Microorganisms useful for fish skin probiotics

Abstract

The present disclosure provides novel microorganisms that protect fish. In one aspect, the present disclosure provides a method for acquiring microorganisms having the capability to protect fish. In one aspect, the present disclosure provides: microorganisms, or the product thereof, having the capability to protect fish; and a composition including the microorganisms or the product thereof. In one embodiment, the microorganisms having the capability to protect fish can be bacteria of the genus pseudomonas. In one aspect, the present disclosure provides: microorganisms, or the product thereof, having the capability to protect fish; and a method for using a composition including the microorganisms or the product thereof to protect the fish.

Description

[Technical Field]

[0001] The present disclosure relates to microorganisms having the ability to protect fish, methods for obtaining such microorganisms, and uses of such microorganisms. [Background technology]

[0002] Antibiotics are attracting international attention as something that should be reduced due to concerns about the emergence of multidrug-resistant bacteria and environmental pollution. In research into disease prevention in farmed fish, attention has been focused on the development of fish disease vaccines as an alternative to antibiotics (Non-Patent Document 1). Although fish disease vaccines have proven effective, they are only effective against specific pathogens in specific fish species and cannot cover a wide range of fish species or pathogens, and the types of vaccines approved for use in the field are limited, making this method insufficient. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ministry of Agriculture, Forestry and Fisheries, Food Safety and Consumer Affairs Bureau, Livestock and Fisheries Safety Management Division, "Regarding the Use of Fisheries Pharmaceuticals," Report No. 29 (January 31, 2016) Summary of the Invention [Means for solving the problem]

[0004] As a result of extensive research, the present inventors have developed a method for obtaining microorganisms capable of protecting fish (e.g., the ability to function as epidermal probiotics for fish) and have found that the microorganisms thus obtained are useful for protecting fish. Microorganisms that may be useful for protecting fish include bacteria of the Pseudomonadaceae family. It has also been found that these microorganisms can colonize fish and / or suppress harmful microorganisms. Therefore, the present disclosure provides the microorganisms thus discovered, their derivatives, and uses thereof. The present disclosure also provides a method for obtaining microorganisms capable of protecting fish.

