Bacterial strains for use as probiotics, their compositions, deposited strains, and methods for identifying probiotic bacterial strains

Abstract

The present invention relates to a bacterial strain or a combination of bacterial strains selected from the group consisting of a Chryseobacterium massilia strain, a Flavobacterium species strain whose genome comprises SEQ ID NO: 1 or has an average nucleotide identity (ANI) value of at least 95% with the Flavobacterium species strain identified by accession number I-5481 deposited at the Collection Nationale De Culture De Microorganismes (CNCM) on January 24, 2020, and mutants thereof, for use in fish as a probiotic. The bacterial strain may have an average nucleotide identity (ANI) value of at least 95% with the Chryseobacterium massilia strain, whose genome comprises SEQ ID NO: 2 or is identified by accession number I-5479, deposited at the CNCM on January 24, 2020, and / or the Flavobacterium species strain, whose genome comprises SEQ ID NO: 1 or is identified by accession number I-5481, deposited at the CNCM on January 24, 2020, or a variant thereof. The probiotic use may be aimed at preventing or minimizing infections by Flavobacterium columnare in fish. The present invention also relates to the deposited bacterial strain, or a probiotic composition, food product, or kit comprising the same, and to a method for identifying bacterial strains that are probiotics against pathogen infections.

Description

[Technical Field]

[0001] The present invention relates to the field of probiotics, particularly in the form of bacterial strains, as active ingredients used as probiotics or as active ingredients suitable for the design of probiotic compositions to be administered to fish. The present invention therefore encompasses the use of such active ingredients for the treatment of said fish, i.e., for preventive and / or beneficial effects on their health. The active ingredients of the present invention have been shown to be particularly beneficial against challenge infections with the bacterium Flavobacterium columnare. The present invention also relates to a method for identifying probiotic bacterial strains. [Background technology]

[0002] Animal resident microbial communities form complex, long-term communities that perform important population-level functions essential for host growth and pathology [1, 2]. Microbiota ecosystems also provide protection against exogenous pathogens by inhibiting pathogen colonization in combination with developing and / or stimulating the host immune system [3-7]. From the perspective of microbial community composition, changes or declines in the diversity of resident microorganisms, a phenomenon commonly referred to as dysbiosis, are often accompanied by compromised susceptibility due to reduced or altered abundance of key microbial community members [7, 8]. These findings have already supported the idea that the addition or augmentation of individual or collective protective bacteria (e.g., probiotics) may minimize microbiota dysbiosis or even prevent infection, thereby restoring host health (9-11).

[0003] While probiotics have demonstrated efficacy in animals and humans, their mechanisms of action are not fully understood, and diversity surveys or low-throughput experimental models provide limited information regarding species contributions to community function [1, 6, 12-14]. Furthermore, characterization of bacterial strains that improve colonization resistance is hampered by the complexity of the host-parasite ecosystem. Recently, zebrafish have emerged as a powerful tool for investigating microbial-microbial and host-microbial interactions [15-20]. Zebrafish can be easily reared in axenic or gnotobiotic conditions with specific bacterial species [15, 21]. Furthermore, zebrafish bacterial populations have become increasingly well characterized, and many phylogenetically distinct zebrafish bacteria can be cultured, making this model system suitable for microbiome manipulation and evaluation of the effects of probiotics on host infection resistance [22-25]. Several studies have used zebrafish to evaluate the effects of exogenous addition of potential probiotics on host infection resistance [23-25]. Various lactic acid bacteria (Lactobacilli spp., Bacillus spp.) have been shown to improve infection outcomes against many zebrafish pathogens (e.g., Aeromonas hydrophila, A. veronii, Streptococcus agalactiae, and Vibrio parahaemolyticus) [26–30], but the reported protection is often partial, highlighting the difficulty of identifying exogenous probiotics that provide sufficient protection.

[0004] We used germ-free, standard zebrafish larvae to mine the native parasitic microbiota for bacterial species protection from Flavobacterium columnare, a Bacteroides pathogen that affects wild and farmed fish species, including carp, channel catfish, goldfish, eel, salmon, and tilapia. [31, 32] We identified two infection resistance scenarios that prevented mortality caused by Flavobacterium columnare, mediated by the Bacteroides pathogen Chryseobacterium massiliae or by a consortium of nine other non-protective bacterial species that formed a protective population. These results provide a powerful approach to mining host microbiota and identifying key members mediating colonization resistance, providing insights into how microbial communities can be modified to protect against pathogens both within and outside aquaculture environments.

