WO2017195073A1

WO2017195073A1 - Probiotics for biological control of pathogenic vibrio sp. in aquaculture

Info

Publication number: WO2017195073A1
Application number: PCT/IB2017/052565
Authority: WO
Inventors: Mohanraj KUMAR
Original assignee: Eiyarkai Three Life Sciences Llp
Priority date: 2016-05-13
Filing date: 2017-05-03
Publication date: 2017-11-16

Abstract

Aspects of the present disclosure provides a novel strain Vibrio sp. DIR21 (accession number MCC 0081) that exhibits quenching of quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture. Further, the isolated Vibrio strain exhibits improvement in the Feed Conversion Ratio (FCR) of crustaceans (shrimp in particular) by way of its protease activity. An aspect of the present disclosure relates to a method of quenching quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture, said method including addition of a strain Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof in said aquaculture to effect quenching of quorum sensing mechanism of said pathogenic bacteria Vibrio harveyi. Another aspect of the present disclosure provides a probiotic composition including a strain Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof, wherein said probiotic composition exhibits quenching of quorum sensing mechanism of Vibrio harveyi in an aquaculture.

Description

PROBIOTICS FOR BIOLOGICAL CONTROL OF PATHOGENIC VIBRIO SP. IN

AQUACULTURE

TECHNICAL FIELD

[0001] The present disclosure generally relates to probiotic(s) for biological control of pathogenic Vibrio sp. in aquaculture. In particular, it pertains to a newly isolated Vibrio sp. that can degrade quorum-sensing signal molecules of the pathogenic bacteria Vibrio sp. apart from exhibiting advantageous protease activity and its utilization for biological control of pathogenic Vibrio sp., specifically Vibrio harveyi, in aquaculture.

BACKGROUND

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Aquaculture also known as fish or shellfish farming is the breeding, hatchery, rearing and harvesting of plants and animals in all types of water environments including ponds, rivers, lakes and the ocean. It includes production of seafood from hatchery fish and shellfish which are grown to market-size in ponds, tanks, cages, or raceways. Around the world, the demand for seafood has increased greatly because people have learned that the seafood in regular diet confer health benefits. Aquaculture provides almost half of the world's fish for human consumption. In 2012, the total world production of fisheries was 158 million tonnes of which aquaculture contributed to 66.6 million tonnes, about 42 percent. The growth rate of worldwide aquaculture has been sustained and rapid, averaging about 8 percent per annum for over thirty years, while the take from wild fisheries has been essentially flat for the last decade.

[0004] However, intensive culture of crustaceans, especially, intensive shrimp culture has led to outbreaks of various diseases, resulting in annual economic losses to the aquaculture industry estimated at billions of dollars worldwide. Major pathogens that are affecting the aquaculture industry include: bacteria, fungi, viruses and parasites. Given that bacteria can survive well in aquatic environment independently of their hosts, bacterial diseases have become major impediments to the aquaculture. For instance, disease is now considered to be the major limiting factor in the shrimp culture sector. [0005] Vibrio species includes some of the most potent pathogens, Vibrio harveyi amongst them being the most prominent pathogen of marine vertebrates and invertebrates. They can cause bacterial infections in fish and shrimp aquaculture farms, and can cause infection and toxicity as a primary problem and production of biofilms and bioluminescence as a secondary problem. They have a "stimulate and respond system" correlated to population density called quorum sensing system. They produce and release low molecular weight signal molecules called autoinducers (AIs) or pheromones. They also have a receptor that can specifically detect the signalling molecule (inducer). When the inducer (AI) binds the receptor, it activates transcription of certain genes, including those for inducer synthesis. When only a few other bacteria of the same kind are in the vicinity, diffusion reduces the concentration of the inducer in the surrounding medium to almost zero, so the bacteria produce little inducer. However, as the population grows, the concentration of the inducer passes a threshold, causing more inducer to be synthesized. This forms a positive feedback loop, and the receptor becomes fully activated that induces the cells in the population to co- ordinately alter gene expression, including those of bioluminescence, type III secretion system, metalloprotease and the like. Autoinducer concentration reaching above threshold level can also result in the formation of structured biofilm that prevents penetration of antibiotics, blocks the immune defence system and reduces the effects of disinfectants and causes antibiotic resistance.

[0006] In the past, varieties of measures were taken to subside and/or eliminate deleterious effects caused by such bacteria. For example, use of antibiotics and/or disinfectants, improvement of hygiene and bio-security, production of immune stimulants such as glucans and the like. Although several of these means could partly solve the problem, most of them are unsustainable for a long term application. In particular, there is a growing concern about the use of antibiotics in aquaculture due to development of resistance. Unlike antibiotics which target the life or growth of the pathogens, active agents that can target virulence of pathogens have less chance for developing resistance by the pathogen. More recently, utilization of probiotic(s) for biological control of one or more pathogens targeting their virulence (e.g. quorum sensing) has caught significant interest of the researchers.

[0007] Laurent et al (Microbiol. Mol. Biol. Rev., Dec. 2000, Vol. 64, No. 4) discloses advantageous effects exhibited by inclusion/utilization of Lactobacillus, Carnobacterium, Vibrio alginolyticus, Bacillus and Pseudomonas as probiotic(s) in aquaculture. Further, the publication identifies challenges involved in successful development of probiotics applicable to commercial use in aquaculture including the development process being a multistep and multidisciplinary process requiring both empirical and fundamental research, full-scale trials and an economic assessment of its use. The development process further becomes complex as not all strains of a particular genus of microorganism exhibits satisfactory probiotic activity and lack of pathogenicity towards the host.

[0008] Thompson et al (The Society for Applied Microbiology, Journal of Applied Microbiology, 109, 2010, 1177-1187) discloses screening of Vibrio gazogenes (NCIMB 2250), Vibrio mediterranei (NCIMB 13228), Vibrio natriegens (NCIMB 2273), Vibrio orientalis (NCIMB 2195), Vibrio proteolyticus (NCIMB 1326), Vibrio scopthalmi (NCIMB 13623) and Vibrio tubiashii (NCIMB 1336) for their inhibitory activity against V. harveyi, V. alginolyticus and V. anguillarum. The publication discloses the potential for utilization of V. gazogenes together with chitin to improve the health and welfare of shrimp under aquaculture conditions.

[0009] From careful scrutinization of these documents amongst others, a person skilled in the art would immediately realize that barring achievement of sporadic success in identifying few strains across different genus of microorganisms that can be effective in providing biological control of one or more pathogens in aquaculture, no strains (or probiotic compositions comprising such strains) could enjoy the widespread economic utility due to one or more shortcomings associated with such strains and/or probiotic compositions. There is, therefore an urgent need to identify strain(s) that can degrade quorum-sensing signal molecules of the pathogenic bacteria of Vibrio sp. (specifically Vibrio harveyi) and exhibits advantageous proteolytic activity to improve feed conversion ratio (FCR) in shrimps, and to develop probiotic composition(s) including such strain(s) for its utilization in biological control of pathogenic Vibrio sp. in aquaculture at the commercial scale.