[0005] Thus, the present disclosure provides: (Item 1) A Pseudomonadaceae bacterium with the ability to protect fish. (Item 2) Item 1. The Pseudomonadaceae bacterium according to item 1, which has the ability to function as a probiotic for fish. (Item 3) 3. The Pseudomonadaceae bacterium according to item 1 or 2, which has the ability to function as a probiotic for fish epidermis. (Item 4) 4. The Pseudomonadaceae bacterium according to any one of items 1 to 3, which has the ability to colonize the epidermis of fish. (Item 5) 5. The Pseudomonadaceae bacterium according to any one of items 1 to 4, which has the ability to improve the survival rate of fish. (Item 6) 6. The Pseudomonadaceae bacterium according to any one of items 1 to 5, wherein the fish are protected by adding the Pseudomonadaceae bacterium to a rearing environment (for example, a rearing liquid, rearing water, etc.) of the fish. (Item 7) 7. The Pseudomonadaceae bacterium according to any one of items 1 to 6, which has the ability to inhibit at least one harmful microorganism. (Item 8) 8. The Pseudomonadaceae bacterium according to Item 7, wherein the harmful microorganism has at least one of the following abilities: an ability to cause a skin disease in fish; an ability to infect fish through the skin; an ability to infect fish through a wound; and an ability to infect fish through contact. (Item 9) 9. The Pseudomonadaceae bacterium according to item 7 or 8, wherein the harmful microorganism is capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, pseudomonadosis of sweetfish, red mouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. (Item 10) The harmful microorganisms include Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas anguilliseptica, Pseudomonas plecoglossicida, Yersinia ruckeri, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum, Tenacibaculum maritimum, Renibacterium salmoninarum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia seriolae, Streptococcus iniae, Lactococcus garvieae, Aeromonas 9. The Pseudomonadaceae bacterium according to item 7 or 8, comprising Pseudomonadaceae, ... (Item 11) The Pseudomonadaceae bacterium according to Item 7 or 8, wherein the harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium columnare, Vibrio anguillarum, Vibrio ordalii, Streptococcus iniae, or a combination thereof. (Item 12) 12. The Pseudomonadaceae bacterium according to any one of items 1 to 11, wherein the performance of the bacterium is determined using zebrafish (Danio rerio) as a standard fish. (Item 13) 13. The Pseudomonadaceae bacterium according to any one of items 1 to 12, wherein the fish is a farmed fish. (Item 14) Item 14. The Pseudomonadaceae bacterium according to Item 13, wherein the farmed fish is eel, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury, or catfish. (Item 15) Item 14. The Pseudomonadaceae bacterium according to Item 13, wherein the farmed fish is a salmonid. (Item 16) Item 14. The Pseudomonadaceae bacterium according to Item 13, wherein the farmed fish is trout. (Item 17) 17. The Pseudomonadaceae bacterium according to any one of items 1 to 16, which is a bacterium of the genus Pseudomonas. (Item 18) 17. The Pseudomonadaceae bacterium according to any one of items 1 to 16, which belongs to the Pseudomonas putida group. (Item 19) 17. The Pseudomonadaceae bacterium according to any one of items 1 to 16, which is Pseudomonas mosselii. (Item 20) KH-ZF1 (accession number: NITE BP-02967), a Pseudomonadaceae bacterium. (Item 21) A fish protecting agent comprising the Pseudomonadaceae bacterium according to any one of items 1 to 20. (Item 22) A probiotic agent for fish, comprising the Pseudomonadaceae bacterium according to any one of items 1 to 20. (Item 23) 23. The probiotic agent according to item 22, which is a fish skin probiotic. (Item 24) 24. The probiotic agent according to Item 22 or 23, wherein the Pseudomonadaceae bacterium has the ability to colonize the epidermis of fish. (Item 25) The fish protecting agent according to Item 21 or the probiotic agent according to any one of Items 22 to 24, which is a protecting agent or probiotic agent for farmed fish. (Item 26) The fish protective agent according to Item 21 or the probiotic agent according to any one of Items 22 to 24, which is a protective agent or probiotic agent for eel, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury, or catfish. (Item 27) The fish protecting agent according to Item 21 or the probiotic agent according to any one of Items 22 to 24, which is a protecting agent or probiotic agent for salmonid fish. (Item 28) The fish protecting agent according to Item 21 or the probiotic agent according to any one of Items 22 to 24, which is a protecting agent or probiotic agent for trout. (Item 29) 21. A method for protecting fish, comprising the step of contacting said fish with the Pseudomonadaceae bacterium according to any one of items 1 to 20. (Item 30) 21. A method for protecting fish, comprising the step of growing the fish in water in which the Pseudomonadaceae bacterium according to any one of items 1 to 20 is present. (Item 31) 1. A method for obtaining microorganisms capable of protecting fish, comprising: (a) obtaining candidate microorganisms from the epidermis of fish; (b) adding the candidate microorganism to a medium containing harmful microorganisms; (c) confirming the inhibition of the harmful microorganism in the culture medium; and (d) when the inhibition of the harmful microorganism in the culture medium is confirmed, a step of identifying the candidate microorganism as a microorganism capable of protecting the fish; A method comprising: (Item 32) 32. The method according to Item 31, wherein in step (c), the inhibition of the harmful microorganisms on the medium is confirmed. (Item 33) Item 32. The method according to Item 31, wherein in step (c), the candidate microorganism and the harmful microorganism are grown on the same medium (e.g., an agar medium or a medium equivalent thereto (e.g., gellan gum)), and a growth inhibition zone in which the harmful microorganism cannot grow is confirmed near the candidate microorganism. (Item 34) 32. The method according to Item 31, wherein in step (c), the inhibition of the harmful microorganism is confirmed in a liquid medium containing the candidate microorganism or its culture supernatant and the harmful microorganism. (Item 35) Item 32. The method according to Item 31, wherein in the step (c), an impregnated product of a culture medium or a culture supernatant of the candidate microorganism or a bottomless cylinder containing the culture medium or the culture supernatant is placed on a medium (e.g., an agar medium or an equivalent medium (e.g., gellan gum)) inoculated entirely with the harmful microorganism, and the inhibition of the harmful microorganism around the impregnated product or the cylinder is confirmed. (Item 36) Item 32. The method according to Item 31, wherein in step (c), after incubating the liquid medium containing the candidate microorganism and the harmful microorganism, the growth of the candidate microorganism and the harmful microorganism are compared to confirm that the growth of the harmful microorganism is more inhibited. (Item 37) Item 32. The method according to Item 31, wherein in the step (c), the candidate microorganism is inoculated onto a medium (e.g., an agar medium or an equivalent medium (e.g., gellan gum, etc.)) that has been inoculated entirely with the harmful microorganism, or onto a medium (e.g., an agar medium or an equivalent medium (e.g., gellan gum, etc.)) that has been inoculated entirely with the harmful microorganism, and it is confirmed that the candidate microorganism forms a colony and that a zone of growth inhibition of the harmful microorganism is observed around the colony. (Item 38) 38. The method according to any one of items 31 to 37, wherein the candidate microorganism is obtained from a colony formed on a culture medium inoculated with scrapings of the fish epidermis. (Item 39) 39. The method according to any one of Items 31 to 38, wherein one or more microorganisms are obtained as microorganisms capable of protecting said fish. (Item 40) 40. The method according to any one of Items 31 to 39, wherein the harmful microorganism has at least one of the following abilities: an ability to cause a skin disease in fish; an ability to infect fish through the skin; an ability to infect fish through a wound; and an ability to infect fish through contact. (Item 41) 40. The method according to any one of items 31 to 39, wherein the harmful microorganism is capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, edwardsiellosis, red spot disease, pseudomonadosis of sweetfish, red mouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. (Item 42) 42. The method according to any one of items 31 to 41, wherein the candidate microorganism is obtained from the epidermis of zebrafish (Danio rerio). (Item 43) 43. A microorganism obtained by the method according to any one of Items 31 to 42. (Item 44) Item 44. The microorganism according to item 43, which belongs to the genus Pseudomonas. (Item 45) Item 44. The microorganism according to item 43, which is a Pseudomonas putida group microorganism. (Item 46) 43. A method for protecting fish, comprising the step of contacting said fish with a microorganism obtained by the method according to any one of items 31 to 42. (Item 47) A strain belonging to the Pseudomonadaceae family, The strain is (i) streaking the strain on NB agar medium and culturing it overnight at 28°C; (ii) then streaking the harmful microorganisms vertically from the strain without touching the strain and incubating overnight at 28°C; and (ii) is characterized by the presence of a clear zone extending over an area of ​​10 mm or more from the streak of the strain, when tested in a method comprising: wherein the harmful microorganism comprises at least one selected from the group consisting of Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 21327), Yersinia ruckeri (NVH 3758), and Yersinia ruckeri (DSMZ 18506); Strains. (Item XX1) A Pseudomonadaceae bacterium with the ability to protect fish. (Item XX2) Any of the above-mentioned Pseudomonadaceae bacteria having the ability to function as a probiotic for fish. (Item XX3) Any of the above-mentioned Pseudomonadaceae bacteria having the ability to function as a probiotic for the epidermis of fish. (Item XX4) A Pseudomonadaceae bacterium according to any one of the above items, which has the ability to colonize the epidermis of fish. (Item XX5) 2. The Pseudomonadaceae bacterium according to any one of the preceding items, which has the ability to improve the survival rate of fish. (Item XX6) The Pseudomonadaceae bacterium according to any one of the above items, wherein the fish are protected by adding the Pseudomonadaceae bacterium to a breeding liquid in a breeding environment for the fish. (Item XX7) The Pseudomonadaceae bacterium according to any of the preceding items, which has the ability to inhibit at least one harmful microorganism. (Item XX8) The Pseudomonadaceae bacterium according to any of the preceding items, wherein the harmful microorganism has at least one of the following abilities: the ability to cause skin diseases in fish, the ability to infect fish through the skin, the ability to infect fish through wounds, and the ability to infect fish through contact. (Item XX9) Any of the Pseudomonadaceae bacteria listed above, wherein the harmful microorganism is capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, pseudomonadosis of sweetfish, red mouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. (Item XX10) The harmful microorganisms include Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas anguilliseptica, Pseudomonas plecoglossicida, Yersinia ruckeri, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum, Tenacibaculum maritimum, Renibacterium salmoninarum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia seriolae, Streptococcus iniae, Lactococcus garvieae, Aeromonas The Pseudomonadaceae bacterium of any of the preceding items, including Pseudomonadaceae, ... (Item XX11) The Pseudomonadaceae bacterium of any of the preceding items, wherein the harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium columnare, Vibrio anguillarum, Vibrio ordalii, Streptococcus iniae, or a combination thereof. (Item XX12) The Pseudomonadaceae bacterium according to any one of the above items, wherein the ability of the bacterium is determined using zebrafish (Danio rerio) as a standard fish. (Item XX13) The Pseudomonadaceae bacterium according to any of the preceding items, wherein the fish is a farmed fish. (Item XX14) The Pseudomonadaceae bacterium according to any of the preceding items, wherein the farmed fish is eel, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury, or catfish. (Item XX15) The Pseudomonadaceae bacterium of any of the preceding items, wherein the farmed fish is a salmonid. (Item XX16) The Pseudomonadaceae bacterium of any of the preceding items, wherein the farmed fish is trout. (Item XX17) A Pseudomonadaceae bacterium according to any of the preceding items, which is a bacterium of the genus Pseudomonas. (Item XX18) Any of the above-mentioned Pseudomonadaceae bacteria belonging to the Pseudomonas putida group, the Pseudomonas fluorescens group, or the Pseudomonas koreensis group. (Item XX19) The Pseudomonadaceae bacterium of any of the above items, which is Pseudomonas mosselii, Pseudomonas marginalis, Pseudomonas koreensis, Pseudomonas protegens, or Pseudomonas parafulva. (Item XX20) A Pseudomonadaceae bacterium, which is KH-ZF1 (accession number: NITE BP-02967), KH-RT1 (accession number: NITE ABP-03222), KH-RT2 (accession number: NITE ABP-03223), KH-RT3 (accession number: NITE ABP-03224), or KH-RT4 (accession number: NITE ABP-03225). (Item XX21) A fish protection agent comprising any of the Pseudomonadaceae bacteria listed above. (Item XX22) A probiotic agent for fish containing any of the Pseudomonadaceae bacteria listed above. (Item XX23) The probiotic agent according to any of the preceding items, which is a probiotic for the epidermis of said fish. (Item XX24) The probiotic agent according to any of the preceding items, wherein the Pseudomonadaceae bacterium has the ability to colonize the epidermis of fish. (Item XX25) Any of the above fish protectants or any of the above probiotic agents, which are protectants or probiotic agents for farmed fish. (Item XX26) Any of the above-mentioned fish protectants or any of the above-mentioned probiotic agents which are protectants or probiotic agents for eel, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury or catfish. (Item XX27) A fish protectant according to any of the above items or a probiotic agent according to any of the above items, which is a protectant or probiotic agent for salmonid fish. (Item XX28) The fish protectant of any of the preceding items or the probiotic agent of any of the preceding items, which is a trout protectant or probiotic agent. (Item XX29) A method for protecting fish, comprising the step of contacting said fish with any of the Pseudomonadaceae bacteria described in the preceding paragraphs. (Item XX30) A method for protecting fish, comprising the step of growing the fish in water containing any of the Pseudomonadaceae bacteria described above. (Item XX31) 1. A method for obtaining microorganisms capable of protecting fish, comprising: (a) obtaining candidate microorganisms from the epidermis of fish; (b) adding the candidate microorganism to a medium containing harmful microorganisms; (c) confirming the inhibition of the harmful microorganism in the culture medium; and (d) when the inhibition of the harmful microorganism in the culture medium is confirmed, a step of identifying the candidate microorganism as a microorganism capable of protecting the fish; A method comprising: (Item XX32) The method according to any one of the preceding items, wherein in step (c), the inhibition of the harmful microorganisms on a solid medium is confirmed. (Item XX33) In step (c), the candidate microorganism and the harmful microorganism are grown on the same medium, and a growth inhibition zone in which the harmful microorganism cannot grow is confirmed near the candidate microorganism. (Item XX34) The method according to any one of the preceding items, wherein in step (c), the inhibition of the harmful microorganism is confirmed in a liquid medium containing the candidate microorganism or its culture supernatant and the harmful microorganism. (Item XX35) Any of the methods described above, wherein in step (c), an impregnated material containing a culture medium or culture supernatant of the candidate microorganism or a bottomless cylinder containing the culture medium or the culture supernatant is placed on a medium inoculated entirely with the harmful microorganism, and the inhibition of the harmful microorganism around the impregnated material or cylinder is confirmed. (Item XX36) Any of the methods described above, wherein in step (c), after incubating the liquid medium containing the candidate microorganism and the harmful microorganism, the growth of the candidate microorganism and the harmful microorganism are compared to confirm that the growth of the harmful microorganism is more suppressed. (Item XX37) In step (c), the candidate microorganism is inoculated onto a medium inoculated entirely with the harmful microorganism or onto a medium in which the harmful microorganism has grown entirely, and it is confirmed that the candidate microorganism forms a colony and that a zone of growth inhibition of the harmful microorganism is observed around the colony. (Item XX38) The method of any of the preceding items, wherein the candidate microorganism is obtained from a colony formed on a medium inoculated with scrapings of the fish's epidermis. (Item XX39) The method according to any of the preceding items, wherein one or more microorganisms are obtained as microorganisms capable of protecting said fish. (Item XX40) Any of the methods described above, wherein the harmful microorganism has at least one of the following abilities: the ability to cause skin diseases in fish, the ability to infect fish through the skin, the ability to infect fish through wounds, and the ability to infect fish through contact. (Item XX41) The method of any of the preceding items, wherein the harmful microorganism is capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, pseudomonad disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (chondrosis, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. (Item XX42) The method of any of the preceding items, wherein the candidate microorganism is obtained from the epidermis of zebrafish (Danio rerio) or trout. (Item XX43) Microorganisms obtained by any of the methods listed above. (Item XX44) Any of the microorganisms listed above that belong to the genus Pseudomonas. (Item XX45) Any of the microorganisms described above, which belong to the Pseudomonas putida group, the Pseudomonas fluorescens group, or the Pseudomonas koreensis group. (Item XX46) A method for protecting fish, comprising the step of contacting said fish with a microorganism obtained by any of the methods described above. (Item XX47) A strain belonging to the Pseudomonadaceae family, The strain is (i) streaking the strain on NB agar medium and culturing it overnight at 28°C; (ii) then streaking the harmful microorganisms vertically from the strain without touching the strain and incubating overnight at 28°C; and (ii) is characterized by the presence of a clear zone extending over an area of ​​10 mm or more from the streak of the strain, when tested in a method comprising: wherein the harmful microorganism comprises at least one selected from the group consisting of Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 21327), Yersinia ruckeri (NVH 3758), and Yersinia ruckeri (DSMZ 18506); Strains. (Item XX48) A strain belonging to the Pseudomonadaceae family, The strain is (i) streaking the strain on NB agar medium and culturing it overnight at 28°C; (ii) then streaking the harmful microorganisms perpendicular to and across the streaks of the strain, and incubating overnight at 28°C; and (ii) when tested in a method comprising: (i) after step (ii), colonies of the strain occupy 50% or more of the area of ​​the streak portion of the harmful microorganism beyond the portion where the streak of the strain intersects with the streak of the strain; wherein the harmful microorganisms include at least one selected from the group consisting of Yersinia ruckeri (NVH 3758), Aeromonas hydrophila (NRIA14), Vibrio anguillarum (NRIA83) and Vibrio ordalii (NRIA90); Strains. (Item XX49) A composition comprising an effective amount of any of the Pseudomonadaceae bacteria described above. (Item XX50) An effective amount of any one of the above Pseudomonadaceae bacteria; and at least one of salts, surfactants, carriers, drying protectants, preservatives, excipients, strengthening agents, antioxidants, dispersants, flocculants and other microorganisms. A composition comprising: (Item XX51) A protective culture fluid for fish, comprising an effective amount of any of the above-mentioned Pseudomonadaceae bacteria and a culture fluid for fish. (Item XX52) A fish protectant comprising an effective amount of any of the above-mentioned Pseudomonadaceae bacteria and a fish farming ingredient. (Item XX53) A kit for protecting fish, comprising an effective amount of any of the above-mentioned Pseudomonadaceae bacteria and ingredients for fish farming. (Item XX54) A method for protecting fish, comprising the step of contacting said fish with an effective amount of any of the Pseudomonadaceae bacteria described above. (Item XX55) A method for protecting fish, comprising the step of growing said fish in water containing an effective amount of any of the Pseudomonadaceae bacteria described above. (Item XX56) The method of any of the preceding items, wherein the fish is protected from at least one harmful microorganism. (Item XX57) Any of the methods described above, wherein the harmful microorganism has at least one of the following abilities: the ability to cause skin diseases in fish, the ability to infect fish through the skin, the ability to infect fish through wounds, and the ability to infect fish through contact. (Item XX58) The method of any of the preceding items, wherein the harmful microorganism is capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, pseudomonad disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (chondrosis, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. (Item XX59) The harmful microorganisms include Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas anguilliseptica, Pseudomonas plecoglossicida, Yersinia ruckeri, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum, Tenacibaculum maritimum, Renibacterium salmoninarum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia seriolae, Streptococcus iniae, Lactococcus garvieae, Aeromonas caviae or a combination thereof. (Item XX60) The method of any of the preceding items, wherein the harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium columnare, Vibrio anguillarum, Vibrio ordalii, Streptococcus iniae, or a combination thereof. (Item XX61) The method according to any one of the preceding items, wherein the contacting step comprises adding the Pseudomonadaceae bacterium to a rearing environment (e.g., rearing liquid, rearing water, etc.) for the fish. (Item XX62) The method according to any of the preceding items, wherein the contacting step is carried out under conditions sufficient for the Pseudomonadaceae bacterium to colonize the epidermis of the fish. (Item XX63) The method of any of the preceding items, wherein the fish are farmed fish. (Item XX64) The method of any of the preceding items, wherein the fish is eel, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury, or catfish. (Item XX65) The method of any of the preceding items, wherein the fish is a salmonid. (Item XX66) The method of any of the preceding items, wherein the fish is a trout. (Item XX67) The method according to any one of the preceding items, wherein the Pseudomonadaceae bacterium is a bacterium of the genus Pseudomonas. (Item XX68) The method according to any one of the preceding items, wherein the Pseudomonadaceae bacterium is a bacterium of the Pseudomonas putida group, the Pseudomonas fluorescens group, or the Pseudomonas koreensis group. (Item XX69) 3. The method of claim 1, wherein the Pseudomonadaceae bacterium is Pseudomonas mosselii, Pseudomonas marginalis, Pseudomonas koreensis, Pseudomonas protegens, or Pseudomonas parafulva. (Item XX70) The method according to any one of the preceding items, wherein the Pseudomonadaceae bacterium is KH-ZF1 (accession number: NITE BP-02967), KH-RT1 (accession number: NITE ABP-03222), KH-RT2 (accession number: NITE ABP-03223), KH-RT3 (accession number: NITE ABP-03224), or KH-RT4 (accession number: NITE ABP-03225). (Item XX71) The method of any of the preceding items, wherein protecting the fish includes protecting the epidermis of the fish. (Item XX72) The method of any of the preceding items, wherein protecting the fish comprises treating or preventing a disease or illness in the fish.