[0005] Indeed, with wild fish stocks reaching biologically unsustainable limits, fish aquaculture is a rapidly growing industry, providing more than half of all fish consumed.

[0101] However, intensive aquaculture fosters the rapid proliferation of pathogens, and high mortality rates in aquaculture facilities pose a significant obstacle to fish meat production. [102-104] These health problems primarily affect immunologically immature fish larvae, for which vaccination is impractical. [105, 106] Prompting the prophylactic and therapeutic use of antibiotics and chemical disinfectants to prevent fish diseases. [107-109] However, the expanded use of antibiotics and chemical disinfectants poses widespread human health concerns, including risks to end-consumer safety, environmental contamination, and the spread of antibiotic resistance. [104, 110] In this respect, the use of probiotics to improve fish health and protect susceptible juveniles represents an economically and ecologically viable alternative to antibiotic treatment [111-113].

[0006] Probiotics are live microorganisms that confer health benefits to the host through mechanisms including growth promotion, immune stimulation, or direct inhibition of pathogenic microorganisms [114-116]. Given the important protective role that the host microbiota plays against pathogenic microorganisms, a process known as colonization resistance [117-119], microbial populations associated with fish are considered an interesting source of probiotic bacteria [120-122]. Indeed, the gastrointestinal tract and mucus of fish are the most common sources of potential fish probiotics [123, 124]. However, probiotic bacteria selection is often empirical or hampered by the lack of repeatability and reproducibility of in vivo approaches, often performed under relatively poorly controlled conditions and with high inter-microbial variability in composition [121, 125].

[0007] To circumvent the experimental difficulties associated with evidence-based identification of fish probiotics, the use of germ-free or well-controlled gnotobiotic hosts is a promising approach [126, 127], and several economically important fish species, including Atlantic cod (Gadus morhua L.)

[0128] , Atlantic halibut (Hippoglossus hippoglossus)

[0129] , European sea bass (Dicentrarchus labrax)

[0125] , or turbot (Scophthalmus maximus)

[0130] , have been successfully reared under germ-free conditions (see [131, 132] for a description). In other studies, germ-free or gnotobiotic-reared laboratory zebrafish (Danio rerio)

[0133] have been successfully reared under germ-free conditions. The effect of exogenous probiotics on host infection was evaluated using the fish, Oncorhynchus rerio [133-136]. However, in many cases, the tested probiotics were exogenous to the host and the short-term microbiota residents, and therefore provided only partial protection from the tested pathogens. Therefore, we developed a novel probiotic that protects rainbow trout (Oncorhynchus mykiss) from infection with Flavobacterium columnare, a fish pathogen responsible for large-scale losses in aquaculture fish species, especially catfish and salmon. We investigated the possibility of members of the zebrafish microbiota (Zebrafish mykiss) being protected from Flavobacterium columnarum infection using a novel protocol for rearing trout larvae under germ-free conditions. Using a novel protocol for rearing trout larvae under germ-free conditions, we showed that germ-free and non-germ-free trout larvae were highly susceptible to Flavobacterium columnarum infection. We then used reconventionalization of germ-free trout to identify bacterial species from the trout microbiota (Flavobacterium spp.) or zebrafish microbiota (Chryseobacterium massilia) that fully restored protection from Flavobacterium columnarum infection.The results show that these new gnotobiotic models make it possible to mine the teleost microbiota and rationally identify fish probiotics that, alone or in combination, may contribute to protection from columnaris disease in rainbow trout and other fish for aquaculture research and management. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Prado et al., Applied Microbiology and Biotechnology, Vol. 104, pp. 1993–2006 (2020) doi.org / 10.1007 / s00253-019-10332-0 [Non-patent document 2] Amir et al., Fish & Shellfish Immunology, Volume 95, December 2019, Pages 464–472, doi.org / 10.1016 / j.fsi.2019.11.011 [Non-patent document 3] Hai NV, Journal of Applied Microbiology 119, pp. 917-935, 2015, doi.org / 10.1111 / jam.12886 [Non-patent document 4] "Columnaris Disease in Fish: A Review With Emphasis on Bacterium-Host Interactions" (DOI: 10.1186 / 1297-9716-44-27, Declerq et al., Veterinary Research 2013, 44:27) Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, the present invention relies on the experiments described herein and proposes new means and tools to address the above problems. In particular, the present invention relates to the provision of relevant probiotic materials for protecting fish from pathogens and their harmful effects. [Means for solving the problem]

