[032] No damage to normal (beneficial) flora, unlike antibiotics where the all the microflora is damaged.
[033] Do not spread antibiotic resistance.
[034] Rapid characterization & development.
[035] The present invention provides a variety of following advantages in aquaculture,
1) Unrestricted use, without limitations because it has no impact on the environment;
2) Zero period of unavailability, that is, the fish may be harvested irrespective of when the products based on phages were applied;
3) Organic production, it permits fish, shrimp, molluscs cultivation under an organic production system.
[036] The present invention provides a variety of following advantages as will be appreciated by a person skilled in art,
[037] A method for producing an antibacterial composition comprising, embedding an aqueous solution of bacteriophages, phage components, or a combination thereof onto a solid or powdered support or encapsulated to produce a composition, and drying the composition to produce an antibacterial composition.
[038] 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.
[039] Methods as mentioned in [037] and [038], wherein the bacteriophage exhibits lytic growth in the bacterial species, strain, serotype or isolate that is different to the pathogenic bacterium.
[040] Methods as mentioned in [037] and [038], wherein the pathogenic bacterium is of the genus Vibrio, Pseudomonas or Aeromonas.
[041] The method as mentioned in [038], wherein the pathogenic bacterium of the genus Vibrio is a pathogen of shrimp or prawns or fish.
[042] A bacteriophage composition comprising one or more than one strain of a stabilized bacteriophage, one or more than one phage component, one or more than one strain of a stabilized bacteriophage and one or more than one phage component encapsulated together, or a combination thereof.
[043] An antibacterial phage composition according to [042], wherein it includes at least one phage strain selected from the group or alone or in any combination of VH1a, VH3, VH17, VH20, VH28, VP13 and MH1 bacteriophages having lytic specificity for the targeted bacteria.
[044] The bacteriophage composition of [042], wherein the one or more than one phage component is selected from the group consisting of a phage tail, a phage protein, and a combination thereof.
[045] The bacteriophage composition of [042], wherein the antibacterial composition additionally includes, optionally, a buffer solution and/or nutrients and/or phage stabilizer and/or thickeners and/or other excipients.
[046] The bacteriophage composition of [042], wherein the stabilized bacteriophage, phage components, or a combination thereof, is encapsulated using a material selected from the group consisting of sugar, sucrose, maltose, trehalose, mannose, mannitol, glycerol or any similar additives, wherein these phages can also be mixed or sprayed onto excipients like but is not limited to sucrose, sorbitol, mannitol, lactose, dextrose starch, dicalcium phosphate, talc, maltodextrin, yeast extract, what bran, soya meal, fish meal, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils.
[047] The bacteriophage composition of [042], further comprising a pharmaceutically acceptable carrier.
[048] The bacteriophage composition of [042], wherein the bacteriophage composition is formulated as a capsule or a tablet.
[049] A composition comprising an animal feed admixed with the bacteriophage composition of [042].
[050] The composition of [049], wherein the animal feed is selected from the group consisting of human feed, a bird feed, a fish feed, a porcine feed, a livestock feed, a poultry feed, a domestic animal feed, and a food for aquaculture.
[051] The bacteriophage composition of [042], wherein the bacteriophage composition is diluted or stored in buffers like saline, Tris, Phosphate buffered saline, salt solutions or any other buffers that can preserve the phages.
[052] The bacteriophage composition of [042], wherein the bacteriophage composition is used as an antiseptic or antibacterial agent.
[053] The bacteriophage composition of [042], wherein the bacteriophage composition is used, as an antibacterial agent in aquaculture.
[054] Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[055] SUBMISSION OF MICROORGANISM AT INTERNATIONAL DEPOSITORY:
[056] The following are the accession numbers of the microbial cultures deposited in MTCC under Budapest treaty:
S. No. Taxonomic Designation Identification Reference MTCC Number Assigned
1. Vibrio harveyi V13 MTCC 25262
2. Siphoviridae siphoviridae VH1a MTCC 25263
Address of the depository: Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of Microbial Technology (IMTECH), Sector-39A, Chandigarh – 160036, India.
[057] DETAILS OF SUBMISSION:
Host: V 13
Date of submission: 11-03-2019
No of samples: 3 stab cultures