OBJECTS OF THE INVENTION

[0010] An object of the present disclosure is to provide a Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio sp.

[0011] Another object of the present disclosure is to provide a Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio harveyi.

[0012] Another object of the present disclosure is to provide a Vibrio strain that can degrade quorum sensing signal molecules of Chromobacterium violaceum.

[0013] Another object of the present disclosure is to provide a non-pathogenic Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio harveyi. [0014] Another object of the present disclosure is to provide a Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio sp. to inhibit bioluminescence and to reduce its virulence.

[0015] Another object of the present disclosure is to provide a Vibrio strain that exhibits protease activity.

[0016] Another object of the present disclosure is to provide a Vibrio strain that improves feed conversion ratio (FCR) in shrimp aquaculture.

[0017] Another object of the present disclosure is to provide a probiotic composition including the Vibrio strain.

[0018] Another object of the present disclosure is to provide a feed additive including the Vibrio strain.

[0019] Another object of the present disclosure is to provide a method for culturing crustaceans using the Vibrio strain.

[0020] Another object of the present disclosure is to provide a method for degrading quorum sensing signal molecules of Vibrio harveyi and reducing the virulence of Vibrio harveyi by addition of the Vibrio strain.

[0021] Another object of the present disclosure is to provide a method for degrading quorum sensing signal molecules of Vibrio harveyi and reducing bioluminescence of Vibrio harveyi by addition of the probiotic composition including Vibrio strain.

[0022] Various objects, features, aspects and advantages of the present invention will become more apparent from the detailed description of the invention herein below along with the accompanying figures in which like numerals represent like components.

SUMMARY

[0023] The present disclosure generally relates to probiotic(s) for biological control of pathogenic Vibrio sp. for their utilization in aquaculture. In particular, it pertains to a newly isolated Vibrio strain that can degrade quorum sensing signal molecules of the pathogenic bacteria Vibrio sp. and exhibits advantageous protease activity, and its utilization for biological control of pathogenic Vibrio sp., specifically Vibrio harveyi, in aquaculture.

[0024] Aspects of the present disclosure provides a novel strain Vibrio sp. DIR21 (accession number MCC 0081) that exhibits quenching of quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture. Further, the strain of Vibrio sp. DIR21 (accession number MCC 0081), in accordance with embodiments of the present disclosure, exhibits improvement in the Feed Conversion Ratio (FCR) of crustaceans by way of its protease activity.

[0025] An aspect of the present disclosure relates to a method of quenching quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture, said method including addition of a strain of Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof in said aquaculture to effect quenching of quorum sensing mechanism of said pathogenic bacteria Vibrio harveyi. In an embodiment, said strain of Vibrio sp. DIR21 or culture broth thereof is formulated into any or a combination of a liquid or semi-solid or dry form, preferably in a liquid or dry powdery form and most preferably in liquid form. In another embodiment, said formulation further includes at least one excipient. In another embodiment, said strain of Vibrio sp. DIR21 or culture broth thereof is added to said aquaculture in a form of feed additive. In another embodiment, said culture broth of strain of Vibrio sp. DIR21 (accession number MCC 0081) is present in a form selected from a group consisting of a concentrate of said culture broth, a diluted form of said culture broth, extract of the said culture broth and a dry product of said culture broth.

[0026] Another aspect of the present disclosure provides a probiotic composition including a strain of Vibrio sp. DIR21 (accession number MCC 0081), wherein said probiotic composition exhibits quenching of quorum sensing mechanism of Vibrio harveyi in an aquaculture. In an embodiment, said composition further includes at least one excipient. In another embodiment, said composition is in a form selected from a group consisting of a liquid or semi-solid or dry form, preferably in a liquid or dry powdery form and most preferably in liquid form. In another embodiment, said strain of Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof further exhibits protease activity.

[0027] Another aspect of the present disclosure provides a composition including a strain of Vibrio sp. DIR21 (accession number MCC 0081), wherein said composition effects quenching of quorum sensing mechanism of Vibrio harveyi in an aquaculture.

[0028] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0030] FIG. 1 illustrates an exemplary photograph depicting quorum quenching in Chromobacterium violaceum by Vibrio sp. DIR21 in accordance with embodiments of the present disclosure.

[0031] FIG. 2 illustrates an exemplary photograph depicting absence of antibiosis on Vibrio harveyi by Vibrio sp. DIR21 in accordance with embodiments of the present disclosure.

[0032] FIG. 3A illustrates an exemplary photograph depicting inoculation (arrow streaking on plates) of various strains of Vibrio sp. to determine their efficacy for bioluminescence quenching of Vibrio harveyi in accordance with embodiments of the present disclosure.

[0033] FIG. 3B illustrates an exemplary bioluminescence image depicting efficacy of various strains of Vibrio sp. for bioluminescence quenching of Vibrio harveyi in accordance with embodiments of the present disclosure.

[0034] FIG. 4 illustrates an exemplary bioluminescence image depicting comparison of efficacy of Vibrio sp. DIR21 with Vibrio parahaemolyticus for quorum quenching of Vibrio harveyi in accordance with embodiments of the present disclosure.

[0035] FIG. 5A illustrates an exemplary photograph depicting absence of haemolytic activity by the Vibrio sp. DIR21 in accordance with embodiments of the present disclosure.

[0036] FIG. 5B illustrates an exemplary image depicting absence of haemolytic activity by the Vibrio sp. DIR21 in accordance with embodiments of the present disclosure.

[0037] FIG. 6 illustrates an exemplary photograph depicting strong protease activity exhibited by the Vibrio sp. DIR21 in accordance with embodiments of the present disclosure.

[0038] FIG. 7 illustrates an exemplary photograph depicting amylase activity exhibited by the Vibrio sp. DIR21 in accordance with embodiments of the present disclosure.

[0039] FIG. 8 illustrates an exemplary graph depicting growth curve for Vibrio sp. DIR21 at different salinity levels in accordance with embodiments of the present disclosure.

[0040] FIG. 9 illustrates an exemplary image depicting purity of the cultures utilized in the experiments in accordance with embodiments of the present disclosure.

[0041] FIG. 10 illustrates an exemplary graph depicting average number of live shrimps under different experimental conditions including live shrimps (post-larvae stage) only (Set

A-l), live shrimps (post-larvae stage) in presence of Vibrio sp. DIR21 (Set B-l), live shrimps

(post-larvae stage) in presence of V. harveyi (Set C-l) and live shrimps (post-larvae stage) in presence of V. harveyi and Vibrio sp. DIR21 (Set D-1) in accordance with embodiments of the present disclosure.