[0006] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0007] The microorganisms disclosed herein can be used as an alternative to antibiotics in fish farming and other fish breeding, thereby reducing the use of antibiotics and lowering the environmental burden. Furthermore, the method for obtaining microorganisms that protect fish disclosed herein can provide new useful microorganisms, thereby achieving more stable, inexpensive, highly productive, and / or diverse fish breeding. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a schematic diagram of an exemplary method for obtaining microorganisms from fish skin: Microbial flora is scraped from the fish skin with a cotton swab, the swab is immersed in sterile water to prepare a microbial liquid, and the microbial liquid is added to a culture medium to isolate the formed colonies. [Figure 2] FIG. 1 is a schematic diagram of an inhibition test using the cross-streak method. [Figure 3]The cross-streak inhibition test results of Example 2 are shown. The left side shows the results for KH-ZF1, and the right side shows the results for other microbial strains. The horizontal streaks are for Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 21327), Yersinia ruckeri (1) (NVH 3758), Yersinia ruckeri (2) (DSMZ 18506), and Escherichia coli DH5α, respectively. [Figure 4] Growth inhibition test on plates using KH-ZF1 from Example 4. The test microorganisms streaked from the center of the plate were Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Yersinia ruckeri (1) (NVH 3758), Yersinia ruckeri (2) (DSMZ 18506), Edwardsiella tarda (1) (NRIA44), Edwardsiella tarda (2) (NRIA51), Vibrio anguillarum (NRIA83), Vibrio ordalii (NRIA90), Streptococcus iniae (NRIA599), and negative controls (non-harmful microorganisms (1) and (2)). The area surrounded by the dashed line indicates the clear zone. [Figure 5] This shows a molecular phylogenetic tree based on the 16S rRNA partial base sequence of KH-ZF1 in Example 5. The line in the upper left indicates the scale bar. The numbers at the branching points of the phylogenetic tree indicate bootstrap values. A T after the name of a microbial strain indicates that the species is the type strain. BSL indicates the biosafety level (BSL1 or higher). [Figure 6]1 shows the results of the colonization test in Example 6. The vertical axis shows the colony-forming units (CFU) per zebrafish, and the horizontal axis shows the time from the addition of KH-ZF1 to the time when the body surface of the zebrafish was rubbed with Mentip. [Figure 7] This is a schematic diagram of the infection control test in Example 7. Five zebrafish (Danio rerio) were placed in 200 ml of rearing water to conduct the test. After rearing at 28°C for two days to allow them to acclimate to these rearing conditions, KH-ZF1 was administered to the rearing water (final bacterial concentration OD600 = 0.01) to expose the zebrafish to the bacterial strain. After rearing at 28°C for 24 hours under these conditions, the muscle at the base of the zebrafish's dorsal fin was injured with a 1.2 mm needle. Immediately after this, the rearing water was replaced, and KH-ZF1 was added again (final bacterial concentration OD600 = 0.01), followed by rearing for another 24 hours. The rearing temperature was then lowered to 20°C and the fish were reared for 24 hours. After that, KH-ZF1 and Yersinia ruckeri (NVH3578) were added to the fish at a bacterial concentration of OD600 = 0.01, and the fish were left at 20°C for 1 hour to attempt infection with Yersinia ruckeri. The rearing water was then replaced, and KH-ZF1 was added (final bacterial concentration D600 = 0.01). The fish were then reared at 20°C and monitored for progress (treatment group). A control group, to which no KH-ZF1 was added, was also observed. [Figure 8] The survival curves for zebrafish treated with pathogenic microorganisms alone (dotted line) or in combination with KH-ZF1 (solid line) are shown. The vertical axis represents cumulative survival rate, and the horizontal axis represents the number of days elapsed since the start of observation. RStudio software was used. [Figure 9] FIG. 1 is a schematic diagram of a competitive superiority test using the cross-streak method. [Figure 10] These are microscopic images of larvae on day 3 exposed to KH-ZF1 (top) or under control conditions (bottom) in Example 9. In the KH-ZF1-exposed group, KH-ZF1 fluorescence was observed on the body surface of the larvae, indicating the establishment of KH-ZF1. [Figure 11]1 shows the results of the survival test of Example 9. The vertical axis shows the survival rate. The horizontal axis shows the elapsed time (days). The results are shown for days 0, 3, 5, 18, 26, 28, and 31 after the start of rearing under KH-ZF1 exposure (left) or control conditions (right). DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0010] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] (definition, etc.) As used herein, "fish" refers to organisms belonging to the subphylum Vertebrata, excluding tetrapods. Fish include, but are not limited to, farmed fish used for food, ornamental fish, and pet fish. In one embodiment, any fish having a skin may be included in the present disclosure.

[0012] As used herein, "microorganisms" refers to organisms including eubacteria and archaea, as well as small (e.g., 1 mm or less, or 0.1 mm or less, or invisible to the naked eye) algae, protozoa, fungi (molds, fungi, etc.), yeast, and animals (protozoa, metazoa, etc.).

[0013] As used herein, the term "harmful microorganisms" refers to microorganisms that deteriorate or may deteriorate the survival, health, flesh quality, recovery from injury, and / or infection state of fish. Harmful microorganisms may also include opportunistic pathogens.

[0014] As used herein, "suppression" of microorganisms (also referred to as "inhibition" in Japanese, but both terms are synonymous in this specification) means a reduction and / or death of microorganisms and / or a slowing down of the growth rate of microorganisms, and also includes slowing down the growth or proliferation of microorganisms and killing microorganisms. Microorganism suppression can be evaluated, for example, based on the transparency of a medium inoculated with microorganisms and the area of ​​the medium that is clear. The ability of a microorganism to suppress or inhibit harmful microorganisms can be measured and identified, for example, by the following tests. Furthermore, if a result showing an inhibitory effect is obtained in any one of the tests, it is understood that this falls within the scope of inhibition in this specification. A test in which a candidate microorganism is added to a medium, and then harmful microorganisms are added to the same medium to check the transparency of the medium and / or the area of ​​the medium that is clear. A test in which candidate microorganisms and harmful microorganisms are grown on the same medium and a growth inhibition zone in which harmful microorganisms cannot grow near the candidate microorganisms is observed. A test to confirm that the growth of harmful microorganisms is reduced by adding the culture supernatant of a candidate microorganism to a liquid medium inoculated with harmful microorganisms. A test in which an impregnated culture medium or culture supernatant of a candidate microorganism is placed on a medium, or the culture medium or culture supernatant of a candidate microorganism is placed in a small bottomless cylinder, and the growth of harmful microorganisms inoculated over the entire medium is confirmed to be suppressed around it. A test in which candidate microorganisms and harmful microorganisms are cultured in the same liquid medium, and then the growth of the two microorganisms is compared to distinguish them, and it is confirmed that the harmful microorganisms are suppressed.

[0015] As used herein, the "skin" of a fish refers to the surface of the body (body surface), eyes, scales, fins, gills, mouth, anus, and genitals of a fish. The skin of the body surface of a fish can be classified into a scale type (scale-type fish) and a mucous type (mucous-type fish).

[0016] As used herein, "protection" of fish refers to improving the survival, health, flesh quality, recovery from injury, and / or infection status (including susceptibility to infection) of fish. The protection of fish can be evaluated, for example, by improving the survival rate of injured fish when they are brought into contact with harmful microorganisms. For example, fish can be protected by adding the Pseudomonadaceae bacteria to the rearing environment of fish (e.g., rearing liquid, rearing water, etc.). Here, the rearing environment refers to any environment in which fish organisms grow, and is not necessarily limited to a rearing liquid, although in the case of fish eggs, there may be little moisture. The ability of a microorganism to protect fish can be measured and identified, for example, by the following test. A test in which fish are subjected to stress and harmful microorganisms are added to a control group, and a test in which harmful microorganisms and candidate microorganisms are added to the fish are compared to confirm an improvement in the survival rate of the fish in the treatment group. - A test to confirm the improvement in the survival rate of fish in the treatment group by first raising fish in the presence of candidate microorganisms for a certain period of time and then comparing the results between a treatment group to which harmful microorganisms are added and a control group to which harmful microorganisms are added without the pretreatment.

[0017] As used herein, "probiotics" refers to compositions containing biological components (e.g., microorganisms in their entirety or parts thereof) that have functions such as acting on harmful microorganisms, forming beneficial microbial flora in fish, maintaining or improving the health of fish skin, and / or stimulating the immune system of fish, or the microorganisms or biological components thereof, or compositions containing these. The ability of a microorganism to function as a probiotic can be measured and identified, for example, by the following test. A test to confirm that the survival rate of fish is improved when they are raised in the presence of a candidate microorganism for a certain period of time, compared to when they are raised in the absence of the candidate microorganism. After raising fish in the presence of candidate microorganisms for a certain period of time, tests are conducted to confirm signs of enhanced immune activity in the fish, such as increased cytokine secretion and improved expression of immune-related genes. A test to confirm that the population of harmful microorganisms in the microflora of fish is reduced by raising fish in the presence of candidate microorganisms for a certain period of time. A test in which fish are raised in the presence of candidate microorganisms for a certain period of time as a pretreatment, and then harmful microorganisms are added to the breeding water to be compared with a control group to which harmful microorganisms were added without pretreatment, to confirm that the population of the harmful microorganisms in the microbial flora of the fish in the treatment group is lower than that of the control group.

[0018] As used herein, "colonization" refers to the ability of a microorganism to remain attached to a target and survive (e.g., grow) for a certain period of time. The ability of a microorganism to colonize fish can be measured and identified, for example, by the following test. A test in which fish are raised in water to which candidate microorganisms have been added, and the number or quantity of candidate microorganisms obtained from the raised fish is confirmed. After raising fish in breeding water to which the candidate microorganisms have been added, the fish are observed under a microscope to confirm the presence of the candidate microorganisms on the fish (e.g., on their skin). After raising fish in breeding water to which candidate microorganisms have been added, the microbial flora of the fish (on their skin, etc.) is analyzed to confirm the presence of the candidate microorganisms in the flora.

[0019] As used herein, the term "fish protection agent" refers to a preparation that contains the microorganism of the present disclosure as an active ingredient and that can protect fish.

[0020] As used herein, the term "probiotic agent" refers to a preparation that contains the microorganism of the present disclosure as an active ingredient and is capable of exerting a probiotic function.

[0021] As used herein, a "derivative strain," "analogous strain," or "mutant strain" preferably, but without limitation, comprises a gene (e.g., 16S rDNA) containing a region substantially homologous to the DNA of the microorganism of interest, and in various embodiments, such a strain has a whole genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical when compared to the whole genome sequence of the original strain by alignment using computer homology programs known in the art. This refers to a microorganism that has been modified by gene mutation, substitution, deletion, and / or addition, such that the derivative still exhibits the biological functions of the original microorganism, although not necessarily to the same degree. For example, genetic mutations can be introduced using any known mutagen, UV, plasma, genome editing technology, etc. In one embodiment, a "derivative strain," "analogous strain," or "mutant strain" is a strain of the same genus and / or species as the original strain. For example, the biological function of such microorganisms can be examined by suitable available in vitro assays described herein or known in the art.

[0022] As used herein, a "purified" biological factor (e.g., a specific strain of organism or intracellular component) refers to a biological factor from which at least a portion of the factors naturally associated with the biological factor have been removed. Thus, the purity of the biological factor in a purified biological factor is typically higher (i.e., more concentrated) than in the state in which the biological factor normally exists. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological factor (e.g., a specific organism strain) is present.

[0023] As used herein, "homology" of genes or nucleotide sequences refers to the degree of identity between two or more gene sequences. Generally, "homology" refers to a high degree of identity or similarity. Therefore, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes are homologous can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, the genes are homologous if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. As used herein, "similarity" of genes or nucleotide sequences refers to the degree of similarity between two or more gene sequences, and refers to a high degree of similarity between sequences that are identical. "Similarity" is a numerical value that takes into account not only identity but also similar bases, where similar bases refer to partial matches in mixed bases (e.g., R=A+G, M=A+C, W=A+T, S=C+G, Y=C+T, K=G+T, H=A+T+C, B=G+T+C, D=G+A+T, V=A+C+G, N=A+C+G+T).

[0024] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. Herein, comparisons of amino acid and nucleotide sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.7.1 (published October 19, 2017). The "identity" value herein generally refers to the value obtained when aligned using the above-mentioned BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value is used as the identity value. When identity is evaluated in multiple regions, the highest value among them is used as the identity value. "Similarity" is a numerical value that takes into account not only identity but also similar amino acids.