Bacteriopahge: VH1a
Date of submission: 11-03-2019
Samples: 3x 5ml of phage lysate
[058]
[059] BRIEF DESCRIPTION OF THE DRAWINGS
[060] Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
[061] FIG. 1 illustrates the REP-PCR with GTG5 primer. Lane M- DNA ladder, lane 1: VH1a, lane 2: VH3, lane 3: VP13, lane 4. VH17, lane 5. MH1, lane 6. VH28.
FIG. 2 illustrates the titer of the phages in different temperature.
[062] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[063] DETAILED DESCRIPTION OF THE INVENTION
[064] It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
[065] The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
[066] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
[067] The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
[068] All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
[069] Example embodiments of the present invention are described with reference to the accompanying figures.
[070] In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
[071] EMBODIMENTS OF THE INVENTION:
[072] This invention presents the isolation, characterization and testing of new native bacteriophages, and their different mixes.
[073] This invention presents the isolation, characterization and testing of new native bacteriophages, and their different mixes, that are capable of infecting and destroying Vibrio, so as to control the infection of this pathogen in species that are important to aquaculture.
[074] This invention provides seven strains of new native isolated bacteriophages, specifically against bacteria belonging to the Vibrio genus, particularly V. harveyi,V. parahemolyticus, V.campbelli, V.alginolyticus species that is effective for the prophylaxis, control and/or treatment of the infection caused by these vibrios and other members of Vibrio genre in all types of species (fish, mollusks, crustaceans) that are important for aquacultures susceptible to this bacterium. The strains were isolated from different marine sources and characterized for storage under different temperatures, salinity, sensitivity to chloroform, morphological structure, RAPD, SDS-PAGE determining the host range.
[075] The invention provides an antibacterial composition that includes at least one phage strain selected from among the 7 strains VH1a, VH3, VH17, VH20, VH28, VP13 and MH1 or any combination of them, to be applied as prophylaxis, control and/or treatment of the infection caused by Vibrio in all types of crops of fish, mollusks and crustaceans susceptible to pathogenic bacterium.
[076] IMPORTANT ATRIBUTES OF THE INVENTION:
[077] Bacterial Strains and isolation of phages
[078] Seawater, Water and larval samples from hatchery, oyster liquid, clams liquid, moribund shrimps were collected from different regions of coastal Karnataka, Andhra Pradesh, Tamil Nadu, Orissa and other parts of India as a source for isolation of Vibrios. Several Vibrio species were isolated, characterized and were used in the study. These bacteria were used to isolate phages. The obtained phages were purified using single plaque purification on agar double layer method.
[079] Host range determination
[080] The host ranges of bacteriophages were determined among a collection of 70 Vibrio species. Bacterial cells were mixed with molten agar and this mixture was poured on solid agar to make double layer agar plates. After solidification, isolated bacteriophage stock solutions were spotted on each plate with different bacterial isolates. After allowing 20 min for the spots to be absorbed, the plates were inverted and incubated for 24 hours at 37°C before the degree of lysis was recorded.
[081] The purified phages were further characterized using RAPD, SDS PAGE and whole genome sequencing. Phages showing good stability and broad host range were further characterized.