[0042] FIG. 11 illustrates an exemplary graph depicting average of % survivors under different experimental conditions including Negative (Reference) control (Set A-l), Positive control (Set B-l), in presence of V. harveyi only (Set C-l) and in presence of V. harveyi and Vibrio sp. DIR21 (Set D-1) in accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION

[0043] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0044] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.

[0045] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0046] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0047] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0048] The present disclosure generally relates to probiotic(s) for biological control of pathogenic Vibrio sp. for their utilization in aquaculture. In particular, it pertains to a newly isolated Vibrio strain that can degrade quorum sensing signal molecules of the pathogenic bacteria Vibrio sp. and exhibits advantageous protease activity, and its utilization for biological control of Vibrio sp., specifically Vibrio harveyi, in aquaculture. Aspects of the present disclosure provides a novel strain Vibrio sp. DIR21 (accession number MCC 0081) that exhibits quenching of quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture. Further, the strain Vibrio sp. DIR21 (accession number MCC 0081), in accordance with embodiments of the present disclosure, exhibits improvement in the Feed Conversion Ratio (FCR) of crustaceans by way of its protease activity.

[0049] In an embodiment, brackish water lake derived samples were collected in the shrimp farms and cultured in a LB sea agar media (refer to Table 2 for media composition). Then, the colonies were observed for grouping, and strains were isolated. Among the isolated strains, strains showing good quorum quenching activity on the representative pathogenic bacteria attacking the cultured fish and crustaceans, including Aeromonas salmonicida, Vibrio harveyi, Vibrio anguillarum, Edwardsiella tarda, Streptococcus iniae, Vibrio parahaemolyticus , and Vibrio alginolyticus, and preferably Vibrio harveyi were selected by primary screening. The primary screened strains having good quorum quenching activity were then screened for their activity of digestive enzymes such as protease and amylase. Among the secondary screened strains, selected was finally Vibrio sp. DIR21 that modulates quorum sensing activity of Vibrio harveyi and it could be observed that, Vibrio sp. DIR21, in accordance with embodiments of the present disclosure, exhibits significant reduction in virulence of V. harveyi in cultured fish and crustaceans, and especially in shrimps.

[0050] The isolated Vibrio sp. DIR21 (with 16S ribosomal RNA gene sequence as provided in SEQ ID - 1) exhibits 99% homology with Vibrio sp. YASM14 16S ribosomal RNA gene, partial sequence. Although, YASM14 (NCBI Accession number: DQ314529.1) was reported as pathogenic strain against fish, Vibrio sp. DIR21 was surprisingly found to be non-pathogenic against shrimps even after sharing 99% homology with YASM14. Further, Vibrio sp. DIR21 proved to be beneficial to shrimps. Accordingly, the newly isolated Vibrio sp. DIR21 was deposited at the Microbial Culture Collection (MCC), Pune on 16^th of February 2016 as "Vibrio sp. DIR21" (accession number MCC 0081). [0051] An aspect of the present disclosure relates to a method of quenching quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture, said method including addition of a strain Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof in said aquaculture to effect quenching of quorum sensing mechanism of said pathogenic bacteria Vibrio harveyi. In an embodiment, said strain Vibrio sp. DIR21 or culture broth thereof is formulated into any or a combination of a liquid or semi-solid or dry form, preferably in a liquid or dry powdery form and most preferably in liquid form. In another embodiment, said formulation further includes at least one excipient. In another embodiment, said Vibrio sp. DIR21 or culture broth thereof is added to said aquaculture in a form of feed additive. In another embodiment, said culture broth of strain Vibrio sp. DIR21 (accession number MCC 0081) is present in a form selected from a group consisting of a concentrate of said culture broth, diluted form of said culture broth, extract of the said culture broth and a dry product of said culture broth.

[0052] In an embodiment, the present disclosure provides a culture broth of the strain, a concentrate of said culture broth, a diluted form of said culture broth or a dry product thereof. Specifically, the culture broth of the present disclosure means a media where the Vibrio sp. DIR21 was cultured, and preferably a culture medium including the strain. As used herein, the culture medium means a medium including nutrients that are required for culturing animal cells, plant cells or bacteria, and the culture broth means a liquid medium where a strain is inoculated and cultured. The culture broth may be a medium including the strain, or a culture filtrate that is prepared by removing the strain from the culture broth where the strain was inoculated and cultured. The concentrate of the culture broth means those prepared by concentrating the culture broth, and the dry product of the culture broth means those prepared by removing water from the culture broth. The drying method may include air drying, natural drying, spray drying, freeze drying or any other method known to a person skilled in the art.

[0053] Another aspect of the present disclosure provides a probiotic composition including a strain Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof, wherein said probiotic composition exhibits quenching of quorum sensing mechanism of Vibrio harveyi in an aquaculture. In an embodiment, said composition further includes at least one excipient. In another embodiment, said composition is in a form selected from a group consisting of a liquid or semi-solid or dry form, preferably in a liquid or dry powdery form and most preferably in liquid form. In another embodiment, said strain Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof further exhibits protease activity. [0054] Another aspect of the present disclosure provides a composition including Vibrio sp. DIR21 (accession number MCC 0081) or broth culture thereof, wherein said composition effects quenching of quorum sensing mechanism of Vibrio harveyi in an aquaculture.