[0010] The present invention therefore relates to a bacterial strain or a combination of bacterial strains for use as probiotics in fish or fish populations, wherein at least one bacterial strain of the bacterial strain or combination is a Chryseobacterium massilia strain, a Chryseobacterium massilia strain in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated, the genome of which has an average nucleotide identity (ANI) of at least 95% with SEQ ID NO: 1, or a strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020. and a Flavobacterium species strain having one or more virulence factor-encoding genes and / or antibiotic resistance genes deleted or inactivated, the genome of which has an average nucleotide identity (ANI) of at least 95% with SEQ ID NO: 1 or a Flavobacterium species strain having an ANI of at least 95% with SEQ ID NO: 1 or identified by accession number I-5481 deposited at the CNCM on January 24, 2020.

[0011] The address of CNCM is Collection Nationale de Culture de Microorganismes, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris CEDEX 15, France.

[0012] SEQ ID NO: 1 is the complete genome sequence of the particular Flavobacterium species strain described herein, as sequenced in the experiments described herein and also available in the ENA (European Nucleotide Archive) under primary accession number ERS4574862 (version 1) and secondary accession number SANEA6847264 (taxpayer number 2730889, scientific name Flavobacterium sp. UGB 4466).

[0013] The 16S rRNA gene sequence of the particular Flavobacterium species strain of SEQ ID NO:1 is disclosed herein in SEQ ID NO:3.

[0014] The terms "16S rRNA" and "16S rDNA" are used interchangeably herein.

[0015] References herein to fish (singular), or to fishes (plural) generally referring to fish populations, are intended to be interchangeable throughout this specification, except where the context or general meaning dictates otherwise.

[0016] Average nucleotide identity (ANI) values ​​can be easily measured by those skilled in the art using general knowledge and available tools, which are well documented in the literature. ANI between two genomes, particularly prokaryotic genomes, is a widely known classification method that emerged in the age of genomics. Prior to this, DNA-DNA hybridization (DDH) was used for nearly 50 years as a criterion for determining prokaryotic species boundaries at the genome level. Other methods are also available, some of which are detailed in this application, including those detailed in the experimental section, such as 16S rRNA gene sequence similarity, recA gene sequence similarity, or rp / C gene sequence similarity. In addition to the explanations provided herein, a list of references for implementing the 16S rRNA gene sequence similarity method is also provided herein, which can be used as a guideline by those skilled in the art.

[0017] Software tools for implementing the average nucleotide identity (ANI) method and calculating ANI values ​​are also readily available to those skilled in the art. They are, in particular, freely available via the Internet. An example can be found at https: / / www.ezbiocloud.net / tools / ani. Available tools are described in detail in the literature. In particular, the percent identity can be conventionally calculated using local or preferably global sequence alignment algorithms and their available computer implementations. In the most preferred embodiment, the percent identity is calculated over the entire length of the compared sequences, which may be the entire genome of the compared strain. When the sequences in the set in question are similar and approximately the same size, a global alignment, which attempts to align all residues in all sequences, is most useful. The computer implementation of the algorithm used is usually accompanied by default parameters in the literature that can be used to run the algorithm. Those skilled in the art can easily adapt it taking into account the purpose or the sequence comparison performed.

[0018] However, whichever algorithm is used, it is recognized that a 95% ANI value is an appropriate cut-off value for classifying the genome of a strain that is sought to distinguish between two species, i.e., to classify within an existing species (annotated in the database) or to define a new species unknown to date.