[082] CHARACTERIZATION OF PHAGES
[083] All the isolated phages were checked on the various isolates and standard strains. Host range studies were done by spot test.
[084] MIC determination was done by microplate assay method.
[085] Phages were grown in bulk, purified and DNA isolated and RAPD was done. SDS PAGE analysis was also carried out.
[086] Based on the above results promising phages were subjected compatibility assay, one step growth curve, temperature, pH and salinity stability assays. Most of the phages were stable upto 50°C and few phages were found to be stable at 80°C also. All of them were tolerant upto 50 ppt salinity and few of them were tolerant upto 70ppt. This is well within the level of 30 ppt used under farming conditions.
[087] THERMAL STABILITY
Thermal stability is one of the crucial parameter to check the stability of the phages. After I hour incubation phage at the respective temperatures it was serially diluted and plated using double layer method.
[088] There phages were checked for their compatibility to be used as cocktail. Few phages were not compatible.
[089] REP-PCR All the isolated phage genomic DNA were subjected to REP-PCR using (GTG)5 primer with the sequence 5’ GTG GTG GTG GTG GTG 3’ (Versalovic et al, 1994). PCR reaction mix for (GTG)5 – PCR contained Assay buffer for Taq polymerase with 1.5 mM MgCl2, 0.2mM each dNTP, 1 µM of (GTG)5 primer, 2U Taq DNA polymerase and 100 ng of template DNA. PCR conditions were 94°C for 5 min, 35 cycles of 94°C for 1 min, 48°C for 1 min and 72°C for 2 min followed by final extension of 72°C for 5 min. PCR products were subjected to agarose gel electrophoresis in 1.5% agarose. The gels were stained with ethidium bromide and photographed.
[090] CULTURE CONDITIONS PHAGE AMPLIFICATION
[091] The seed inoculum was added into LB media. The culture flask was incubated at 37°C at 120 rpm till it reaches to 4 OD. Phage lysate was added from the characterized working phage bank (WPB) to the culture.
[092] Incubation was continued at 37°C at 120 rpm for 8 hours.
[093] The lysate was centrifuged at 7000 rpm for 60 min. The supernatant was collected and Filter-sterilized.
[094] Titration by agar overlay method
[095] 990 µl of LB was taken in 1.5 ml vial and 10 µl of phage lysate was added, vial was closed tightly and mixed thoroughly to obtain 10-2.
[096] The same process was repeated to prepare dilution up to 10-10. Dilution 10-8 /10-9 /10-10 were used for plating.
[097] In a tube, specific host and 1 ml phage dilution was added. Incubated for 5 min at room temperature.
[098] 6 ml of soft agar was added into above tube containing phage and host mixture. Mixed well and poured on LB hard agar plate.
[099] The plate was kept for 10 min on flat surface of laminar air flow bench for solidification.
[0100] After solidification, the plate was incubated at 37°C for overnight.
[0101] Next day the plaque was counted and the PFU (Plaque Forming unit) was calculated by using below formula.
No. of plaque × Dilution
[0102] PFU/ml = __________________________
Volume plated in ml

[0103] In a preferred embodiment, the antibacterial composition additionally includes, optionally, a buffer solution and/or nutrients and/or phage stabilizer and/or thickeners and/or other excipients. The phages are stabilized by lyophilization with or without stabilizers such as sugar, sucrose, maltose, trehalose, mannose, mannitol, glycerol or any similar additives. These phages can also be mixed or sprayed onto excipients like but is not limited to sucrose, sorbitol, mannitol, lactose, dextrose starch, dicalcium phosphate, talc, maltodextrin, yeast extract, what bran, soya meal, fish meal, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils. These phages can also be diluted or stored in buffers like saline, Tris, Phosphate buffered saline, salt solutions or any other buffers that can preserve the phages.
[0104] EXAMPLE EMBODIMENTS OF THE INVENTION:
[0105] Bacterial Strains and isolation of phages
[0106] Seawater, Water and larval samples from hatchery, oyster liquid, clams liquid, moribund shrimps were collected from different regions of coastal Karnataka, Andhra Pradesh, Tamil Nadu, Orissa and other parts of India as a source for isolation of Vibrios. Several Vibrio species were isolated, characterized and were used in the study. These bacteria were used to isolate phages. The obtained phages were purified using single plaque purification on agar double layer method.
[0107] Host range determination
[0108] The host ranges of bacteriophages were determined among a collection of 70 Vibrio isolates. Bacterial cells were mixed with molten agar and this mixture was poured on solid agar to make double layer agar plates. After solidification, isolated bacteriophage stock solutions were spotted on each plate with different bacterium isolates After allowing 20 min for the spots to be absorbed, the plates were inverted and incubated for 24hour at 37°C before the degree of lysis was recorded.
[0109] The purified phages were further characterized using RAPD, SDS PAGE and whole genome sequencing. Phages showing good stability and broad host range were further characterized.
[0110] Sequencing, analysis and annotation of phage genomes was carried out.
[0111] To isolate phage DNA, phages were propagated as described above. Phage DNA was isolated by extraction with phenol: chloroform: isoamyl alcohol (25:24: 1, V/V), ethanol precipitation and resolution in water. Whole genome sequencing was done, and the BLAST algorithm was used to determine the similarity to described genes in the National Center for Biotechnology Information [NCBI] database. The genomes were scanned for potential open reading frames (ORFs).