[0025] In one embodiment of the present disclosure, the numerical value of "70% or more" for identity or the like may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more, or may be within a range of any two of these starting values. The "identity" is calculated by the percentage of homologous bases between two or more base sequences according to known methods such as those described above. Specifically, before calculating the percentage, the base sequences of the base sequences to be compared are aligned, and gaps are introduced into some of the base sequences, if necessary, to maximize the percentage of identical base nucleic acids. Alignment methods, percentage calculation methods, comparison methods, and related computer programs are well known in the art (e.g., BLAST, etc., as described above). Unless otherwise specified, "identity" herein can be expressed as a value measured by NCBI's BLAST. When comparing base sequences with BLAST, the algorithm used is Blastp, which can be used with the default settings. Measurement results are expressed numerically as positives or identities. In this case, when "similarity" is used instead of "identity," the numerical value also takes into account those that fall under the definition of "similar" "bases" described herein.

[0026] As used herein, the term "biological function," when referring to a microorganism, refers to a specific function that the microorganism may have, such as, but not limited to, protecting fish (e.g., improving fish survival rate). In the present disclosure, examples include, but are not limited to, improving fish survival rate as well as suppressing harmful microorganisms. As used herein, a biological function can be exerted by a corresponding "biological activity." As used herein, "biological activity" refers to an activity that a microorganism may have in a certain environment, and includes activities that exert various functions (e.g., improving fish survival rate). Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that affect a response (i.e., have a measurable effect in response to some exposure or stimulus), and can include, for example, a measure of the amount of an upstream or downstream protein or other similar function of the microorganism of the present disclosure after some stimulus or event.

[0027] As used herein, the "amount" of an analyte in a sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in a volume of the sample. However, the amount also contemplates a relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample may be an amount that is greater than a control level or normal level of the analyte that is normally present in the sample.

[0028] As used herein, the term "about" refers to the indicated value plus or minus 10%, unless otherwise specified.

[0029] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., a composition comprising the microorganism of the present disclosure, additional components, buffer solutions, instructions, etc.) are provided, typically separated into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit advantageously includes instructions or manuals describing how to use or process the provided parts (e.g., a composition comprising the microorganism, additional components, etc.). When the term "kit" is used herein, the kit typically includes instructions describing how to use the microorganism, composition, etc. of the present disclosure.

[0030] As used herein, "instructions" refers to instructions to the user on how to use the present disclosure. The instructions contain wording that instructs on how to use the present disclosure. If necessary, the instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, or the Food and Drug Administration (FDA) or the Department of Agriculture (USDA) in the United States), and it is clearly stated that the instructions have been approved by the regulatory agency. The instructions may be provided in paper form, but are not limited thereto, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, email, etc.).

[0031] As used herein, "ingredients for fish farming" refers to any ingredient that is not expected to have the effects of the microorganisms of the present disclosure on the fish to be farmed, but that plays a certain role when combined with the microorganisms of the present disclosure, and examples thereof include carriers (including water), salts, surfactants, carriers, drying protectants, preservatives, antibacterial agents, excipients, fortifiers, antioxidants, stabilizers, diluents, buffers, binders, dispersants, flocculants, other microorganisms, etc.

[0032] As used herein, the term "fish culture liquid" refers to a fluid component of fish culture, and may be a solution or a suspension, or a mixture of a fluid component and a solid component (which may or may not be dissolved). Typically, the fish culture liquid may be a liquid that simulates the environment in which fish live, such as a river, ocean, or lake, or a concentrated solution thereof.

[0033] As used herein, the term "protective culture fluid" refers to a fluid for protecting fish, which contains the microorganisms of the present disclosure and a culture fluid for culturing fish. Typically, fish are raised in the protective culture fluid or a fluid diluted at any dilution factor (e.g., about 2 to 1,000,000 times).

[0034] (Preferred embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0035] (useful microorganisms) In one aspect, the present disclosure provides novel microorganisms capable of protecting fish and / or suppressing harmful microorganisms. In particular, the microorganisms disclosed herein are capable of improving the survival rate of fish, colonizing fish (e.g., their epidermis), and / or suppressing harmful microorganisms. In one embodiment, fish can be protected by adding the microorganisms disclosed herein to the rearing environment (e.g., rearing solution, rearing water, etc.) of fish. The microorganisms disclosed herein are also expected to have effects such as direct action against harmful microorganisms, formation of a microbial flora in fish (e.g., their epidermis), maintenance and / or improvement of the health of fish (e.g., their epidermis), and stimulation of fish immunity. Furthermore, application of the microorganisms disclosed herein to fish eggs and / or fry is expected to improve the hatching rate and / or survival rate of fry.

[0036] In one embodiment, the Pseudomonadaceae bacterium of the present disclosure is a specific strain that, when tested in a method comprising the steps of (i) streaking the strain linearly on an NB agar medium and culturing the strain overnight at 28°C, and (ii) then streaking harmful microorganisms vertically from the strain without touching the strain and culturing the strain overnight at 28°C, can be characterized by the presence of a clear zone extending over a range of, for example, 10 mm or more (3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more) from the streak of the strain after step (ii), wherein the harmful microorganism is selected from the group consisting of Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 21327), Yersinia ruckeri (NVH 3758), and Yersinia ruckeri (DSMZ 18506).

[0037] In one embodiment, the microorganism of the present disclosure is a bacterium of the family Pseudomonadaceae. Pseudomonadaceae microorganisms may have at least one of the following characteristics: they have cytochrome C oxidase, are non-fermentative, have the Entner-Doudoroff pathway via glyceraldehyde-6-phosphate dehydrogenase and aldolase, are spore-producing, are motile, and produce the fluorescent pigment pyoberdin. The family Pseudomonadaceae includes the genera Azomonas, Azomonotrichon, Azorhizophilus, Azotobacter, Cellvibrio, Chryseomonas, Flavimonas, Mesophilobacter, Permianibacter, Pseudomonas, Rhizobacter, Rugamonas, Serpens, and Thiopseudomonas. In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Pseudomonas. Pseudomonas organisms may have at least one of the following characteristics: they are rod-shaped, they are Gram-negative, they have one or several polar flagella that confer motility, they are aerobic, they do not form spores, they are catalase-positive, they are oxidase-positive, they do not form gas in the Hugh-Leifson test with glucose, they are beta-hemolytic on blood agar, they are indole-negative, they are methyl red-negative, they are Voges-Proskauer-negative, and they are citrate-positive. Genus Pseudomonas includes abietaniphila, acidovorans, aestusnigri, aeruginosa, agarici, alcaligenes, alcaliphila, aminovorans, amygdali, andropogonis, anguil liseptica, antarctica, antimicrobica, argentinensis, arsenicoxydans, asplenii, asturiensis, aurantiaca, aureofaciens, avellanae, avenae, avenae subsavenae, avenae subscitrulli, avenaesubskonjaci, azotifigens, azotoformans, baetica, balearica, bauzanensis, beijerinckii, benzenivorans, beteli, borbori, boreopolis, brassicacearum, brassicacearum subsbrassicacearum, brassicacearum subsneoaurantiaca, brenneri, caeni, cannabina, carboxydohydrogena, caricapapayae, caryophylli, cattleyae, cedrina, cedrina subscedrina, cedrina subsfulgida, cepacia, chengduensis, chloritidismutans, chlororaphis, chlororaphis subsaurantiaca, chlororaphis subsaureofaciens, chlororaphis subschlororaphis, chlororaphissubspiscium, cichorii, cissicola, citronellolis, cocovenenans, composti, congelans, corrugata, costantinii, cremoricolorata, cuatrocienegasensis, deceptionensis, delafieldii, delhiensis, diminuta, doudoroffii, duriflava, echinoides, elongata, entomophila, extremaustralis, extremorientalis, facilis, ficuserectae, flava, flavescens, flectens, fluorescens, fragi, frederiksbergensis, fulva, fuscovaginae, gelidicola, geniculata, gessardii, gladioli, glathei, glumae, graminis, grimontii, guangdongensis, guariconensis, guguanensis, guineae, halophila, helmanticensis, hibiscicola, hussainii, huttiensis, indica, indigofera, iners, japonica, jessenii, jinjuensis, kilonensis, knackmussii, koreensis, kunmingensis, kuykendallii, lanceolata, lemoignei, libanensis, lini, litoralis, lundensis, lurida, lutea, luteola, mallei, maltophilia, mandelii, marginalis, marina, marincola, mediterranea, meliae, mendocina, mephitica, meridiana, mesophilica, migulae, mixta, mohnii, monteilii, moorei, moraviensis, mosselii, mucidolens, multiresinivorans, nautica, nitroreducens, oleovorans, oleovoranssubslubricantis, oleovorans subsoleovorans, orientalis, oryzihabitans, otitidis, pachastrellae, palleroniana, palleronii, panacis, panipatensis, parafulva, paucimobilis, pelagia, peli, perfectomarina, pertucinogena, phenazinium, pickettii, pictorum, plantarii, plecoglossicida, poae, pohangensis, prosekii, protegens, proteolytica, pseudoalcaligenes, pseudoalcaligenes subscitrulli, pseudoalcaligenes subskonjaci, pseudoalcaligenes subspseudoalcaligenes, pseudoflava, pseudomallei, psychrophila, psychrotolerans, punonensis, putida, pyrrocinia, radiora, reinekei, resinovorans, rhizosphaerae, rhodesiae, rhodos, rubrilineans, rubrisubalbicans, sabulinigri, saccharophila, salegens, salomonii, saponiphila, savastanoi, segetis, seleniipraecipitans, simiae, solanacearum, spinosa, stanieri, straminea, stutzeri, synxantha, syringae, syringae subssavastanoi, syringaeSpecies include, for example, subssyringae, syzygii, taeanensis, taeniospiralis, taetrolens, taiwanensis, testosteroni, thermotolerans, thivervalensis, tolaasii, toyotomiensis, tremae, trivialis, tuomuerensis, umsongensis, vancouverensis, veronii, vesicularis, viridiflava, vranovensis, woodsii, xanthomarina, xiamenensis, xinjiangensis, zeshuii, etc. In one embodiment, the microorganism of the present disclosure may be of the Pseudomonas putida group. The Pseudomonas putida group may include Pseudomonas mosselii, Pseudomonas fulva, Pseudomonas cremocolorata, Pseudomonas entomophila, Pseudomonas parafulva, Pseudomonas monteilii, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, and Pseudomonas putida, and the bacteria included in this group may be expected to have similar abilities to each other (Anzai Y et al., Int J Syst Evol Microbiol. 2000 Jul;50 Pt 4:1563-89.). In one embodiment, the microorganism of the present disclosure may be the Pseudomonas fluorescens group. The Pseudomonas fluorescens group includes Pseudomonas antarctica, Pseudomonas azotoformans, Pseudomonas blatchfordae, Pseudomonas brassicacearum, Pseudomonas brenneri, Pseudomonas cedrina, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas gessardii, PseudomonasIn one embodiment, the microorganism of the present disclosure may be the Pseudomonas koreensis group, although examples of bacteria that may be included in this group include Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas meridiana, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas panacis, Pseudomonas protegens, Pseudomonas proteolytica, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii. The Pseudomonas koreensis group may include Pseudomonas koreensis, Pseudomonas mandelii, and Pseudomonas lemonnieri (Andreani et al., Food Microbiol. 2014 May;39:116-26.), and bacteria in this group may be expected to have similar abilities to each other. The Pseudomonas koreensis group may also include some Pseudomonas fluorescens, and so these bacteria may be expected to have similar abilities to each other. In one embodiment, the microorganism of the present disclosure is Pseudomonas mosselii, Pseudomonas marginalis, Pseudomonas koreensis, Pseudomonas protegens, or Pseudomonas parafulva. The present inventors have discovered this by examining the inhibitory ability of harmful microorganisms. The new microorganism was identified as Pseudomonas mosseri and deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, and received on June 3, 2019, with a certificate of accession issued on June 17, 2019. The accession number is NITE ABP-02967, and the accession number is NITE BP-02967. Furthermore, the present inventors have identified four additional useful species of Pseudomonadaceae microorganisms and deposited them with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation, and received on May 26, 2020. , the certificate of acceptance was issued on June 29, 2020. The receipt numbers for these four microorganisms are NITE ABP-03222, NITE ABP-03223, NITE ABP-03224, and NITE ABP-03225, respectively. The accession numbers are NITE BP-03222, NITE BP-03223, NITE BP-03224, and NITE BP-03225, respectively. In one embodiment, the microorganism of the present disclosure is Pseudomonas bacterium KH-ZF1( Contract Number: NITE BP-02967), KH-RT1 (Receipt number: NITE B P-03222), KH-RT2( Contract Number: NITE B P-03223), KH-RT3( Contract Number: NITE B P-03224) or KH-RT4( Contract Number: NITE B P-03225) or its derivatives.