[0019] The identification of the specific bacterial strains of the invention described herein was performed by whole-genome-based identification using the TrueBac ID system (v1.92, DB: 20190603) [https: / / www.truebacid.com / ;

[66] . The core components of the TrueBac ID genome system consist of (1) a dedicated reference database, named the TrueBac database, which is curated to hold the most up-to-date nomenclature, 16S rRNA gene, and genome sequences of standard / reference strains, and (2) an optimized bioinformatics pipeline that achieves the identification of genome sequences of interest using average nucleotide identity (ANI). The algorithmic identification scheme using WGS works as follows: First, a search of three genes, 16S rRNA, recA, and rplC, extracted from the whole-genome assembly was used to identify the collection of most phylogenetically closely related taxa. Next, the gene-based search was supplemented with an additional, faster whole-genome-based search using the Mash tool (https: / / github.com / marbl / mash). The top hits from these four searches were then pooled and ANI calculated using the MUMmer tool. Species-level identification was based on an algorithm cutoff of 95% ANI when possible or when 16S rRNA gene sequence similarity was greater than 99% (to accurately identify species and subsequently identify novel Flavobacterium strains).

[0020] Thus, one skilled in the art can readily determine ANI values ​​using the MUMmer tool described above and herein.

[0021] According to certain embodiments, Chryseobacterium massilia as described herein or used within the scope of the present invention alternatively, or in addition to any other characteristic described herein, has a genome having an average nucleotide identity (ANI) of at least 95% or more, preferably 96% or more, with SEQ ID NO:2.

[0022] SEQ ID NO:2 is the complete genome sequence of the specific Chryseobacterium massilia strain described herein, sequenced in the experiments described herein and also available in the ENA (European Nucleotide Archive) database under primary accession number ERS4385998 (version 1) and secondary accession number SANEA6623857 (taxpayer number 204089, scientific name Candidatus Chryseobacterium massiliae).

[0023] In certain embodiments, the strain is a mutant of the above strains in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated.

[0024] The 16S rRNA gene sequence of the particular Chryseobacterium massilia strain of SEQ ID NO:2 is disclosed herein in SEQ ID NO:4.

[0025] For the specific Flavobacterium species and Chryseobacterium massilia strains described herein, the closest taxon to which the specific strains described herein correspond is provided in the table below.

[0026] [Table 1]

[0027] Thus, the Flavobacterium sp. bacterial strain of the present invention (also referred to herein as Flavobacterium sp. 4466) is found to have an average nucleotide identity (ANI) of 94.65% with Flavobacterium spartansii ATCC BAA-2541 strain, the sequence of which can be found in the GenBank database under accession number MUHG01000041.1 (version 1) - https: / / www.ncbi.nlm.nih.gov / nuccore / MUHG01000041.1. Thus, the strain whose sequence is disclosed in SEQ ID NO: 1 constitutes a novel Flavobacterium sp. not previously described.

[0028] Thus, in certain embodiments, the present invention is directed to or utilizes a strain of Flavobacterium species having an average nucleotide identity (ANI) value of at least 95% or greater with the bacterial strain Flavobacterium spartansii ATCC BAA-2541, taken as the reference sequence, which may be used as a probiotic in fish or fish populations, as described herein.

[0029] The specific Flavobacterium species bacterial strain described herein also shares 97.80% 16S rRNA gene sequence similarity with Flavobacterium spartansii ATCC BAA-2541 bacterial strain.

[0030] The 16S rRNA gene sequence of the Flavobacterium spartansii ATCC BAA-2541 bacterial strain is disclosed herein in SEQ ID NO:5.

[0031] Thus, in a particular embodiment, a Flavobacterium species strain is of interest or is utilized whose genome comprises a 16s rDNA sequence having at least 98% sequence identity with SEQ ID NO: 5, taken as the reference sequence.

[0032] In certain embodiments, the present invention is directed to or utilizes a Flavobacterium species strain whose genome has an average nucleotide identity (ANI) value of at least 95% or more with the Flavobacterium spartansii ATCC BAA-2541 bacterial strain, taken as the reference sequence, and a 16s rDNA sequence having a sequence identity of at least 98% with SEQ ID NO: 5, taken as the reference sequence.

[0033] In certain embodiments, the strain is a mutant of any one of the above strains in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated.

[0034] In particular embodiments, the Flavobacterium spp. strain of the invention or described in any embodiment herein maintains the properties of the Flavobacterium spp. strain identified by accession number I-5481 deposited at the Collection Nationale de Culture de Microorganismes (CNCM) on January 24, 2020, in particular the biological properties described herein, and / or maintains the properties of the Flavobacterium spp. strain of SEQ ID NO: 1, in particular the biological properties described herein.