[0112] Bacteriophage therapy of Vibrio infected post larvae
[0113] Disinfected tanks were used for phage therapy. For disinfection, the tanks were initially taken out and washed with tap water and detergent and rinsed repeatedly, 2-3 times in tap water. Subsequently, they were disinfected with Sodium Hypochlorite containing 100ppm chlorine, and rinsed with tap water. The tanks were dried under sunlight for one day.
[0114] The tanks were filled with 10 litres of 30ppt sea water and 500 post larvae (PL) were released to each tank. They were fed on micro - particulate diet and maintained for acclimatization for two days. During this period larval activity, feeding and major water quality parameters such as ammonia, nitrite, alkalinity and hardness were recorded and ascertained soundness of the system to undertake the experiment.
[0115] The tanks were set in the following order:
[0116] Tank 1- Control without the application of bacteria (Vibrio harveyi) or phage.
[0117] Tank 2- Test - Application of Vibrio harveyi.
[0118] Tank 3- Test - Application of Vibrio harveyi + phage cocktail
[0119] Tank 4 Test - Application of Vibrio harveyi + Antibiotic (Ciprofloxacine).
[0120] Phage cocktail and antibiotic were added at 0hr, 24hr, 48hr and 72hrs.
[0121] TCBS agar plates were used for plating the samples. The larval survival was recorded and expressed as relative per cent survival.
[0122] Results
[0123] Bacterial strains
[0124] Over 70 strains of vibrio have been isolated and 35 strains were used as representative strains for phage host range. Series V1-V24, VH4, LB1-43 are Vibrio harveyi/Vibrio campbelli, series AV are Vibrio parahemolyticus and series C1-C5 are reference Vibrio harveyi and series C6-C7 are reference Vibrio prahemolyticus strains from CAIM, Mexico.
[0125] Host range determination
[0126] Spot test is used for screening of host range of the phages. In this test, eight different amplified phage lysates can be tested at the same time by spotting a drop of the amplified phage lysate on the bacterial lawn in a Petri dish. The results are read after overnight incubation and are scored as +++ for clear zone, ++ for turbid zone, + very turbid zone and – for no zone. The results of some phage isolates are presented in Table 1.
[0127] Table 1. Susceptibility of the representative Vibrio strains to infection with phage isolates as assessed by the spot assay