[0055] In an embodiment, the present disclosure provides a composition including the newly isolated Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof, the concentrate thereof, the diluted form thereof or the dry product thereof as an active ingredient, and at least one excipient. As used herein, the term "excipient" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. For formulation of the composition into a liquid preparation, a pharmaceutically acceptable carrier which is sterile and biocompatible may be used such as saline, sterile water, sea water, buffered saline, albumin infusion solution, dextrose solution, maltodextrin solution, Carboxy methyl cellulose (CMC), gum Arabic, alginate (sodium alginate), glycerol, mixtures of one or more thereof and the like as known to a person skilled in the art. If necessary, other conventional additives can also be added including antioxidants, buffers, bacteriostatic agents, and the like known to a person skilled in the art. In an embodiment, any or a combination of sodium benzoate (0.03% to 0.05%), benzoic acid (0.03% to 0.05%), benzyl alcohol (0.03%), Chlorhexidine digluconate (0.05 to 0.15%), sodium thiosulphate (0.5 to 3%), sodium chloride (0.1 to 5%), sodium citrate (0.5 to 3%), Triclosan (1 to 3 mg/L), Cinnamon oil (0.05 to 0.3 ml/L), Tea Tree Oil (0.05 to 0.3 ml/L), Neam oil (0.05 to 0.3 ml/L), Walnut oil (0.05 to 0.3ml/L), Cinnamaldehyde (0.05 to 0.3ml/L) and methparaben (0.02 to 0.1%) can be utilized to exert the microbicidal or microbistatic effect. However, a person skilled in the art should appreciate that these agents can be used in any other concentration to serve its intended purpose without departing from the scope and spirit of the present invention. Alternatively, any other microbicidal or microbistatic agents, as known to or appreciated by a person skilled in the pertinent art, can be used in the composition without departing from the scope and spirit of the present invention. In an embodiment, any or a combination of gum arabic, carboxymethyl cellulose, xanthan gum, guar gum, sodium alginate and glycerol can be used as thickeners in the composition. In an embodiment, thickeners can be present in about 1% to about 4% of the final composition. Further, diluents, dispersants, surfactants, binders and lubricants can be additionally added to the composition to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions, or pills, capsules, granules, or tablets. [0056] In an embodiment, the composition includes about 5% V/V to about 30% V/V of culture broth with Vibrio sp. DIR21, about 5 g/L to about 20 g/L trisodium citrate, about 5 g/L to about 25 g/L carboxy methyl cellulose, about 0.05 ml/L to about 0.5 ml/L cinnamaldehyde, about 50 mg/L to about 200 mg/L tartrazine and 3% NaCl solution in water to make up the final volume. In a preferred embodiment, the composition comprises 10% V/V of culture broth with Vibrio sp. DIR21, 10 g/L trisodium citrate, 15g/L carboxy methyl cellulose, 0.2ml/L cinnamaldehyde, 150mg/L tartrazine and 3% NaCl solution in water to make up the final volume.

[0057] In an embodiment, the composition can find utility as a probiotic composition. Probiotics live in the gastrointestinal tract of the host (fish or crustaceans) to inhibit harmful bacteria and proliferation of pathogenic bacteria. In addition, beneficial digestive enzymes produced by such probiotics can facilitate absorption and utility of nutrients to improve a feed conversion ratio.

[0058] Examples of the oral dosage forms including the Vibrio strain or culture broth thereof of the present disclosure as an active ingredient may include tablets, troches, lozenges, aqueous or emulsive suspensions, powder, liquid, gel or granules, emulsions, hard or soft capsules, syrups, elixirs and the like known to a person skilled in the art. For formulation such as tablets and capsules, useful are a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrant such as corn starch or sweet potato starch, a lubricant such as magnesium stearate, calcium stearate, sodium stearylfumarate, or polyethylene glycol wax. For capsules, a liquid carrier such as a lipid may be further used in addition to the above- mentioned compounds.

[0059] In an embodiment, the present disclosure provides a feed additive including the newly isolated Vibrio sp. strain or the culture broth thereof, the concentrate thereof, diluted form thereof or the dry product thereof. The isolated Vibrio sp. DIR21 may be directly added during the feed preparation. The Vibrio sp. DIR21 in the feed of the present disclosure may be in a liquid or semi-solid or dry form, and preferably in a liquid or dry powdery form. The drying method may include any or a combination of air drying, natural drying, spray drying, freeze drying, and the like as known to a person skilled in the art. The Vibrio sp. DIR21 of the present disclosure may be mixed in a powder or liquid form at any ratio of 0.01% to 99% by weight (or V W as the formulation may be), and preferably at 0.1% to 10% by weight (of V W), based on the feed weight. In addition, the feed for aquaculture may further include other additives (excipients) to improve its preservation, in addition to the Vibrio sp. DIR21 of the present disclosure. The feeds containing the Vibrio sp. DIR21 of the present disclosure may include plant-based feeds such as grains, nuts, food processing byproducts, algae, fibers, oil, starches, meals, and grain byproducts, and animal-based feeds such as proteins, inorganic substances, fat, minerals, fat, single-cell proteins, soybean meal, zooplankton, squid meal and fish meals, but are not limited thereto.

[0060] In an embodiment, the probiotic composition including the Vibrio sp. DIR21 can further include additives (excipients) for preventing quality deterioration, such as binders, emulsifiers and preservatives, and additives (excipients) for increasing utility, such as amino acids, vitamins, enzymes, flavorings, non-protein nitrogen, silicates, buffering agents, extracts, and oligosaccharides, but is not limited thereto. In addition, the probiotic composition including the Vibrio sp. DIR21 may further include feed premixes, but not limited thereto.

[0061] In an embodiment, the present disclosure provides a method for culturing fish or crustaceans, including the step of treating aquaculture farm of fish or crustaceans using the newly isolated Vibrio strain, the culture broth thereof, the concentrate thereof, or the dry product thereof.

[0062] As described above, the newly isolated Vibrio sp. DIR21 of the present disclosure exhibits a quorum quenching activity and can also inhibit symbiosis, virulence, bioluminescence and biofilm formation. Thus, the strain can be used to prevent diseases caused by common pathogenic bacteria in aquaculture, thereby allowing the culturing of fish or crustaceans with safety. The term "aquaculture" as used in the present disclosure generally relates to aqua-farming, the farming of aquatic organisms such as crustaceans, molluscs, aquatic plants and the like (specifically crustaceans) in variety of environments including but not limited to tanks, lakes, ponds, or any other natural or man-made aquatic reservoirs that can be suitable for breeding, hatchery, rearing and harvesting of the said organism(s).

[0063] In an embodiment, the present disclosure provides a method for preventing biofilm formation that is a source of pollution in fish or crustacean farms using the newly isolated Vibrio strain, the culture broth thereof, the concentrate thereof, or the dry product thereof, or a method for preventing diseases caused by pathogens in animals excluding humans using the newly isolated Vibrio strain, the culture broth thereof, the diluent thereof, the concentrate thereof, the derivatives thereof or the dry product thereof.

[0064] In an embodiment, the present disclosure provides an agent for improving water quality, including the newly isolated Vibrio sp. DIR21, the culture broth thereof, the dilutent thereof the concentrate thereof, the derivatives thereof or the dry product thereof. The newly isolated Vibrio sp. DIR21 of the present disclosure inhibits bioluminescence by Vibrio harveyi, reduces virulence of Vibrio harveyi and reduces the content of ammonia present in the aquaculture environment, by the way of augmenting the feed digestion by its protease activity. To improve water quality, the newly isolated Vibrio sp. DIR21 of the present disclosure may be separately prepared in the form of an agent for improving water quality, or the strain and/or the probiotic composition may be directly sprayed. The Vibrio sp. DIR21 in the agent for improving water quality of the present disclosure may be in a liquid or a semisolid or a dry form, and preferably in a liquid or dry powdery form.