[0038] In one embodiment, the microorganism of the present disclosure is Pseudomonas bacterium KH-ZF1( Contract Number: NITE BP-02967), KH-RT1( Contract Number: NITE B P-03222), KH-RT2( Contract Number: NITE B P-03223), KH-RT3( Contract Number: NITE B P-03224) or KH-RT4( Contract Number: NITE BThe microorganism is a derivative of Pseudomonas bacteria KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4 (P-03225). Here, the derivative does not necessarily have to be a strain derived from Pseudomonas bacteria KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4, but refers to a microorganism that exhibits the biological functions of Pseudomonas bacteria KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4, although not necessarily to the same degree. In one embodiment, the microorganism that is a derivative of the present disclosure exhibits biological functions selected from the ability to protect fish, the ability to colonize fish (e.g., on the epidermis), and the ability to inhibit harmful microorganisms, similar to Pseudomonas bacteria KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4, but the degree of the biological functions may differ from that of KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4. In one embodiment, the microorganisms of the present disclosure may be Pseudomonas bacteria, more specifically, Pseudomonas mosselii, Pseudomonas marginalis, Pseudomonas koreensis, Pseudomonas protegens, or Pseudomonas parafulva. In one embodiment, the ability of the microorganisms of the present disclosure (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4) to protect fish, suppress or inhibit harmful microorganisms, colonize fish, and function as probiotics may be confirmed by any of the tests described herein or modifications thereof for confirming each of these abilities.

[0039] In one embodiment, the microorganisms of the present disclosure (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4) have the ability to suppress harmful microorganisms, and this ability can be confirmed by placing an autoclave-sterilized paper disk (product number 49005010; Toyo Roshi) in the center of NB agar medium (for example, the composition described in Example 1) or an equivalent medium (for example, gellan gum, etc.) in a 9 cm diameter Petri dish, soaking the paper disk with 20 μl of a cell suspension containing the microorganisms of the present disclosure in sterilized water at an OD600 of 1.0, leaving the paper disk standing at 20°C for 2 days, and then inserting a mentip (cotton part) at an angle of about 20 to 30° to the surface of the agar medium (or an equivalent medium (for example, gellan gum, etc.)) while applying a force sufficient to not indent the surface of the medium, and then immersing the mentip in the culture medium (OD600) of the harmful microorganisms. 600 By moving a paper disc (which has been immersed in a broth containing 2-3) and allowed to soak in the culture solution, and then wiping off excess water), approximately 100 μL of bacterial culture solution is streaked radially from near the paper disc without touching the microorganisms of the present disclosure, and culturing at 20°C for 2 days, a clear zone of 0.1 mm or more, 0.2 mm or more, 0.5 mm or more, 0.7 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 12 mm or more, 15 mm or more, 17 mm or more, 20 mm or more, 25 mm or more, 30 mm or more, 35 mm or more, or 40 mm or more can be observed, which can be confirmed by the observation of a clear zone. The presence of a clear zone can be determined visually by those skilled in the art.

[0040] In one embodiment, the microorganisms of the present disclosure (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4) may have a competitive advantage over harmful microorganisms. For example, the competitive advantage over harmful microorganisms can be measured by placing a mentip (cotton part) at an angle of about 20 to 30° to the surface of an agar medium (or an equivalent medium (e.g., gellan gum)) on the culture medium (OD 20) of the microorganisms of the present disclosure while applying a force sufficient to prevent the surface from being indented. 600Approximately 100 μL of bacterial culture solution is streaked by moving a mentip (submerged in a broth containing 2-3) to allow the culture solution to soak in, and excess water is wiped off), and after overnight incubation at 28°C, harmful microorganisms are streaked by moving a mentip perpendicular to the streak so that it intersects with the candidate microorganism streak (the same streaking method is used except that the culture solution of the harmful microorganism is used instead of the culture solution of the microorganism disclosed herein). The candidate microorganisms are then mixed and incubated overnight at 28°C. This can be confirmed by observing that colonies of the candidate microorganism occupy 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more of the area of ​​the harmful microorganism streak beyond the point where the candidate microorganism streak intersects with the streak of the harmful microorganism. Due to their competitive advantage over harmful microorganisms, the microorganisms disclosed herein can exhibit particularly high inhibitory capabilities against harmful microorganisms.

[0041] In one embodiment, the microorganisms of the present disclosure (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4) have the ability to colonize fish (e.g., zebrafish (Danio rerio)), and this ability was confirmed by placing six fish and the microorganisms of the present disclosure into which a fluorescent protein had been introduced at an OD600 of 0.01 in 100 ml of rearing water (1 L of ultrapure water to which 3 g of Instant Ocean had been added), rearing them at 20°C, and after 6 and 24 hours, rubbing the epidermis (e.g., body surface) of the fish with a mentip, immersing the mentip in 0.5 ml of sterilized rearing water and stirring to prepare a suspension, which was then cultured in a Difco (trademark) Pseudomonas Isolation System. When the cells are plated onto 9 cm agar (Becton Dickinson Japan, Tokyo) and incubated at 28°C for 12 hours, the colony-forming units (CFUs) based on fluorescence are confirmed to have increased by 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 150% or more, 200% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, or 1000% or more at 24 hours compared to 6 hours.

[0042] In one embodiment, the microorganisms of the present disclosure (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3, or KH-RT4) have the ability to improve the survival rate of fish, and this ability was confirmed by placing five fish (e.g., zebrafish (Danio rerio)) in 200 ml of rearing water (3 g of Instant Ocean added to 1 L of ultrapure water), rearing them at 28°C for two days, replacing the rearing water, adding the microorganisms of the present disclosure to an OD600 of 0.01, rearing them at 28°C for 24 hours, and then adding an anesthetic solution (400 mg of tricaine, 1 M The fish were anesthetized by transferring them to a solution prepared by adding 2 ml of a stock solution (adjusted to pH 7) of 2.1 ml of Tris (pH 9) and 97.9 ml of ultrapure water to 100 ml of ultrapure water, and then a wound was made in the muscle at the base of the dorsal fin of the fish with a syringe needle (Terumo syringe needle, NN-1838R, 18G (1.20 mm), blade type R·B (blade angle 12°), needle length 38 mm). The wounded fish were returned to the changed rearing water, and the microorganisms disclosed herein were added to an OD600 of 0.01. The fish were then reared at 28°C for 24 hours, the temperature was lowered from 28°C to 20°C, and the fish were reared for 24 hours. The rearing water was then replaced, and the microorganisms disclosed herein and harmful microorganisms (e.g., Yersinia ruckeri) was added to the fish so that the OD600 was 0.01, and the fish were kept at 20°C for 1 hour. The water was replaced, and the microorganisms of the present disclosure were added so that the OD600 was 0.01. The fish were then kept at 20°C and monitored over time. The survival rates of the treatment group were compared over 8 days with those of a control group, which differed from the treatment group only in that no microorganisms of the present disclosure were used. The survival rate of the treatment group was found to be statistically significantly higher than that of the control group. Statistical methods generally used include those found in biostatistics textbooks. For example, a long-rank test can be used; a P value of 0.05 or less or 0.01 or less can be used to indicate a significant difference.

[0043] Those skilled in the art can use the above criteria appropriately to test derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4 to obtain derivatives having the above biological functions (and their degrees).

[0044] In one embodiment, the microorganisms disclosed herein (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4) are microorganisms obtained by the methods for obtaining microorganisms described below.

[0045] In another aspect, the present disclosure provides a strain belonging to the family Pseudomonadaceae bacteria, wherein the strain is characterized in that, when tested by a method comprising the steps of: (i) streaking the strain linearly on an NB agar medium and culturing the strain overnight at 28°C; and (ii) thereafter streaking harmful microorganisms vertically from the strain without touching the strain and culturing the strain overnight at 28°C, after step (ii), a clear zone is present over a range of 10 mm or more from the streak of the strain, wherein the harmful microorganisms are selected from Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 21327), Yersinia ruckeri (NVH 3758), and Yersinia ruckeri (DSMZ The present invention provides a bacterial strain comprising at least one species selected from the group consisting of: 18506. The conditions (abilities) that these strains should have and the conditions for testing them are provided in the present specification and examples, and those skilled in the art can identify the bacterial strain by referring to publicly known information as appropriate.

[0046] (Method for obtaining useful microorganisms) In one aspect, the present disclosure provides a method for obtaining microorganisms capable of protecting fish. This method may include the steps of (a) obtaining a candidate microorganism from the epidermis of fish, (b) adding the candidate microorganism to a culture medium containing harmful microorganisms, (c) confirming the suppression of the harmful microorganisms in the culture medium, and (d) obtaining the candidate microorganism as a microorganism capable of protecting fish if the suppression of the harmful microorganisms in the culture medium is confirmed. Because fish often come into direct contact with microorganisms in water, and therefore frequently suffer from skin infections, targeting microorganisms on the epidermis of fish may be useful for efficiently obtaining microorganisms capable of protecting fish. Using microorganisms originally present on the epidermis of fish may enable safe, simple, and / or environmentally friendly protection of fish. The epidermis of the fish from which the candidate microorganisms are obtained may be any surface of the body, eyes, scales, fins, gills, mouth, anus, and genitals, and in one embodiment, it is the surface of the body.

[0047] In one embodiment, the confirming step (c) can be carried out by confirming the ability of the microorganisms of the present disclosure (including derivatives of KH-ZF1) to protect fish, suppress or inhibit harmful microorganisms, colonize fish, and function as probiotics using any of the tests described herein or modifications thereof. In one embodiment, the confirming step (c) can be carried out by confirming the suppression of harmful microorganisms in a culture medium. In one embodiment, the confirming step (c) can be carried out by growing the candidate microorganism and the harmful microorganism in the same culture medium and confirming a growth inhibition zone in which the harmful microorganism cannot grow near the candidate microorganism. In one embodiment, the confirming step (c) can be carried out by confirming the suppression of harmful microorganisms in a liquid culture medium containing the candidate microorganism or its culture supernatant and the harmful microorganism. In one embodiment, the confirming step (c) can be carried out by placing an impregnated product of the culture medium or culture supernatant of the candidate microorganism or a bottomless cylinder containing the culture medium or culture supernatant on an agar medium or equivalent medium (e.g., gellan gum, etc.) that has been inoculated entirely with harmful microorganisms, and confirming the suppression of harmful microorganisms around the impregnated product or cylinder. In one embodiment, the confirming step (c) can be carried out by incubating a liquid medium containing the candidate microorganism and harmful microorganisms, comparing the growth of the candidate microorganism and harmful microorganism, and confirming that the growth of the harmful microorganism is more suppressed. In one embodiment, the confirming step (c) can be carried out by inoculating the candidate microorganism on an agar medium or equivalent medium (e.g., gellan gum, etc.) that has been inoculated entirely with harmful microorganisms, or on an agar medium or equivalent medium (e.g., gellan gum, etc.) that has been completely grown with harmful microorganisms, and confirming that the candidate microorganism forms a colony and that a zone of growth inhibition or a clear zone due to the death of the harmful microorganism is observed around the colony. Different types of microorganisms cultured in the same liquid medium can be distinguished by means of colony shape, color, the use of selective media, etc.For example, methods for distinguishing and examining populations include counting individual colonies by plate culture if the colonies differ in color or shape, directly counting each microorganism under a microscope if different morphologies are observed under a microscope, designing gene primers that can distinguish each microorganism and performing real-time PCR, fluorescent in situ hybridization (FISH), etc. Methods for identifying candidate microorganisms under a microscope include the well-known FISH method and methods that utilize the fluorescence of fluorescent proteins previously introduced into candidate microorganisms.

[0048] In one embodiment, candidate microorganisms can be obtained from colonies formed on a culture medium inoculated with scrapings (e.g., scrapings with a cotton swab) of fish skin (e.g., body surface). One or more microorganisms can be obtained as microorganisms capable of protecting fish. In one embodiment, microorganisms capable of protecting fish can be obtained from the skin of zebrafish (Danio rerio). In one embodiment, microorganisms capable of protecting fish can be obtained from the skin of salmonid fish (e.g., trout (e.g., rainbow trout (Oncorhynchus mykiss)). In one embodiment, microorganisms capable of protecting fish can be obtained from the skin of scale-type fish.