HOST fVH3 fVH17 fVH28 fVH1a fVP13 fMH1 fVH20
V1 +++ - - - - - -
V3 + + ++ ++ - - -
V4 +++ + - +++ - - -
V6 +++ +++ +++ +++ +++ - +
V11 +++ - +++ +++ - ++ -
V12 +++ +++ +++ +++ +++ +++
V13 +++ +++ +++ +++ +++ +++ +++
V14 +++ +++ +++ +++ +++ ++ +++
V15 +++ - +++ +++ - - -
V16 +++ +++ +++ +++ +++ ++ +++
V17 +++ +++ +++ +++ - - +++
V18 ++ +++ ++ ++ + +++ +++
V19 +++ +++ +++ +++ ++ +++ -
V20 +++ +++ +++ +++ - +++ -
V21 +++ +++ +++ +++ +++ - +++
V22 +++ +++ +++ +++ - - -
V23 +++ +++ +++ +++ +++ ++ ++
AV4 ++ +++ ++ ++ +++ +++ ++
AV7 ++ +++ +++ +++ - - +++
AV9 +++ +++ - ++ ++ - ++
AV10 +++ + - + - - +
AV12 - ++ + +++ - - +++
C1 +++ ++ +++ +++ - - +++
C2 + - + - - - -
C3 +++ - - - - - -
C4 - - - +++ - - +++
C5 + - +++ ++ - - ++
C6 - + - - - -
C7 - ++ - ++ - - ++
LB1 +++ ++ +++ +++ +++ +++
LB4 +++ +++ +++ +++ +++ +++ +++
LB9 ++ - - - - - -
LB12 + +++ +++ +++ - ++ +++
LB43 + - - - -
VH4 +++ +++ +++ +++ +++ +++ +++
[0128] +++- Clear zone, ++- Slightly turbid zone, + - Turbid zone, - No zone
[0129] As can be seen from table 1, the phages have individually very strong lytic power, and combinations (or cocktails) of these bacteriophages may be produced that are able to kill all of the tested Vibrio strains, thereby producing broad spectrum antibacterial compositions.
[0130] FIG 1 shows the REP-PCR with GTG5 primer
[0131] This invention provides seven strains of phages isolated from places related to aquaculture or marine environments that are specific against bacteria belonging to the Vibrio genre, in particular the V. harveyi and V. parahemolyticus species, which is efficient in the prophylaxis, control and/or treatment of the infection caused by Vibrio in all types of fish, mollusk and crustacean species that are important to the aquaculture industry susceptible to this bacterium. This invention also describes antibacterial compositions based on the strains of previous bacteriophages, and optionally excipients and formulations of the antibacterial compositions to be used in the prophylaxis, control and/or treatment of the infection caused by Vibrio in all types of fish, mollusk and crustacean species of importance to the aquaculture industry susceptible to this bacterium. The compositions and formulations of this invention are added directly to the water in which the species to be treated are being cultivated, or rather, they are used to associate them to controlled liberation matrixes or to include them in the food.
[0132] Temperature Stability

Table 1: The titer of the phages in different temperature is given in the table and chart (FIG 2)

PHAGE ROOM
TEMPERATURE
(pfu/ml)
37°C
(pfu/ml)
50°C
(pfu/ml)
60°C
(pfu/ml)
70°C
(pfu/ml)
80°C
(pfu/ml)
90°C
(pfu/ml)
F1a
2.3×1011
1.2×1011
8×1010 5.56×1010 3.2×108 6.08×108 no plaque
fVH3 1.67×108 2.14×108 4×106
1.08×106 No plaque No plaque No plaque
fVH20 2×109 4.14×108 1.06×108 5.12×106 1.62×106 2.04×104 No plaque
F VH28 2.82×109 2.5×109 1.68×109 1.14×108 8.0×104 1.6×104 2.9×102
fVH17 3.64×1010 2.86×1010 2.1×109 No plaque No plaque No plaque No plaque
fMH1 5.4×109 5.2×109 1.65×108 No plaque No plaque No plaque No plaque
FVP13 1×1011 4.13×1010 3.86×1010 5.12×107 8.64×106 4×105 2.84×104

1.
Control tank
(No bacteria and no phage) 0hours NIL 4×103 _ 100
24 hours NIL 3×104 _ 94
48 hours NIL 9.6×104 _ 90
72 hours NIL 90

2.
Only bacteria 0hours NIL 2×106 4.2×105 100
24 hours NIL 2.8×106 6.8×105 73
48 hours NIL 4.9×105 3×104 61
72 hours NIL 55

3.
Bacteria + phage cocktail 0hours 100µl/L 2×106 4.2×105 100
24 hours 100µl/L 2.6×104 1.6×102 96
48 hours 100µl/L 1.1×104 2×101 91
72 hours 100µl/L 90