[0035] [Table 2]

[0036] The Chryseobacterium massilia bacterial strain of the present invention has an average nucleotide identity (ANI) of 95.85% with the Chryseobacterium massilia strain CCUG51329, whose sequence can be found in the GenBank database - https: / / www.ncbi.nlm.nih.gov / assembly / GCF_003385535.1 / under accession number ASM338553 (version 1).

[0037] Thus, in certain embodiments, the present invention is directed to or utilizes a Chryseobacterium massilia strain whose genome has an average nucleotide identity (ANI) value of at least 96% or greater with the reference sequence of the bacterial strain Chryseobacterium massilia CCUG51329, which may be used as a probiotic in fish or fish populations, as described herein.

[0038] The particular Chryseobacterium massilia strain described herein also shares 98.86% 16S rRNA gene sequence similarity with the Chryseobacterium massilia CCUG51329 bacterial strain.

[0039] The 16S rRNA gene sequence of the Chryseobacterium massilia CCUG51329 bacterial strain is disclosed herein in SEQ ID NO:6.

[0040] According to a particular embodiment, the present invention is directed to or utilizes a Chryseobacterium massilia strain having an average nucleotide identity (ANI) value of at least 96% with the Chryseobacterium massilia CCUG51329 bacterial strain, taken as the reference sequence, and comprising a 16s rDNA sequence having at least 99.9% sequence identity with SEQ ID NO: 6, taken as the reference sequence.

[0041] Alternatively, the present invention targets or utilizes a Chryseobacterium massilia strain whose genome has an average nucleotide identity (ANI) value of at least 97% or more with the Chryseobacterium massilia CCUG51329 bacterial strain, which is taken as the reference sequence.

[0042] In certain embodiments, the strain is a mutant of any one of the above strains in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated.

[0043] In certain embodiments, the Chryseobacterium massilia strain of the invention or described in any embodiment herein maintains the properties of the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the Collection Nationale de Culture de Microorganismes (CNCM) on January 24, 2020, in particular the biological properties described herein, and / or maintains the properties of the Chryseobacterium massilia strain of SEQ ID NO: 2, in particular the biological properties described herein.

[0044] According to certain embodiments, the ANI value of the Flavobacterium species strain used within the present invention relative to the Flavobacterium species strain identified by accession number I-5481 deposited at the Collection Nationale de Culture de Microorganismes (CNCM) on January 24, 2020, is as disclosed in any embodiment herein and / or may reach 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0045] Methods other than ANI values ​​for defining Flavobacterium species encompassed by the present invention are detailed herein. Those skilled in the art will understand that all of these methods can easily define appropriate subsets of Flavobacterium species strains in the context of the present invention. Table 1 below shows examples of corresponding data in the process of verifying the taxonomic identity of protist genomes, although the classification methods are different.

[0046] As defined herein, strains for use within the scope of the present invention may be defined by a set of parameters, such as an ANI value of 95% or greater, and a so-called "16S" value of 97% or greater, 98% or greater, or 99% or greater, combinations of which are encompassed herein by the use of the term "and / or" to combine parameters, depending on all values ​​and embodiments described herein.

[0047] According to a particular embodiment, the Chryseobacterium massilia strain used within the scope of the present invention is the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the Collection Nationale de Culture de Microorganismes (CNCM) on January 24, 2020, and / or a Chryseobacterium massilia strain having an average nucleotide identity (ANI) value of at least 95% or more with SEQ ID NO: 2, preferably at least 96%.

[0048] With reference to the deletion or inactivation of one or more virulence factor-encoding genes and / or antibiotic resistance genes in the strain under consideration, a feature that can be associated with any embodiment described herein, it is believed that a person skilled in the art can easily select the gene or genes that have been deleted or inactivated and, if necessary, modify the resulting strain according to the explanations provided herein and the general knowledge of the person skilled in the art regarding the attenuation of bacterial strains or the inactivation of resistance genes.

[0049] According to one embodiment, the bacterial strain of the present invention according to any embodiment described herein is non-pathogenic to healthy fish. The bacterial strain may be naturally non-pathogenic or may be non-pathogenic due to genetic and / or chemical attenuation.