[0065] For the agent for improving water quality, a carrier which is sterile and biocompatible may be used such as saline, sea water sterile water, buffered saline, albumin infusion solution, dextrose solution, maltodextrin solution, glycerol, and mixtures of one or more thereof. If necessary, other conventional additives may be added such as antioxidants, buffers, bacteriostatic agents, and the like. Further, diluents, dispersants, surfactants, binders and lubricants may be additionally added to the composition to prepare formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0066] If the agent for improving water quality is used, the water quality of the aquaculture farm can be improved. Specifically, the agent for improving water quality may be added to the aquaculture farm before aquaculture or during aquaculture. Preferably, it may be added to the aquaculture farm before aquaculture and left for a predetermined period. Alternatively, it can be intermittently added to said aquaculture to suit the needs. Consequently, the newly isolated Vibrio sp. DIR21 of the present disclosure can be used to degrade quorum-sensing signal molecules, thereby inhibiting bioluminescence, virulence and biofilm formation. In addition, the agent for improving water quality can be added during aquaculture once or more so as to prevent additional biofilm formation and to further reduce bioluminescence and virulence.

[0067] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art. EXAMPLES

[0068] Example 1 - Quorum quenching assay using reporter strain Chromobacterium violaceum.

[0069] Culture media to be used in the experiment was prepared in required quantity according to the composition as provided in Table 1. The culture media was then sterilized by autoclaving and poured in the Petri dishes/plates. In the experiment, Chromobacterium violaceum was used as the reporter strain that exhibits intense violet pigmentation through quorum sensing. NG-9 (a Vibrio sp.) was utilized as a negative control. The culture media was arrow streaked with Chromobacterium violaceum (shown as 102 in Figure 1), Chromobacterium violaceum & NG-9 (shown as 104 in Figure 1) and Chromobacterium violaceum & Vibrio strain DIR21 (shown as 106 in Figure 1) as illustrated in Figure 1. The inoculated Petri dishes were kept in the incubation chamber at 25 °C and intensity of colour (violet pigmentation) was observed after 30 hrs. Figure 1 illustrates an exemplary photograph depicting quorum quenching in Chromobacterium violaceum by Vibrio sp. DIR21. As can be observed, intensity of the violet color in Chromobacterium violaceum streaked in vicinity of Vibrio sp. DIR21 (shown as 106 in Figure 1) is very low in comparison to the intensity of the violet color in Chromobacterium violaceum streaked in vicinity of NG-9 (negative control) or to the intensity of violet colour in Chromobacterium violaceum streaked alone. Results of this experiment clearly indicate quorum quenching ability of Vibrio sp. DIR21 against Chromobacterium violaceum.

Table 1: Composition of culture media used for Quorum quenching assay

[0070] Example 2 - Assay for antagonism against Vibrio harveyi

[0071] Culture media to be used in the experiment was prepared in required quantity according to the composition as provided in Table 2. The culture media was then sterilized by autoclaving and poured in the Petri dishes/plates. As illustrated in Figure 2, Vibrio sp. DIR21 was streaked in the culture media and incubated at Room Temperature (25 °C) for 24 hrs. After 24 hrs, Vibrio harveyi was streaked (204 of Figure 2) perpendicularly to (without touching) Vibrio sp. DIR21 (202 of Figure 2) and the plate was incubated at 25^°C for 12 hrs. Results of the experiment, as illustrated in Figure 2, confirms that Vibrio sp. DIR21 does not interfere with growth of Vibrio harveyi i.e. absence of antibiosis against Vibrio harveyi by Vibrio sp. DIR21. Rather, Vibrio sp. DIR21 controls the Vibrio harveyi infection only through quorum quenching.

Table 2: Composition of culture media used for antagonism assay

[0072] Example 3 - Screening of different strains of Vibrio sp. to determine their efficacy for bioluminescence quenching of Vibrio harveyi.

[0073] Culture media to be used in the experiment was prepared in required quantity according to the composition as provided in Table 3A and 3B. Autoclave the "Part A" of the media and to the sterile "Part A" of the medium add the below sterile medium components (from Table 3B) individually before "Part A" of the medium solidifies. Mix the contents gently and pour onto sterile petri plates.

Table 3 - Composition of culture media used for bioluminescence quenching assay

Table 3A

Part A

Components Quantity (for 1 Litre of culture medium)

Peptone 10 gram

Yeast extract 5 gram

Sodium sulphate 3.25 gram

Calcium chloride 1.8 gram

Potassium chloride 0.55 gram Agar 15 gram

Sea water (filtered) Make up the volume till 950 ml

Table 3B

Components (Stock solution prepared in Volume to be added from respective distilled water) stock solution to make 1 Litre of

"culture medium 2" along with "Part

A"

Sodium bicarbonate (32 g/Litre stock solution) 5 ml from stock solution

Potassium Bromide (16 g/Litre stock solution) 5 ml from stock solution

Strontium chloride (hexahydrate) (8.4 g/Litre 5 ml from stock solution

stock solution)

Ammonium chloride (0.4 g/Litre stock solution) 5 ml from stock solution

Sodium nitrate (0.32 g/Litre stock solution) 5 ml from stock solution

Boric acid (4.4 g/Litre stock solution) 5 ml from stock solution

Sodium metasilicate (0.8 g/Litre stock solution) 5 ml from stock solution

Monosodium phosphate (1.6 g/Litre stock 5 ml from stock solution

solution)

Sodium Fluoride (0.48 g/Litre stock solution) 5 ml from stock solution

Ferric citrate (20 g/Litre stock solution) 5 ml from stock solution

[0074] Vibrio sp. to be screened (DIR11, DIR21, DIR13, SP33) were arrow streaked on the culture media and incubated for 24 hrs at 25 ^°C. After 24 hrs incubation, V. harveyi was arrow streaked in vicinity of each Vibrio sp. to be screened. Figure 3A illustrates an exemplary photograph depicting inoculation (by parallel arrow streak method) of various strains of Vibrio sp. to determine their efficacy for bioluminescence quenching of Vibrio harveyi. As illustrated, 302 indicates arrow streak of V. harveyi only, 304 indicates arrow streaks of V. harveyi and Negative control (this negative control was obtained while screening for the probiotic), 306 indicates arrow streaks of V. harveyi and SP33, 308 indicates arrow streaks of V. harveyi and Vibrio sp. DIR21, 310 indicates arrow streaks of V. harveyi and Vibrio sp. DIR11 and 312 indicates arrow streaks of V. harveyi and Vibrio sp. DIR13. Figure 3B illustrates an exemplary bioluminescence image depicting efficacy of various strains of Vibrio sp. for bioluminescence quenching of Vibrio harveyi. As can be inferred from the bioluminescence image shown in Figure 3B, Vibrio sp. DIR21, shown at 308, exhibits significant reduction in bioluminescence due to quorum quenching in comparison to other strains of Vibrio species.