[0049] In one embodiment, a harmful microorganism to be confirmed for inhibition can be selected that is suspected to have at least one of the following abilities: causing skin disease in fish (e.g., farmed fish), transmitting through the skin, infecting through wounds, and transmitting through contact. For example, a person skilled in the art can appropriately select a microorganism that has the ability to cause skin disease in fish (e.g., farmed fish), transmitting through the skin, infecting through wounds, and / or transmitting through contact by referring to "Fish Pathology" (edited by Kodama Hiroshi, Midori Shobo, Tokyo, 2012) and "Infectious and Parasitic Diseases of Fish and Shellfish" (edited by Wakabayashi and Muroga, and written by Ogawa Kazuo, Koseisha Kouseikaku, Tokyo, 2004), etc.

[0050] In one embodiment, the harmful microorganism may be selected to be capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, pseudomonas disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (chondrosis, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. The typical causative microorganisms for each fish disease are as follows: Vibriosis (Listonella anguillara (formerly Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida); furunculosis (Aeromonas salmonicida); atypical Aeromonas salmonicida infection (Aeromonas salmonicida); Aeromonas hydrophila infection (Aeromonas hydrophila); Edwardsiellosis (Edwardsiella tarda); red spot disease (Pseudomonas anguilliseptica); Pseudomonas disease in sweetfish (Pseudomonas plecoglossicida); redmouth disease (Yersinia ruckeri); bacterial gill disease (Flavobacterium branchiophilum); columnaris disease (gill rot, tail rot, fin rot) (Flavobacterium columnare); coldwater disease (Flavobacterium psychrophilum); gliding bacteriosis (Tenacibaculum maritimum); bacterial kidney disease (Renibacterium salmoninarum); mycobacteriosis (Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei); nocardiosis (Nocardia seriolae); streptococcosis (Streptococcus iniae). Yersinia ruckeri is the causative agent of redmouth disease, which is known to cause pandemics in salmonids.In one embodiment, the harmful microorganisms may include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium columnare, Vibrio anguillarum, Vibrio ordalii, Streptococcus iniae, or a combination thereof.

[0051] (Composition containing microorganisms) In one aspect, the present disclosure provides a composition comprising the microorganism of the present disclosure. The microorganism of the present disclosure can be produced by culturing using any suitable method. In one embodiment, the composition is a fish protectant. In one embodiment, the composition is a probiotic agent for fish (e.g., the skin). Use of the fish protectant or probiotic agent of the present disclosure can improve at least one of the survival, health condition, flesh quality, recovery from injury, and infection status (including susceptibility to infection) of fish.

[0052] (Applicable to) In one embodiment, fish to which the microorganisms or compositions of the present disclosure are applied include, but are not limited to, farmed fish, ornamental fish, etc. Farmed fish include, but are not limited to, eel, sweetfish, yellowtail, trout (e.g., rainbow trout (Oncorhynchus mykiss)), sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury, and catfish. The microorganisms of the present disclosure can be obtained from and / or applied to the skin of fish, which can be of the scale type or slime type, such as eels and catfish, which are slime-type fish. In one embodiment, the fish to which the microorganisms or compositions of the present disclosure are applied can be salmonids (e.g., trout). Salmonids are an example of scale-type fish. The microorganisms or compositions of the present disclosure can be used at any stage of fish development, such as eggs, hatchlings, fry, adults, sexually mature fish, etc.

[0053] (Usage form) The form of the microorganism or composition of the present disclosure may be, for example, a liquid state or a solid state. Examples of liquid-state microorganisms or compositions include a microbial culture solution, and a solution in which microorganisms are collected from the culture solution by centrifugation or the like and then re-dispersed in water, a buffer solution, or a culture solution. Examples of solid-state microorganisms or compositions include those dehydrated by centrifugation or pressing, those in a paste or mayonnaise state that is intermediate between a solid and a liquid, and those obtained by drying (e.g., vacuum drying or freeze-drying). Examples of solid forms include powders, granules, tablets, and the like. The composition may also be provided in a state in which the microorganism or culture supernatant is immobilized on a carrier.

[0054] In one embodiment, the microorganism or composition of the disclosure comprises about 1 x 10 8 cells / mL, approximately 1×10 7 cells / mL, approximately 1×10 6 cells / mL, approximately 1×10 5 cells / mL, approximately 1×10 4 cells / mL, approximately 1×10 3 cells / mL, approximately 1×10 2 It can be added to breeding water to give a density of 10 cells / mL or about 10 cells / mL and used for fish.

[0055] (Applicable environment) The microorganisms or compositions of the present disclosure can be used in any suitable environment. In one embodiment, the microorganism or composition of the present disclosure may be used in any temperature environment, such as 0 to 100°C, 5 to 70°C, 10 to 50°C, 15 to 40°C, 20 to 35°C, less than 70°C, less than 60°C, less than 50°C, less than 40°C, less than 30°C, less than 25°C, less than 20°C, less than 15°C, less than 10°C, less than 5°C, less than 0°C, about 70°C, about 60°C, about 50°C, about 40°C, about 30°C, about 25°C, about 15°C, about 10°C, about 5°C, or about 0°C.

[0056] In one embodiment, the microorganism or composition of the present disclosure can be used in any pH environment, such as pH 3 to 13, pH 4 to 12, pH 5 to 11, pH 6 to 10, pH 7 to 9, pH 5.5 to 8.5, about pH 3, about pH 4, about pH 5, about pH 6, about pH 7, about pH 8, about pH 9, about pH 10, about pH 11, about pH 12, or about pH 13.

[0057] In one embodiment, the microorganisms or compositions of the present disclosure may be used in an environment with any dissolved oxygen concentration (DO), such as about 0.05 mg / L, about 0.1 mg / L, about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 3 mg / L, about 5 mg / L, about 8 mg / L, or about 10 mg / L, and in one embodiment, may be used in an environment with a dissolved oxygen concentration (DO) of up to about 8 mg / L, which is close to saturation or suitable for fish farming.

[0058] In one embodiment, the microorganisms or compositions of the present disclosure may be used in an environment with any salt (e.g., sodium chloride) concentration, such as about 0 g / L, about 0.05 g / L, about 0.1 g / L, about 0.5 g / L, about 0.7 g / L, about 1 g / L, about 1.5 g / L, about 2 g / L, about 2.5 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 7 g / L, or about 10 g / L.

[0059] In one embodiment, the microorganisms or compositions of the present disclosure may be used in the presence of salts, surfactants, light, electric current, agitation, aeration, or any combination thereof.

[0060] In one embodiment, the microorganism or composition of the present disclosure may be used with a carrier capable of immobilizing the microorganism. The use of such a carrier can effectively prevent washout. The material of the carrier is not particularly limited as long as it is capable of immobilizing the microorganism. Examples include carbon fiber (PAN-based, pitch-based, phenolic resin-based, etc.), polyethylene resin, polypropylene resin, polyurethane resin, polystyrene resin, polyvinyl chloride resin, polyvinyl acetate resin, polyvinyl alcohol resin, polyethylene glycol resin, acrylic resin, gelatin, sodium alginate, carrageenan, dextrin, ceramics, silicon, metal, charcoal, activated carbon, minerals (zeolite, diatomaceous earth, etc.), glass, glass waste, and composites thereof. To increase the immobilization rate and the efficiency of microbial activity, it is preferable to use a porous or fibrous carrier. Alternatively, the microorganism may be encapsulated in a gel-like carrier. The shape of the carrier may be, for example, a cube, a rectangular parallelepiped, a cylinder, a sphere, a disk, a sheet, a film, or the like. For information on microbial immobilization technology, see, for example, "Wastewater Treatment by Microbial Immobilization Methods" (edited by Ryuichi Sudo, Industrial Water Research Association) and "Water Treatment by Microbial Immobilization Methods - Carrier Immobilization Method, Entrapment Immobilization Method, Biological Activated Carbon Method (New Water Treatment Series (1))" (written by Kazuhiro Mochizuki, Katsutoshi Hori, and Hideki Tatemoto, NTS Co., Ltd.).

[0061] In one embodiment, the microorganisms or compositions of the present disclosure may be used alone or in combination with other microorganisms without the use of a carrier, allowing the microorganisms to form flocs or granules by themselves. Additionally, nucleating materials or microorganisms that promote the formation of flocs or granules may be used.

[0062] (additional ingredients) In one embodiment, the microorganism or composition of the present disclosure may be used in combination with additional components, which may be added to the composition or may be used separately from the microorganism or composition, and if used separately, may be provided as a kit.

[0063] In one embodiment, the additional components include, but are not limited to, components that enhance the activity of the microorganisms used, surfactants, drying protectants, components for maintaining the microorganisms for a long period of time, preservatives, excipients, strengthening agents, antioxidants, dispersants, flocculants, and other microorganisms, and any suitable components can be used.

[0064] In another embodiment, the additional component may include any component that can be used when contacting or applying to fish, such as a fish culture liquid or a fish culture component. When such an additional component is included, the composition can be used as is when used in fish culture, allowing the fish to be raised while being protected. The fish culture liquid, the fish culture component, etc. may be provided as part of a composition, or may be used separately from the microorganism or composition, or may be provided as a kit.

[0065] (Method using microorganisms) In one aspect, the present disclosure provides a method for protecting fish, comprising contacting the fish with a microorganism or composition of the present disclosure. In one embodiment, the method for protecting fish comprises raising the fish in water containing the microorganism of the present disclosure. The fish to be protected can be any of the fish described herein to which the microorganism or composition of the present disclosure can be applied. In one embodiment, the microorganism or composition of the present disclosure can be applied to the same species of fish as the fish from which the microorganism was obtained from its skin, or to a different species of fish. In one embodiment, the microorganism or composition of the present disclosure can be applied to fish raised in the same environment (e.g., a fish farm) as the fish from which the microorganism was obtained from its skin, or to fish raised in a different environment. The method for protecting fish of the present disclosure can be carried out in any of the environments described herein to which the microorganism or composition of the present disclosure can be applied. The method for protecting fish of the present disclosure can use any of the additional components described herein that can be used in combination with the microorganism or composition of the present disclosure.

[0066] In one embodiment, the disclosed method includes treating or preventing a disease or illness in fish. Alternatively, the disclosed method includes protecting the skin of fish. Targeted fish diseases or illnesses include, for example, skin diseases and infectious diseases, such as vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, sweetfish pseudomonas disease, redmouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), coldwater disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis.

[0067] In some embodiments, the microorganism or composition of the present disclosure comprises an effective amount of the Pseudomonadaceae bacterium of the present disclosure, or is provided so as to provide an effective amount upon contact. In one embodiment, the microorganism or composition of the present disclosure may be provided in water in which an effective amount of the Pseudomonadaceae bacterium of the present disclosure is present, and fish may be grown therein.

[0068] In this way, by providing an effective amount of the microorganism or composition of the present disclosure, fish are protected from at least one harmful microorganism. The harmful microorganism that can be protected by the method of the present disclosure can be any of those described herein, such as a microorganism that has at least one of the following abilities: causing skin diseases in fish, transmitting infection through the skin, infecting fish through wounds, and infecting fish through contact. Specific examples include harmful microorganisms that can cause vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Edwardsiellosis, red spot disease, pseudomonas disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), coldwater disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. Such harmful microorganisms include Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas anguilliseptica, Pseudomonas plecoglossicida, Yersinia ruckeri, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum, Tenacibaculum maritimum, Renibacterium salmoninarum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia seriolae, Streptococcus iniae, Lactococcus garvieae, Aeromonas caviae or a combination thereof, etc.

[0069] Therefore, a method for protecting fish, which includes a step of contacting the microorganisms or compositions of the present disclosure with fish or a step of raising fish in water containing the microorganisms of the present disclosure, may include a step of testing the fish for harmful microorganisms before, during, or after contacting the fish with or raising the fish with the microorganisms or compositions. Testing for such harmful microorganisms can be carried out by techniques known in the art, and samples may be collected directly from the fish or from the rearing solution during rearing.