[0050] Methods for attenuating pathogenic bacteria are known in the art. Genetic attenuation can be achieved by inactivating one or more genes involved in the bacterial metabolic pathways, more specifically one or more pathogenic mechanisms of the bacteria, and / or one or more genes involved in or responsible for the production of bacterial virulence factors.

[0051] According to one embodiment, the bacterial strain is attenuated by partial or complete deletion of one or more genes, even more particularly one or more genes involved in or responsible for the production of virulence factors of said bacterial strain, which may be as listed in Tables 7 and 8 herein, respectively, for the strain considered (see Results section).

[0052] More specifically, the isolated Chryseobacterium species was shown to contain five putative virulence factors, including several proteins involved in capsule biosynthesis, the heat shock protein HtpB subunit, the KatA catalase, and the ClpP protease proteolytic subunit. According to certain embodiments, the Chryseobacterium species of the present invention have one or several of the genes contained in the above Chryseobacterium species, particularly genes exhibiting the above-mentioned involvement, partially or completely deleted (as can be confirmed by the explanations and tools presented and described in the Results and Methods sections of this specification).

[0053] Flavobacterium sp. 4466 has been shown to contain capsule-encoding genes, sialic acid synthase, type IV and type VI secretion system effectors, and catalase as potential virulence factors. According to particular embodiments, the Flavobacterium sp. 4466 strain of the present invention has one or several of the genes contained in said Flavobacterium sp., particularly genes indicative of the above involvement, partially or completely deleted (as can be confirmed by the explanations and tools presented and described in the Results and Methods section herein).

[0054] For all of these embodiments, exemplary representative genes, the contents of which are described herein, are set forth in Table 6. It will be appreciated that one or more of the genes detailed in this table are deleted or partially deleted in the mutant genes of the present invention.

[0055] The same applies to antibiotic resistance genes. According to one embodiment, which may be added to other embodiments described in the present application, the bacterial strain is modified by partial or complete deletion of one or more genes, and even more particularly, one or more genes involved in the antibiotic resistance of said bacterial strain. The genes may be as listed in Table 6 herein for each strain considered (see Results section).

[0056] Flavobacterium sp. 4466 has been shown to contain genes encoding resistance to carbapenems, licosamides, streptogramins, pleuromutilins, and fluoroquinolone antibiotics. According to particular embodiments, the Flavobacterium sp. 4466 strain of the present invention has one or several of the genes contained in said Flavobacterium sp., particularly genes indicative of the above involvement, partially or completely deleted (as can be confirmed by the explanations and tools presented and described in the Results and Methods section herein).

[0057] In either case, the partial deletion is effected to an extent sufficient to inactivate the function of the gene.

[0058] For ease of description herein, bacterial strains in which one or more virulence factor-encoding genes or antibiotic resistance genes have been deleted or inactivated may also be referred to herein as "mutants." However, the mutants of the present invention retain the functional properties of their parent strains. In particular, they retain their probiotic effects.

[0059] Unless the context or general meaning dictates otherwise, when reference is made herein to bacterial strains, their variants as defined above are systematically included in the definition of said bacterial strain or combination or community of strains (it being understood that variants of one strain are combined with other non-mutated strains or other mutant strains, according to all possible combinations thereof).

[0060] According to certain embodiments applicable throughout the specification, the bacterial strain or strains (and their mutants) are combined with an acceptable carrier or delivery vehicle, and optionally adjuvant components, in a single composition or separate compositions comprising, consisting essentially of, or consisting of a mixture of different bacterial strains. According to certain embodiments applicable throughout the specification, the bacterial strain or strains of interest within the scope of the present invention are used for the purposes described herein or in the compositions described herein, provided that the compositions, in particular Flavobacterium columnare bacteria, maintain the bacterial strains and their probiotic properties, particularly for any of the uses described herein, in particular against infections by the pathogens described herein and their consequences, and / or for the fish or fish population to be treated as described in any embodiment herein.

[0061] According to certain embodiments, when only one bacterial strain is used, said bacterial strain is administered to a host in need thereof without an acceptable carrier or delivery vehicle (or adjuvant component, if applicable), or in a composition according to any embodiment as defined above and described herein.