[0075] Example 4 - Co-culture experiment to validate quenching of bioluminescence of Vibrio harveyi by Vibrio sp. DIR21

[0076] Culture media to be used in the experiment was prepared in required quantity according to the composition as provided in Table 4. 30ml each of culture media was poured in different flasks of 250 ml capacity, covered with cotton plug and autoclaved. One flask with the media was used as a blank (no inoculation). Other flasks were inoculated by addition of inoculums (overnight cultures) such that maximum amount of the inoculum culture does not exceed 10% of the media (i.e. 3 ml inoculums culture addition in 30 ml media). For Vibrio harveyi inoculation onto respective culture medium, overnight culture was inoculated (3.3% of culture medium) only after 2 hours from inoculation of other cultures. Accordingly, one flask was inoculated with Vibrio parahaemylyticus , one flask was inoculated with V. harveyi, one flask was inoculated with Vibrio sp. DIR21, one flask was inoculated with V. harveyi and V. parahaemylyticus (as negative control) and one flask was inoculated with V. harveyi and Vibrio sp. DIR21. All flasks were incubated at 25°C for about 10.5 hours. 1.5ml of the respective culture was separately transferred into separate wells of a black 6 well plate. Figure 4 illustrates an exemplary bioluminescence image depicting the efficacy of Vibrio sp. DIR21 for quenching the bioluminescence of Vibrio harveyi. As illustrated, 406 indicates sterile culture media, 412 indicates culture of V. harveyi, 404 indicates culture of V. parahaemolyticus , 402 indicates culture of V. parahaemolyticus and V. harveyi, 410 indicates culture of Vibrio sp. DIR21, and 408 indicates the culture of Vibrio sp. DIR21 and V. harveyi. As can be inferred from the bioluminescence image as illustrated in Figure 4, Vibrio sp. DIR21 exhibits excellent quenching of bioluminescence of Vibrio harveyi.

Table 4 Composition of culture media for quenching of bioluminescence in co- culture assay

Components Quantity (for 1 Litre of culture medium)

Peptone 10 gram

Yeast extract 5 gram

Sea Water (filtered) Make up to 1 litre [0077] Example 5 - Assay for confirmation of absence of haemolytic activity by Vibrio sp. DIR21

[0078] Sterilize the media components (Table 5) by autoclaving. Bring the media to hand bearable warm condition (care should be taken that media does not get solidified before pouring onto petri plates) and add 75 ml of anti -coagulated (with 4.5 mM of EDTA) goat blood. Gently swirl to mix and pour onto sterile petri plates. Then Vibrio sp. DIR21 and V. harveyi were streaked onto culture media, as illustrated in Figure 5 A, and incubated at 25 ^°C for 36 hrs. As illustrated, 502 indicates V. harveyi and 504 indicates Vibrio sp. DIR21. Figure 5A illustrates an exemplary photograph depicting absence of haemolytic activity by the Vibrio sp. DIR21. FIG. 5B illustrates an exemplary image depicting absence of haemolytic activity by the Vibrio sp. DIR21. As can be inferred from Figure 5A and Figure 5B, Vibrio sp. DIR21 exhibits no haemolytic activity qualifying for its utility in probiotic composition(s) for aquaculture.

Table 5 Composition of culture media for haemolytic activity

[0079] Example 6 - Assay for protease activity of Vibrio sp. DIR21.

[0080] The culture media for protease assay was prepared according to the composition as provided in Table 6. The media content was Sterilized by autoclaving. When the above medium was cooled down to hand bearable temperature, pH of the medium was adjusted to 8 using IN NaOH. lOg/Litre casein was added and the medium was gently heated in boiling water for 5 minute, the medium was poured onto sterile petri plates. Overnight cultures were streaked onto culture media and observed after 42 hours of incubation at 25^°C. Figure 6 illustrates an exemplary photograph depicting strong protease activity exhibited by the Vibrio sp. DIR21. As illustrated, 602 indicates Vibrio sp. DIR21 and 604 indicates V. harveyi. Table 6 Composition of culture media for protease assay

[0081] Example 7 - Assay for amylase activity of Vibrio sp. DIR21

[0082] The culture media for amylase activity assay was prepared according to the composition as provided in Table 7. the above media content was gently heated in boiling water for 10 minutes. When the starch was dissolved completely, the medium was sterilized by autoclaving. the medium was poured onto sterile petri plates for utilization in the experiment. Overnight cultures were streaked onto culture media and observed after 42 hrs of incubation at 25 ^°C. Finally, the plate was flooded with 1% iodine solution. Figure 7 illustrates an exemplary photograph depicting amylase activity exhibited by the Vibrio sp. DIR21. As illustrated, 702 indicates Vibrio sp. DIR21 and 704 indicates V. harveyi.

Table 7 Composition of culture media for amylase activity

[0083] Example 8 - Assay for growth of Vibrio sp. DIR21 at different salinity.

[0084] Base media was prepared by addition of 3g peptone and 1.5g yeast extract in 300ml distilled water and 50ml was transferred to 6 different 250ml flasks. Then, NaCl was added to each flask to make required salt concentration (e.g. 0.1%, 0.3%, 1%, 3%, 4% and 5%). Initial Target OD of 0.2 was achieved by inoculating the culture (containing Vibrio sp. DIR21) to fresh media. Mother culture was prepared in said base media with 3% NaCl. Spectrophotometer readings were taken in triplicate. Optical Density of the media at different salt concentration at different time points during incubation at 25 ^°C was noted and the same are provided in Table 8. Average Optical Density (OD) at different salt concentration (salinity) is shown in Table 9 below.

Table 8 - Optical Density (OD) at different salt concentration (salinity)

NaCl Time Points (in hrs)

Concentration 0 2 5 8 9 11 13 15 17 25 (%)

0.193 0.207 0.267 0.356 0.387 0.42 0.455 0.468 0.468 0.481

0.1 0.193 0.207 0.267 0.337 0.387 0.42 0.455 0.443 0.468 0.481

0.193 0.2 0.267 0.327 0.376 0.408 0.455 0.42 0.468 0.481

Average 0.193 0.204 0.267 0.34 0.383 0.416 0.455 0.442 0.468 0.481

0.187 0.221 0.318 0.42 0.481 0.508 0.552 0.537 0.568 0.568

0.3 0.187 0.221 0.318 0.408 0.481 0.508 0.552 0.522 0.568 0.568

0.193 0.221 0.301 0.397 0.468 0.494 0.552 0.508 0.568 0.568 average 0.189 0.221 0.312 0.408 0.476 0.503 0.552 0.522 0.568 0.568

0.193 0.284 0.431 0.568 0.657 0.657 0.721 0.698 0.744 0.823

1 0.193 0.275 0.42 0.552 0.638 0.657 0.721 0.698 0.744 0.823

0.193 0.284 0.42 0.537 0.638 0.657 0.721 0.698 0.744 0.823 average 0.193 0.281 0.423 0.552 0.644 0.657 0.721 0.698 0.744 0.823