4.
Bacteria + antibiotics 0hours 10mg/L 2×106 4.2×105 100
24 hours 10mg/L 3.28×104 _ 94
48 hours 10mg/L 2.1×104 _ 90
72 hours 10mg/L 89

BACTERIOPHAGE THERAPY RESULT
[0133] Table 2: Shows the result of Bacteriophage therapy

[0134] Formulation:
[0135] These phages can also be mixed or sprayed onto excipients like but is not limited to sucrose, sorbitol, mannitol, lactose, dextrose starch, dicalcium phosphate, talc, maltodextrin, yeast extract, wheat bran, soya meal, fish meal, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils.
[0136] These phages can also be diluted or stored in buffers like saline, Tris, Phosphate buffered saline, salt solutions or any other buffers that can preserve the phages.
[0137] Bacteriophage therapy of Vibrio infected post larvae
[0138] Disinfected tanks were used for phage therapy. For disinfection, the tanks were initially taken out and washed with tap water and detergent and rinsed repeatedly, 2-3 times in tap water. Subsequently, they were disinfected with Sodium Hypochlorite containing 100ppm chlorine, and rinsed with tap water. The tanks were dried under sunlight for one day.
[0139] The tanks were filled with 10 litres 30 ppt sea water and 500 post larvae (PL) were released to each tank. They were fed on micro - particulate diet and maintained for acclimatization for two days. During this period larval activity, feeding and major water quality parameters such as ammonia, nitrite, alkalinity and hardness were recorded and ascertained soundness of the system to undertake the experiment.
[0140] The tanks were set in the following order:
[0141] Tank 1- Control without the application of bacteria (Vibrio harveyi) or phage.
[0142] Tank 2- Test - Application of Vibrio harveyi.
[0143] Tank 3- Test - Application of Vibrio harveyi + phage cocktail
[0144] Tank 4 Test - Application of Vibrio harveyi + Antibiotic (Ciprofloxacine).
[0145] Phage cocktail and antibiotic were added at 0hr, 24hr, 48hr and 72hrs.
[0146] TCBS agar plates were used for plating the samples. The larval survival was recorded and expressed as relative per cent survival.
[0147] FLOW CHART OF PROTOCOL FOR ISOLATION AND SELECTION OF BACTERIOPHAGES
i. Isolation of vibrio isolates
[0148] Seawater, Water and larval samples from hatchery, oyster liquid, clams liquid, moribund shrimps were collected from different regions of coastal Karnataka, Andhra Pradesh, Tamil Nadu, Orissa and other parts of India as a source for isolation of vibrio species
[0149] Isolates were characterized and identified by standard biochemical methods.
[0150] Identification of the isolates was confirmed by toxR PCR and sequencing.
[0151] ii. Isolation of phages
[0152] Phages specific to the vibrio strains were isolated. All the isolated phages were subjected 3 passages using agar overlay method and purified.
[0153] iii. Characterization of phages
[0154] All the isolated phages were checked on the various isolates and standard strains. Host range studies were done by spot test.
[0155] Minimum inhibitory concentrations (MIC) determination was done by microplate assay method.
[0156] Phages were grown in bulk, purified and DNA isolated. RAPD (Random Amplified Polymorphic DNA) and SDS PAGE analysis was carried out. Phages for further study were selected from the groups which were showing different pattern.
[0157] Based on the above results promising phages were subjected to compatibility assay, one step growth curve, temperature, pH and salinity stability assays. Most of the phages were stable upto 50°C and few phages were found to be stable at 80°C also. All of them were tolerant upto 50 ppt (parts per thousand) salinity and few of them were tolerant upto 70ppt. This is well within the level of 30ppt used under farming conditions.
[0158] These phages were checked for their compatibility to be used as cocktail. Few phages were not compatible.
[0159] iv. Preparation of formulation
[0160] Cocktails were made with compatible phages and the different formulations were tested.
[0161] Different phage formulations were tried.