[0062] According to a particular embodiment, when a combination of different bacterial strains is used, said bacterial strains are capable of infecting a host in need thereof: i. as individual bacterial strains without an acceptable carrier or delivery vehicle (or adjuvant component, if applicable); or ii. as a mixture of different bacterial strains present in a single composition, or iii. in different compositions each comprising, consisting essentially of, or consisting of at least one bacterial strain; or iv. As an assembly of at least one individualized (single) bacterial strain and different compositions each comprising, consisting essentially of, or consisting of at least one bacterial strain. It is administered.

[0063] "Individualized" or "single" bacterial strain means a bacterial strain without an acceptable carrier or delivery vehicle (or adjuvant component, if applicable).

[0064] Those skilled in the art will readily appreciate that the bacterial strains of the present invention having the desired protective properties described herein can be directly contacted with fish or fish populations to function as probiotics, for example, by adding the bacterial strains of the present invention directly to the water in which the fish are kept.

[0065] Pharmaceutical formulations can also be formulated. Examples of formulations for delivering probiotics include capsules, liquids, powdered beads, tablets, etc. In certain embodiments, the bacterial strains of the present invention are administered in liquid formulations. In certain embodiments, the bacterial strains of the present invention are administered in powder formulations.

[0066] Alternatively, the bacterial strains of the present invention may conveniently be fed to fish with their feed to achieve their probiotic effect, as known in the art, or when included in a food product, the bacterial strains of the present invention may be fed to fish with fish meal (i.e., fed together with fish meal, i.e., simultaneously without mixing before feeding the fish) or may be included in a fish food product. In a particular embodiment, the bacterial strains of the present invention are encapsulated.

[0067] In certain embodiments, the bacterial strains of the present invention are administered in or along with a food-based product, and the carriers or delivery vehicles described herein are adapted accordingly, according to conventional practice in the art.

[0068] According to certain embodiments, a probiotic effect is achieved when the bacterial strains of the present invention are delivered to the gastrointestinal tract (GIT) of treated fish. The present application demonstrates colonization of the GIT of treated fish.

[0069] According to certain embodiments, a probiotic effect is achieved when the bacterial strain is viable (live bacteria), or at least viable, or capable of being rehydrated and / or resuscitated and / or activated to become viable upon contact with the intended host.

[0070] According to certain embodiments applicable throughout the specification, the bacterial strains of the present invention are live bacteria.

[0071] Notwithstanding the above, and according to certain embodiments, the bacterial strains of the present invention are lyophilized. Lyophilized commercial preparations are advantageous for storage and transportation.

[0072] In certain embodiments, the bacterial strains of the present invention are encapsulated. Probiotic encapsulation enhances the stability of probiotics, facilitates handling and storage of probiotic cultures, and is a convenient means of protecting bacteria from harmful conditions (e.g., exposure to oxygen, freezing temperatures, or acidic environments) during production, storage, and, if applicable, during transit through the gastrointestinal tract. It can also be applied to the direct administration of bacteria to fish breeding water. Thus, "encapsulated" means that the bacteria have undergone an encapsulation process, which is defined as packaging cells, particularly live cells, in a shell material to protect them from unfavorable environmental conditions and, optionally, for sustained release under intestinal conditions. Several methods for encapsulating probiotics are known in the art, including spray drying, extrusion, emulsification or phase separation, freeze-drying, and ionotropic gelation. These methods are not limited to those listed here. Probiotic encapsulation technology (PET) generally involves immobilizing microorganisms in semipermeable and / or biocompatible materials. Those skilled in the art can easily find examples and explanations of encapsulation in the literature, for example, Prado et al., Applied Microbiology and Biotechnology, Vol. 104, pp. 1993-2006 (2020) doi.org / 10.1007 / s00253-019-10332-0, or Amir et al., Fish & Shellfish Immunology, Vol. 95, December 2019, pp. 464-472, doi.org / 10.1016 / j.fsi.2019.11.011, or Hai NV, Journal of Applied Microbiology, 119, pp. 917-935, 2015, doi.org / 10.1111 / jam.12886. Thus, the carriers or delivery vehicles described herein include those corresponding to the encapsulation of the bacterial strains of the present invention.

[0073] Conversely, the bacterial strain or combination of bacterial strains of the present invention may be present in a composition consisting essentially of, or consisting of, at least one bacterial strain and an acceptable carrier or delivery vehicle, and optionally an adjuvant component.