0.18 0.267 0.468 0.619 0.721 0.744 0.795 0.769 0.853 1.238

3 0.18 0.284 0.468 0.619 0.721 0.721 0.823 0.744 0.853 1.238

0.18 0.275 0.443 0.619 0.721 0.721 0.823 0.744 0.853 1.238 average 0.18 0.275 0.459 0.619 0.721 0.728 0.813 0.752 0.853 1.276

0.2 0.2 0.337 0.443 0.537 0.568 0.638 0.602 0.677 0.853

4 0.2 0.2 0.327 0.443 0.537 0.568 0.638 0.602 0.657 0.853

0.193 0.2 0.337 0.443 0.552 0.552 0.619 0.602 0.657 0.853 average 0.197 0.2 0.333 0.443 0.542 0.562 0.631 0.602 0.663 0.853

0.2 0.167 0.229 0.275 0.366 0.337 0.387 0.356 0.42 0.522

5 0.2 0.173 0.236 0.284 0.356 0.337 0.387 0.356 0.42 0.522

0.2 0.173 0.236 0.284 0.366 0.337 0.397 0.356 0.408 0.522 average 0.2 0.171 0.233 0.281 0.362 0.337 0.390 0.356 0.416 0.522 Table 9 - Average Optical Density (OD) at different salt concentration (salinity)

[0085] Figure 8 illustrates an exemplary graph depicting growth curve for Vibrio sp. DIR21 at different salinity levels. As can be inferred from the graph, salt concentration ranging from about 0.3% (or 3 parts per thousand (ppt) or 3 gram/L) to about 4% (or 40 parts per thousand or 40 gram/L) can support good growth for Vibrio sp. DIR21 and maximum growth was achieved at 30 parts per thousand (3%) among the conditions tested. Further, it could be inferred that Vibrio sp. DIR21 shows wide range of salt tolerance, which is desirable property in terms of its utilization at commercial scale as it can work for shrimp farms with salinity level ranging from 3ppt to 40ppt, sufficient to cover most of the aquaculture pond salinities. During the experiment, purity of the culture was validated by performing gram staining. Figure 9 illustrates an exemplary image depicting purity of the cultures (gram negative bacteria, 30 ppt salinity) utilized in the experiments.

[0086] Example 9 - In vivo experiment of Vibrio sp. DIR21 with Shrimp at PL (post larvae) stage.

[0087] Shrimps at PL 12 (post larvae day 12) stage were received and maintained at 18 ppt (pars per thousand) salinity level in an aquaculture. Shrimps were fed with the commercial feed twice a day. After 4 days of adaptation, approximately 50 shrimps were transferred to different cylinders (tanks) i.e. Set A, Set B, Set C and Set D (experiment conducted in triplicate) each containing approximately 5.5L water with 18 ppt salinity. Each tank/cylinder was sufficiently aerated using an air pump and each tank/cylinder was covered to prevent entry of any particulate matters and spillage. Detail of the experimental setup is as provided in Table 10 bel Table 10 - Experimental details for In-vivo test

[0088] Vibrio sp. DIR21 culture was grown for 26hrs in conical flask in culture media 3 (Refer to Table 4 for media composition) at room temperature. OD₆₀₀ reading of 26hr culture was 1.442. 35 ml (0.64%) of this culture was added to specific cylinders/tanks (Set B & Set D) with shrimp. For cylinders/tanks in which culture is not added, corresponding quantity of LB sea media was added. After 24 hrs, shrimps were challenged with culture containing V. harveyi. Overnight culture (16 hrs) of V. harveyi in LB sea broth (refer Table 4 for media composition) with OD600 of 0.236 was added in an amount of 5.5ml (to achieve 0.1% final concentration) to the cylinders/tanks labelled as Set C and Set D. Further, 5.5ml of sterile media was added to the cylinders/tanks labelled as Set A and Set B. The in vivo study was continued by feeding them with commercial feed, as per requirement of shrimp feeding pattern (but equal quantity of feed added to all test cylinders, to keep uniformity in quantity of feed provided). Feeding was done twice a day. Aeration was given all throughout the study. No water exchange was done throughout the study. After 90 hrs, number of shrimps survived during the study was counted in each tank, details of which are as provided in the Table 11 below.

Table 11 - Results of the In-vivo test

s. Set No. No. of shrimps No. of shrimps Avg. SD of %

No. (Initial seeding) (survivors) surviv of % survivors ors surviv

ors

Cou Avg Std. Cou Avg. Std.

nt Dev. nt Dev.

1 Set A - 1 56 51 5.567 56 46.3 8.504 100 90.52 8.769 (triplicate 1)

2 Set A - 1 45 40 88.88

(triplicate 2)

3 Set A - 1 52 43 82.69

(triplicate 3)

4 Set B - 1 47 49. 2.081 39 43 3.464 82.97 87.07 3.652 (triplicate 1) 3

5 Set B - 1 51 45 88.23

(triplicate 2)

6 Set B - 1 50 45 90

(triplicate 3)

7 Set C - 1 47 49. 3.214 22 24 5.291 46.8 48.35 7.588 (triplicate 1) 3

8 Set C - 1 48 20 41.66

(triplicate 2)

9 Set C - 1 53 30 56.6

(triplicate 3)

10 Set D - 1 49 52. 3.055 45 43.6 5.131 91.83 83.6 10.559 (triplicate 1) 3

11 Set D - 1 55 48 87.72

(triplicate 2)

12 Set D - 1 53 38 71.69

(triplicate 3)

[0089] FIG. 11 illustrates an exemplary graph depicting average number of live shrimps under different experimental conditions including Negative control (Set A-1), Positive control (Set B-l), in presence of V. harveyi only (Set C-l) and in presence of V. harveyi and Vibrio sp. DIR21 (Set D-1). FIG. 12 illustrates an exemplary graph depicting average of % survivors under different experimental conditions including Negative control (Set A-1), Positive control (Set B-l), in presence of V. harveyi only (Set C-l) and in presence of V. harveyi and Vibrio sp. DIR21 (Set D-1). Finally, it could be concluded that the Vibrio sp. DIR21 provides protections to the shrimps against Vibrio harveyi. [0090] Further, it could be noted during the In-vivo test that presence of Vibrio sp. DIR21 improves FCR (Feed Conversion Ratio) as feed left over in Vibrio sp. DIR21 treated shrimp culture was l/3^rd less when compared to negative control (without Vibrio sp. DIR21). This observation affirms that the Vibrio sp. DIR21 improves digestion of shrimps.

ADVANTAGES OF THE INVENTION

[0091] The present disclosure provides a Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio sp.

[0092] The present disclosure provides a Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio harveyi.

[0093] The present disclosure provides a non-pathogenic Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio harveyi.