[0162] v. Lab scale animal studies:
[0163] Phages were tested individually in the lab scale animal challenge study. 100 Post larvae were challenged with the bacteria and the respective phages. All the tanks with phages shows marked survivability when compared to control tank which contained only bacteria. All these studies prove that protection by phages was as good as antibiotics.
[0164] vi. Field Trials:
[0165] Two field trials were carried out.
[0166] Both the trials showed that the larval survivability increased markedly with the bacteriophage therapy.
[0167] Major advantage of the current invention is non-invasive method of detection.
[0168] This is a novel Non-chemical anti-microbial formulation
[0169] No damage to the normal beneficial bacteria
[0170] Extremely safe and specific
[0171] Active against antibiotic resistant bacteria
[0172] Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
[0173] While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
[0174] Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
[0175] It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
[0176] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[000] References
1) Brock, J. A., and Lightner, D. V. 1990, Diseases of Crustacea. Diseases caused by microorganisms.
2) O. Kinne. John Wiley, vol. 3, pp. 245-349, In Diseases of marine animals, Ed. by O. Kinne. John Wiley, New York.
,CLAIMS: CLAIMS:
I/WE CLAIM:
1) A method for producing an antibacterial composition comprising, embedding an aqueous solution of bacteriophages, phage components, or a combination thereof onto a solid or powdered support or encapsulated to produce a composition, and drying the composition to produce an antibacterial composition.
2) 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.
3) The method as claimed in claim 1 and claim 2, wherein the bacteriophage exhibits lytic growth in the bacterial species, strain, or serotype. The method as claimed in claim 1 and claim 2, wherein the pathogenic bacterium is of the genus Vibrio.
4) The method of Claim 1, wherein the pathogenic bacterium of the genus Vibrio is a pathogen of shrimp or prawns or fish.
5) A bacteriophage composition comprising one or more than one strain of a stabilized bacteriophage, one or more than one phage component, one or more than one strain of a stabilized bacteriophage and one or more than one phage component encapsulated together, or a combination thereof.
6) A bacteriophage composition according to claim 5, wherein it includes at least one phage strain selected from the group or alone or in any combination of bacteriophages having lytic specificity for the targeted bacteria, wherein the bacteriophage is VH1a.
7) The bacteriophage composition of claim 5 and claim 6, wherein the one or more than one phage component is selected from the group consisting of a phage tail, a phage protein, and a combination thereof.
8) The bacteriophage composition of claim 5 and claim 6, wherein the bacteriophage composition additionally includes, optionally, a buffer solution and/or nutrients and/or phage stabilizer and/or thickeners and/or other excipients
9) The bacteriophage composition of claim 5 and claim 6, wherein the stabilized bacteriophage, phage components, or a combination thereof, is encapsulated using a material selected from the group consisting of sugar, sucrose, maltose, trehalose, mannose, mannitol, glycerol or any similar additives, wherein these phages can also be mixed or sprayed onto excipients like but is not limited to sucrose, sorbitol, mannitol, lactose, dextrose starch, dicalcium phosphate, talc, maltodextrin, yeast extract, what bran, soya meal, fish meal, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils.
10) The bacteriophage composition of claim 5, further comprising a pharmaceutically acceptable carrier.
11) The bacteriophage composition of claim 5, wherein the bacteriophage composition is formulated as a capsule or a tablet.
12) A composition comprising an animal feed admixed with the bacteriophage composition of claim 5.
13) The bacteriophage composition of claim 5 and claim 6, wherein the bacteriophage composition is diluted or stored in buffers like saline, Tris, Phosphate buffered saline, salt solutions or any other buffers that can preserve the phages.
14) The bacteriophage composition of claim 5 and claim 6, wherein the bacteriophage composition is used as an antiseptic or antibacterial agent.
15) The bacteriophage composition of claim 5 and claim 6, wherein the bacteriophage composition is used, as an antibacterial agent in aquaculture.
16) The composition of claim 12, wherein the animal feed is selected from the group consisting of human feed, a bird feed, a fish feed, a porcine feed, a livestock feed, a poultry feed, a domestic animal feed, and a food for aquaculture.