[0070] In a preferred embodiment, once the type of harmful microorganism present is examined and the type of harmful microorganism is identified, preliminary experiments may be conducted to determine the optimal conditions for administering the microorganism or composition of the present disclosure. Such preliminary experiments may include, for example, confirming the effectiveness against the harmful microorganisms present and the effective concentration. The microorganism or composition of the present disclosure to be administered may also include confirming in advance the safety of the microorganism or composition to be administered to the fish species to be protected. Such safety confirmation may include determining the acceptable concentration and other administration conditions, and any method known in the art may be used. Once such conditions, such as the acceptable concentration, are determined, actual contact and rearing may be carried out taking into account the appropriate administration conditions and the appropriate effective concentration.

[0071] In another embodiment, in a method for protecting fish, comprising contacting fish with a microorganism or composition of the present disclosure or rearing fish in water containing the microorganism of the present disclosure, the Pseudomonadaceae bacteria of the present disclosure are provided under conditions sufficient for colonization on the epidermis of the fish. The conditions sufficient for colonization on the epidermis of the fish may be determined in advance, and in such cases, the contact or rearing is carried out under those conditions. Alternatively, a preliminary experiment may be carried out to determine the conditions sufficient for colonization on the epidermis of the fish during contact or rearing, and any method known in the art may be used.

[0072] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and include, for example, Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., and Vahaboglu, H. 2003. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J. Med. Microbiol. 52:403-408; Marie-Ange Teste, Manon Duquenne, Jean M Francois and Jean-Luc Parrou 2009. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Molecular Biology 10:99; Bergey's Manual of Systematic Bacteriology, Bergey's Manual of Systematic of Archaea and Bacteria, etc., which are incorporated herein by reference in relevant parts (possibly in their entirety).

[0073] (Note) In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when "within a range" of "two values" is specified, the range includes the two values ​​themselves.

[0074] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth. The present disclosure has been described above with reference to preferred embodiments for ease of understanding.

[0075] The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Example]

[0076] Examples are described below. When necessary, the handling of organisms used in the following examples complied with the standards stipulated by Nagoya University, regulatory authorities, and the Cartagena Protocol. While the specific reagents used were those listed in the examples, equivalent products from other manufacturers (Sigma-Aldrich, Fujifilm, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science Inc., Kanto Chemical, Funakoshi, Tokyo Kasei, Merck, etc.) can also be used.

[0077] Example 1: Isolation of Microorganisms from Zebrafish Epidermis Adult zebrafish (Danio rerio) were obtained from Mie University and Masuko Suikei and maintained in our laboratory. The fish were maintained in aquaria containing water different from that of the source until the experiment. CROSS MINI (NWC-341; NISSO) aquaria were used. Zebrafish were fed twice daily with Tetramin Super (17653; Spectrum Brands Japan) using a Tetra Auto Feeder (AF-3; Spectrum Brands Japan) every 12 hours. Lighting was provided by a Tetra LED Mini Light (73333; Spectrum Brands Japan), which was turned on and off every 12 hours using a digital timer (PT70DW; REVEX). Water temperature was maintained at 28°C using a Safe Cover Heat Navi SH80 (7775; Gex).

[0078] Zebrafish were anesthetized by swimming in 2 ml of tricaine solution (4.0 mg / ml tricaine, 0.021 M Tris) per 100 ml of ultrapure water, and the mucous membrane on the body surface was scraped with a Mentip (1p1504; Nippon Cotton Swab, Tokyo). The Mentip was immersed in 1 ml of ultrapure water and vortexed to obtain a suspension. This suspension, along with a 5-fold dilution of this suspension with ultrapure water, was sprayed in 200 μl aliquots onto Nutrient Broth Agar (NB medium), Enriched Cytophaga Agar, Zobell 2216E Agar, and Tryptone Soya Broth Agar (TSA medium). The agar plates were then cultured at 28°C for 2 days (Figure 1). The resulting 124 colonies were then subjected to the cross-streak method. Composition of various media [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5]

[0079] (Example 2: Screening for useful bacteria by the Cross Streak method) The pathogenic bacteria used were Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 21327), Yersinia ruckeri (1) (NVH 3758), and Yersinia ruckeri (2) (DSMZ 18506). Escherichia coli DH5α was used as a representative nonpathogenic bacterium. These bacteria were precultured overnight and used for screening.

[0080] The cross streak method was performed with reference to the method of Toth et al. (Erika M. Toth, Andrea K. Borsodi, Tamas Felfoeldi, Balazs Vajna, Rita Sipos, Karoly Marialigeti. 2013. Practical Microbiology: based on the Hungarian practical notes entitled "Mikrobiologiai Laboratoriumi Gyakorlatok". Marialigeti EMTaK, editor. Budapest: Eoetvoes Lorand University.) To evaluate growth inhibition of harmful microorganisms, candidate microorganisms obtained from each colony in Example 1 were streaked linearly with a Mentip on NB agar medium (composition described in Example 1) in a 9 cm diameter plate and cultured overnight at 28°C. Subsequently, harmful microorganisms were streaked with a Mentip perpendicular to the candidate microorganism streak without touching the candidate microorganism, and cultured overnight at 28°C. Growth inhibition ability was determined to be present when a clear zone was present over an area of ​​10 mm or more from the candidate microorganism streak (Figure 2). The growth inhibitory abilities of various candidate microorganisms are shown below. [Table 1] KH-ZF1 had particularly excellent inhibitory activity against harmful microorganisms (Figure 3).

[0081] (Example 3: Purification of isolated useful bacteria) The isolated KH-ZF1 was streaked onto NB agar medium (composition described in Example 1) and cultured at 28°C for 2 days. Five colonies were then selected and cultured overnight in liquid NB medium at 28°C, and the culture medium was used to perform the cross-streak method described above. One colony that showed growth inhibition in the cross-streak method was selected, streaked onto agar medium, and cultured at 28°C for 2 days. This procedure was repeated five or more times.

[0082] Example 4: Ability of beneficial microorganisms to inhibit harmful microorganisms The harmful microorganisms used in the microbial inhibition tests were Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Yersinia ruckeri (1) (NVH 3758), Yersinia ruckeri (2) (DSMZ 18506), Edwardsiella tarda (1) (NRIA44), Edwardsiella tarda (2) (NRIA51), Vibrio anguillarum (NRIA83), Vibrio ordalii (NRIA90), and Streptococcus iniae (NRIA599). Two non-harmful microorganisms were also tested on the same plates as negative controls. Overnight cultures of these microorganisms were used.

[0083] An autoclave-sterilized paper disk (49005010; Toyo Roshi, Tokyo) was placed in the center of NB agar medium in a 9 cm diameter Petri dish, and 20 μl of KH-ZF1 cell suspension (KH-ZF1 was cultured in NB medium (composition described in Example 1), then centrifuged at 5000 × g for 10 minutes, the precipitated cells were collected, and resuspended in sterilized water to an OD600 of 1.0) was impregnated into the paper disk and allowed to stand at 20°C for 2 days. The test microorganism was then streaked radially from the KH-ZF1 addition site with a Mentip, without touching the KH-ZF1, and cultured at 20°C for 2 days. This streaking was specifically performed as follows. The culture solution (OD 600 =2-3) The cotton part of the mentip was immersed in 2 mL of the culture medium, and excess water was wiped off. The mentip was then moved at an angle of approximately 20-30° to the surface of the agar medium, applying enough force so as not to indent the surface, to streak approximately 100 μL of the bacterial culture medium radially from near the paper disc on the dish, taking care not to touch the grown KH-ZF1. The results are shown in Figure 4. Selective inhibition of harmful microorganisms compared to non-harmful microorganisms was observed.

[0084] (Example 5: Identification of useful bacteria) Species identification of KH-ZF1 by 16S ribosomal RNA (rRNA) was commissioned to Techno Suruga Lab (Shizuoka Prefecture) and was carried out as follows. DNA was extracted from the cultured KH-ZF1 strain using achromopeptidase (Fujifilm Wako Pure Chemical Industries, Osaka, Japan). PCR amplification was performed using Tks Gflex DNA Polymerase (Takara Bio, Shiga, Japan). Cycle sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Primers 9F and 1510R were used for PCR amplification, and primers 9F, 515F, 1099F, 536R, 1242R, and 1510R were used for sequencing. Sequencing was performed using an ABI PRISM 3130 x1 Genetic Analyzer System (Applied Biosystems, USA). Nucleotide sequences were determined using ChromasPro 2.1 (Technelysium, Australia).

[0085] For BLAST homology searches, we used the analysis software ENKI (Techno Suruga Lab, Shizuoka Prefecture), the database DB-BA14.1 (Techno Suruga Lab, Shizuoka Prefecture), and the international nucleotide sequence database (DDBJ / ENA (EMBL) / GenBank). For molecular phylogenetic analysis, we estimated phylogenetic trees using the neighbor-joining method, used a Kimur-2-parameter base substitution model, and evaluated the reliability of the tree structure using the bootstrap method (1000 iterations).

[0086] As a result, KH-ZF1 showed 99.8% homology to Pseudomonas mosselii (CIP105259) in a BLAST search against DB-BA, and 99.9% homology to Pseudomonas mosselii (PtA1) in a BLAST search against the international nucleotide sequence database, and was identified as Pseudomonas mosselii. 16S rRNA portion of KH-ZF1 ofA molecular phylogenetic tree based on the base sequences is shown in Figure 5.

[0087] (Example 6: Fish skin adhesion test) Six zebrafish (Danio rerio) were placed in each Erlenmeyer flask with 100 ml of rearing water (1 L of ultrapure water plus 3 g of Instant Ocean). KH-ZF1 cells transfected with mCherry were added to the rearing water to an OD600 of 0.01. The zebrafish were reared at 20°C. Six and 24 hours after KH-ZF1 addition, the zebrafish's body surface (both sides) was scraped with a mentip. The mentip was then immersed in 0.5 ml of sterile rearing water and agitated to suspend the zebrafish. This suspension was plated onto 9 cm Difco™ Pseudomonas Isolation Agar plates (Becton Dickinson Japan, Tokyo) and incubated at 28°C for 12 hours. Colony-forming units (CFU) of KH-ZF1 cells were counted based on fluorescence.

[0088] KH-ZF1 cells transfected with a fluorescent protein (mCherry) were prepared as follows. Transposon vector pBSL::mCherry km r Using E. coli WM6026 carrying the gene, the gene was introduced into KH-ZF1 by conjugation and cultured on Pseudomonas isolation agar medium supplemented with kanamycin. r The gene-transfected KH-ZF1 was obtained.

[0089] The results are shown in Figure 6. It was observed that KH-ZF1 adhesion to the body surface of zebrafish increased over time.

[0090] (Example 7: Infection prevention test) The infection control ability of KH-ZF1 was investigated. The test procedure is as follows. 200 ml of rearing water (3 g of Instant Ocean per 1 L of ultrapure water) was placed in a 500 ml Erlenmeyer flask, and five zebrafish (Danio rerio) were placed per flask. The zebrafish were reared at 28°C for two days. The rearing water in the Erlenmeyer flask was replaced, and KH-ZF1 was added to an OD600 of 0.01. The zebrafish were reared at 28°C for 24 hours. The zebrafish were anesthetized by transferring them from the Erlenmeyer flask to an anesthetic solution (400 mg of tricaine, 2.1 ml of 1 M Tris (pH 9), and 2 ml of a stock solution (adjusted to pH 7) in 97.9 ml of ultrapure water, added to 100 ml of ultrapure water). A wound was made in the muscle at the base of the dorsal fin of each zebrafish using a syringe needle (Terumo syringe needle, NN-1838R, 18G (1.20 mm), R / B blade (12° blade angle), 38 mm needle length). The water in the Erlenmeyer flask was replaced, the injured zebrafish was placed in, and KH-ZF1 was added to an OD600 of 0.01. The zebrafish were then reared at 28°C for 24 hours. The temperature was then lowered from 28°C to 20°C, and the zebrafish were reared for 24 hours. The water in the Erlenmeyer flask was replaced, and KH-ZF1 and Yersinia ruckeri (NVH3758) were added to an OD600 of 0.01, and the zebrafish were then reared at 20°C for 1 hour. The water in the Erlenmeyer flask was replaced, KH-ZF1 was added to an OD600 of 0.01, and the zebrafish were kept at 20°C while monitoring their survival (no water replacement was performed thereafter) (Figure 7). Air was pumped into the Erlenmeyer flask with an air pump, and the flask was covered with plastic wrap to prevent evaporation of the water. In addition to the treatment group administered with KH-ZF1, a control group was also tested in which no KH-ZF1 was added in the above procedure.