[0094] The present disclosure provides a Vibrio strain that can degrade quorum sensing signal molecules of pathogenic bacteria Vibrio sp. and inhibits biofilm formation, bioluminescence and virulence.

[0095] The present disclosure provides a Vibrio strain that exhibits protease activity.

[0096] The present disclosure provides a Vibrio strain that improves feed conversion ratio in shrimp aquaculture.

[0097] The present disclosure provides a probiotic composition including the Vibrio strain.

[0098] The present disclosure provides a feed additive including the Vibrio strain.

[0099] The present disclosure provides a method for culturing crustaceans using the Vibrio strain.

[00100] The present disclosure provides a method for degrading quorum sensing signal molecules of Vibrio harveyi and for inhibiting bioluminescence and virulence by addition of the Vibrio strain.

[00101] The present disclosure provides a method for degrading quorum sensing signal molecules of Vibrio harveyi and inhibiting virulence, bioluminescence and biofilm formation by addition of the probiotic including Vibrio strain. SEQUENCE LISTING

<110> KUMAR, Mohanraj

<120> PROBIOΉCS FOR BIOLOGICAL CONTROL OF PATHOGENIC VIBRIO SP. IN AQUACULTURE

<160> 1

<210> 1

<211> 1510

<212> RNA

<213> Vibrio sp. DIR21

<400> 1

TAGAGTTTTG ATCATGGCTC AGATTGAACG CTGGCGGCAG GCCTAACACA TGCAAGTCGA 60

GCGGAAACGA GTTATCTGAA CCTTCGGGGA ACGATAACGG CGTCGAGCGG CGGACGGGTG 120

AGTAATGCCT AGGAAATTGC CCTGATGTGG GGGATAACCA TTGGAAACGA TGGCTAATAC 180

CGCATAATGC CTACGGGCCA AAGAGGGGGA CCTTCGGGCC TCTCGCGTCA GGATATGCCT 240

AGGTGGGATT AGCTAGTTGG TGAGGTAATG GCTCACCAAG GCGACGATCC CTAGCTGGTC 300

TGAGAGGATG ATCAGCCACA CTGGAACTGA GACACGGTCC AGACTCCTAC GGGAGGCAGC 360 AGTGGGGAAT ATTGCACAAT GGGCGCAAGC CTGATGCAGC CATGCCGCGT GTGTGAAGAA 420

GGCCTTCGGG TTGTAAAGCA CTTTCAGTCG TGAGGAAGGT AGTGTAGTTA ATAGCTGCAT 480

TATTTGACGT TAGCGACAGA AGAAGCACCG GCTAACTCCG TGCCAGCAGC CGCGGTAATA 540

CGGAGGGTGC GAGCGTTAAT CGGAATTACT GGGCGTAAAG CGCATGCAGG TGGTTTGTTA 600

AGTCAGATGT GAAAGCCCGG GGCTCAACCT CGGAATTGCA TTTGAAACTG GCAGACTAGA 660

GTACTGTAGA GGGGGGTAGA ATTTCAGGTG TAGCGGTGAA ATGCGTAGAG ATCTGAAGGA 720

ATACCGGTGG CGAAGGCGGC CCCCTGGACA GATACTGACA CTCAGATGCG AAAGCGTGGG 780

GAGCAAACAG GATTAGATAC CCTGGTAGTC CACGCCGTAA ACGATGTCTA CTTGGAGGTT 840

GTGGCCTTGA GCCGTGGCTT TCGGAGCTAA CGCGTTAAGT AGACCGCCTG GGGAGTACGG 900

TCGCAAGATT AAAACTCAAA TGAATTGACG GGGGCCCGCA CAAGCGGTGG AGCATGTGGT 960

TTAATTCGAT GCAACGCGAA GAACCTTACC TACTCTTGAC ATCCAGAGAA CTTTTCAGAG 1020 ATGAATTGGT GCCTTCGGGA ACTCTGAGAC AGGTGCTGCA TGGCTGTCGT CAGCTCGTGT 1080

TGTGAAATGT TGGGTTAAGT CCCGCAACGA GCGCAACCCT TATCCTTGTT TGCCAGCGAG 1140

TAATGTCGGG AACTCCAGGG AGACTGCCGG TGATAAACCG GAGGAAGGTG GGGACGACGT 1200

CAAGTCATCA TGGCCCTTAC GAGTAGGGCT ACACACGTGC TACAATGGCG CATACAGAGG 1260

GCAGCCAACT TGCGAGAGTG AGCGAATCCC AAAAAGTGCG TCGTAGTCCG GATCGGAGTC 1320

TGCAACTCGA CTCCGTGAAG TCGGAATCGC TAGTAATCGT GGATCAGAAT GCCACGGTGA 1380

ATACGTTCCC GGGCCTTGTA CACACCGCCC GTCACACCAT GGGAGTGGGC TGCAAAAGAA 1440

GTAGGTAGTT TAACCTTCGG GAGGACGCTT ACCACTTTGT GGTTCATGAC TGGGGTGAAG 1500

TCGTACAGGG 1510

Claims

We Claim:

1. An isolated Vibrio strain DIR21 having accession number MCC0081.

2. A composition comprising an isolated Vibrio strain as claimed in claim 1.

3. The composition as claimed in claim 2, wherein said composition is in form of a solid, a liquid, or a semi-solid.

4. The composition as claimed in any of the claims 2 and 3, wherein said composition further comprises at least one additive.

5. A method of modulating feed conversion ratio in an aquaculture, said method comprising the step of contacting a strain Vibrio sp. DIR21 (accession number MCC 0081) as claimed in claim 1 or culture broth thereof with said aquaculture to modulate feed conversion ratio there within.

6. The method as claimed in claim 5, wherein said aquaculture comprises any or a combination of fish and crustacean, and wherein said crustacean comprises shrimp.

7. A method of inhibiting mortality rate of any or a combination of fish and crustaceans in an aquaculture, the method comprising the step of contacting a strain Vibrio sp. DIR21 (accession number MCC 0081) as claimed in claim 1 or culture broth thereof with said aquaculture to inhibit mortality rate thereof.

8. The method as claimed in claim 7, wherein said crustacean comprises shrimp.

9. A method of quenching quorum sensing mechanism of a pathogenic bacteria Vibrio harveyi in an aquaculture, said method comprising the step of contacting a strain Vibrio sp. DIR21 (accession number MCC 0081) or culture broth thereof with said aquaculture containing the pathogenic bacteria Vibrio harveyi to effect quenching of quorum sensing mechanism of the pathogenic bacteria Vibrio harveyi.

10. The method of claim 9, wherein said culture broth of strain Vibrio sp. DIR21 (accession number MCC 0081) is present in a form selected from a group consisting of a concentrate of said culture broth, a diluted form of said culture broth, a dry product of said culture broth and an extract of said culture broth.