[0091] KH-ZF1 and Yersinia ruckeri (NVH3758) used in the test were prepared as follows. Preparation of KH-ZF1 KH-ZF1 cultured on a plate was inoculated into a 15 ml tube containing 2 ml of NB medium and cultured with shaking at 28°C for 24 hours. 100 μl of this culture was inoculated into a 100 ml Erlenmeyer flask containing 10 ml of NB medium and cultured with shaking at 28°C for 24 hours. The culture was centrifuged at 8000 × g for 5 minutes, and the supernatant was discarded to collect the cells. The cells were resuspended in breeding water, washed, centrifuged at 8000 × g for 5 minutes, and the supernatant was discarded to collect the cells (this process was repeated three times). The cells were resuspended in breeding water and the OD600 was measured. Preparation of Yersinia ruckeri (NVH3758) Yersinia ruckeri cultured on a plate was inoculated into a 15 ml tube containing 2 ml of LB medium and cultured with shaking at 28°C for 24 hours. 100 μl of this culture was inoculated into a 100 ml Erlenmeyer flask containing 10 ml of LB medium and cultured with shaking at 28°C for 12 hours. The culture was centrifuged at 8000 × g for 5 minutes, and the supernatant was discarded to collect the bacteria. The bacteria were resuspended in breeding water, washed, centrifuged at 8000 × g for 5 minutes, and the supernatant was discarded to collect the bacteria (this process was repeated three times). The bacteria were resuspended in breeding water and the OD600 was measured.

[0092] The results are shown in Figure 8. A log-rank test revealed a significant difference in survival rate between the treatment group and the control group, with P = 0.004.

[0093] (Example 8: Useful microorganisms obtained from rainbow trout skin) The mucous membrane on the body surface of adult rainbow trout (Oncorhynchus mykiss) cultivated at Ure Fish Farm (Aichi Prefecture) and Ishii Fish Farm (Gifu Prefecture) was scraped off. Colonies were formed on an agar medium in the same manner as in Example 1.

[0094] Strains obtained from these colonies were evaluated for growth inhibitory activity and competitive advantage.

[0095] The growth inhibitory activity was evaluated in the same manner as in Example 2 and Figure 2. The pathogenic bacteria used were Yersinia ruckeri (NVH 3758), Aeromonas hydrophila (NRIA14), Vibrio anguillarum (NRIA83), and Vibrio ordalii (NRIA90). The streaking method was the same as in Example 4.

[0096] Competitive advantage was evaluated as follows (see also Figure 9). Yersinia ruckeri, Aeromonas hydrophila, Vibrio anguillarum, and Vibrio ordalii were used as pathogenic bacteria (harmful microorganisms) (the same strains as those used in the growth inhibitory activity test). These bacteria were pre-cultured overnight and used for screening. Candidate microorganisms obtained from each colony were streaked linearly onto NB agar medium (composition described in Example 1) in a 9-cm diameter plate with a Mentip and cultured overnight at 28°C. The Mentip was then moved perpendicular to the streak to intersect with the streak of the candidate microorganism, streaking the harmful microorganism. The candidate microorganisms were mixed with the harmful microorganisms and cultured overnight at 28°C. A competitive advantage was determined if the candidate microorganism grew preferentially in the area beyond the streak of the candidate microorganism on the streak of the Mentip carrying the harmful microorganism. The streak method was the same as in Example 4.

[0097] As a result, KH-RT1, KH-RT2, KH-RT3 and KH-RT4 had excellent inhibitory ability against harmful microorganisms, as shown in the table below. [Table 2] [Table 3]

[0098] Next, DNA was extracted from the cultures KH-RT1, KH-RT2, KH-RT3, and KH-RT4. Based on the DNA sequence, we performed sequence comparison with the V1-V3 region of the 16S ribosomal RNA gene. KH-RT1 was identified as Pseudomonas marginalis, KH-RT2 as Pseudomonas koreensis, and KH-RT3 as Pseudomonas protegens. KH-RT4 is thought to be a Pseudomonas microorganism, specifically Pseudomonas parafulva. Further useful strains can be isolated in a similar manner.

[0099] Example 9 The protective effect of beneficial bacteria on larvae was tested. KH-ZF1 cultured at 28°C was added to the rearing water to achieve an OD600 of 0.01. Sixty newly hatched rainbow trout larvae were reared in this rearing water at 15°C with aeration. Seventy-four newly hatched rainbow trout larvae were reared under control conditions without KH-ZF1. On day 3 (after counting the number of survivors), some larvae were collected for microscopic observation twice between days 5 and 18. On day 18, some larvae were collected for microscopic observation and exposure testing, and the rearing water was collected for CFU counts. The rearing water was not changed. Survival rates were checked on days 0, 3, 5, 18, 26, 28, and 31 (the larvae collected for observation were not returned and were excluded from the count).

[0100] The results are shown in Figures 10 and 11. On the third day after exposure, KH-ZF1 was observed to be established on the surface of the larvae (Figure 10). The addition of KH-ZF1 improved the survival rate of the larvae, with a particularly high improvement observed from day 26 onwards. KH-ZF1 was confirmed to have a high protective effect on fish larvae. Similar protective effects can be observed with KH-RT1, KH-RT2, KH-RT3, KH-RT4 or other strains.

[0101] Other embodiments Example 10 Harmful microorganisms are administered to both groups of rainbow trout, which are divided into a treatment group administered with KH-RT1, KH-RT2, KH-RT3, KH-RT4 or other strains, and a control group that is not administered with the strains, and it is confirmed that the survival rate of the rainbow trout in the treatment group is higher than that of the control group.

[0102] Example 11 Similar to KH-ZF1, KH-RT1, KH-RT2, KH-RT3, KH-RT4, or other strains are prepared by introducing a fluorescent dye gene. These strains are administered to rainbow trout, which are then raised for a period of time. Microorganisms are periodically scraped off the epidermis with a Mentip, and the CFU of the strains are counted based on the fluorescent coloration in the same manner as in Example 6 to confirm their colonization on the fish epidermis.

[0103] Example 12 Similar to Example 7, rainbow trout are injured and reared in rearing water (treatment group) containing KH-RT1, KH-RT2, KH-RT3, KH-RT4, or other strains, and harmful microorganisms (e.g., Yersinia ruckeri). Improved survival rates are confirmed compared to a control group in which KH-RT1, KH-RT2, KH-RT3, or KH-RT4 was not added to the rearing water.

[0104] (Note) While the present disclosure has been illustrated by preferred embodiments thereof, it is understood that the scope of the present invention is to be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein.

[0105] (Note) This application claims the benefit of priority to Japanese Patent Application No. 2019-106436, filed on June 6, 2019, the entire contents of which are incorporated herein by reference. [Industrial Applicability]

[0106] The present disclosure provides microorganisms and methods for protecting fish, thereby achieving more stable, inexpensive, highly productive and / or diverse fish breeding, and / or reducing the environmental burden by reducing the use of existing drugs, etc. [Accession number]

[0107] KH-ZF1 (Accession number: NITE BP-02967) KH-RT1( Contract Number: NITE B P-03222) KH-RT2( Contract Number: NITE B P-03223) KH-RT3( Contract Number: NITE B P-03224) KH-RT4( Contract Number: NITE B P-03225)

Claims

1. A Pseudomonadaceae bacterium or a culture thereof, the bacterium being KH-ZF1 (accession number: NITE BP-02967), KH-RT1 (accession number: NITE BP-03222), KH-RT2 (accession number: NITE BP-03223), KH-RT3 (accession number: NITE BP-03224), or KH-RT4 (accession number: NITE BP-03225).

2. A fish protecting agent comprising the Pseudomonadaceae bacterium according to claim 1 or a culture thereof.

3. A probiotic agent for fish, comprising the Pseudomonadaceae bacterium according to claim 1 or a culture thereof.

4. The probiotic agent according to claim 3, which is a probiotic for the epidermis of the fish.

5. The probiotic agent according to claim 3 or 4, wherein the Pseudomonadaceae bacterium has the ability to colonize the epidermis of fish.

6. The fish protecting agent according to claim 2 or the probiotic agent according to any one of claims 3 to 5, which is a protecting agent or probiotic agent for farmed fish.

7. The fish protecting agent according to claim 2 or the probiotic agent according to any one of claims 3 to 5, which is a protecting agent or probiotic agent for eel, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, flounder, tilapia, pufferfish, yellowtail, grouper, mackerel, saury, or catfish.

8. The fish protecting agent according to claim 2 or the probiotic agent according to any one of claims 3 to 5, which is a protecting agent or probiotic agent for salmonid fish.

9. The fish protecting agent according to claim 2 or the probiotic agent according to any one of claims 3 to 5, which is a protecting agent or probiotic agent for trout.

10. 10. A method for protecting fish, comprising contacting said fish with the Pseudomonadaceae bacterium of claim 1.

11. A method for protecting fish, comprising the step of growing said fish in water in which the Pseudomonadaceae bacterium of claim 1 is present.

12. 1. A method for obtaining microorganisms capable of protecting fish, comprising: (a) obtaining candidate microorganisms from the epidermis of fish; (b) adding the candidate microorganism to a medium containing harmful microorganisms; (c) confirming the inhibition of the harmful microorganism in the culture medium; and (d) when the inhibition of the harmful microorganism in the culture medium is confirmed, a step of identifying the candidate microorganism as a microorganism capable of protecting the fish; A method comprising:

13. 13. The method according to claim 12, wherein in step (c), the inhibition of the harmful microorganisms on a solid medium is confirmed.

14. 13. The method according to claim 12, wherein in step (c), the candidate microorganism and the harmful microorganism are grown on the same medium, and a growth inhibition zone in which the harmful microorganism cannot grow is confirmed near the candidate microorganism.

15. The method according to claim 12, wherein in step (c), the inhibition of the harmful microorganism is confirmed in a liquid medium containing the candidate microorganism or its culture supernatant and the harmful microorganism.

16. The method according to claim 12, wherein in step (c), a bottomless cylinder containing an impregnated material containing a culture medium or culture supernatant of the candidate microorganism or the culture medium or the culture supernatant is placed on a medium inoculated entirely with the harmful microorganism, and the inhibition of the harmful microorganism around the impregnated material or cylinder is confirmed.

17. The method according to claim 12, wherein in step (c), after incubating the liquid medium containing the candidate microorganism and the harmful microorganism, the growth of the candidate microorganism and the harmful microorganism are compared to confirm that the growth of the harmful microorganism is more inhibited.

18. The method according to claim 12, wherein in step (c), the candidate microorganism is inoculated onto a culture medium inoculated entirely with the harmful microorganism or onto a culture medium in which the harmful microorganism has grown entirely, and it is confirmed that the candidate microorganism forms a colony and that a zone of growth inhibition of the harmful microorganism is observed around the colony.

19. The method according to any one of claims 12 to 18, wherein the candidate microorganisms are obtained from colonies formed on a culture medium inoculated with scrapings of the fish's epidermis.

20. The method according to any one of claims 12 to 19, wherein one or more microorganisms are obtained as microorganisms capable of protecting said fish.

21. The method according to any one of claims 12 to 20, wherein the harmful microorganism has at least one of the following abilities: an ability to cause a skin disease in fish; an ability to infect fish through the skin; an ability to infect fish through a wound; and an ability to infect fish through contact.

22. The method according to any one of claims 12 to 20, wherein the harmful microorganism is capable of causing vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, edwardsiellosis, red spot disease, pseudomonas disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis.

23. The method according to any one of claims 12 to 22, wherein the candidate microorganism is obtained from the epidermis of zebrafish (Danio rerio) or trout.

24. 24. A method for protecting fish, comprising obtaining a microorganism by the method of any one of claims 12 to 23, and contacting said microorganism with said fish.

Images