[0074] According to all embodiments described herein, the compositions allow the bacterial strain or combination of bacterial strains of the present invention to be present in a formulation adapted for the intended purpose, with a carrier or delivery vehicle selected to meet the formulation requirements. Formulations can include liquid or powder formulations, capsules, beads, tablets, appropriately formulated encapsulated bacterial strains (e.g., in a liquid), and / or food products if food is added.

[0075] It will be appreciated that those skilled in the art will be readily able to identify suitable carriers or delivery vehicles to include in the "compositions" or "formulations." Details are provided above and herein.

[0076] According to certain embodiments applicable throughout the specification, when a combination of different bacterial strains is used, said bacterial strains are administered to a host in need thereof simultaneously or separately in any order, or sequentially in any order.

[0077] According to certain embodiments applicable throughout the specification, the bacterial strain of the present invention is an isolated bacterial strain.

[0078] "Probiotic" means that a bacterial strain or combination thereof exerts a beneficial effect, preferably a beneficial effect on the health status, of the organism to which it is administered. Probiotics confer a health benefit to the host, particularly when administered to the host non-exclusively as a food ingredient or as an adjunct to a food ingredient (and thus not necessarily administered strictly as a feed). The beneficial effect may be achieved through the interaction of the bacterial strains or combinations thereof according to the present invention with the microflora of the host to which they are administered. The "probiotic" characteristic, used as an adjective to describe the suitability of the bacterial strains, active ingredients, compositions, and other food or liquid products described herein, means that they have the intended function encompassed by the definition of "probiotic."

[0079] In particular embodiments, beneficial effects demonstrated herein include colonization of the gastrointestinal tract with the bacterial strains of the present invention, as well as protection and improved survival of the host following administration of the bacterial strains of the present invention.

[0080] According to certain embodiments, "probiotic" or "probiotic" effect refers to the ability of probiotics to colonize the gastrointestinal tract of a host to which they are administered.

[0081] According to another specific embodiment, which may be in addition to the above-mentioned features, "probiotic" or "probiotic effect" refers to the ability to protect a host to which it is administered from adverse health effects due to infection with another pathogen, e.g., the Flavobacterium columnare pathogen. In this regard, adverse health effects due to infection with the Flavobacterium columnare pathogen are fully described herein. Any effect that even partially suppresses such adverse effects on the host can be said to contribute to or constitute a probiotic effect. Thus, one skilled in the art can determine whether a probiotic effect has been achieved by observing the resulting beneficial health effects compared to no treatment.

[0082] According to another specific embodiment, which may be added to other characteristics, including those described above, "probiotic" or "probiotic effect" refers to the ability to confer improved survival to a host or host population after administration of the bacterial strain of the present invention. The experimental section herein also provides an explanation of how improved survival is conferred to a host or host population after administration of the bacterial strain of the present invention. Thus, one skilled in the art can determine whether a probiotic effect based on this criterion has been achieved by observing the improved survival effect obtained on the host or host population, compared to no treatment. The data presented herein demonstrate that the present invention confer beneficial effects on the health of the host to which it is administered, improving survival even after challenge with a pathogen.

[0083] According to the present invention, the host is a fish, particularly a bony fish (ie Teleostei infraclass), preferably a fish suspected of being affected by columnaris disease.

[0084] In certain embodiments, the fish are selected from one or more of the species listed in the table below, and are generally farmed fish.

[0085] [Table 3A]

[0086] [Table 3B]

[0087] [Table 3C]

[0088] According to certain embodiments, the fish may be eels (Anguilla spp.), salmon (Oncorhynchus spp. and Salmo spp.), tilapia (Oreochromis spp.), hybrid striped bass (Morone chrysops x M. saxatilis), walleye (Stitzostedion vitreum), channel catfish, cetrachids (e.g., largemouth bass (Micropterus salmoides)), bait minnows (Pimephales promelas), goldfish (Carassius auratus), carp (Cyprinus carpio), or other fish. carpio), as well as ornamental fish (tropical fish species, for example, black molly (Poecilia sphenops) and small fish (Xiphophorus maculatus).

[0089] This fish is generally known as a target species of the pathogen Flavobacterium columnare.

[0090] In certain embodiments, the fish is rainbow trout (synonymous with Oncorhynchus mykis throughout this specification).

[0091] According to certain embodiments, the treated fish may be at any stage of its development, for example, eggs, larvae, particularly those with an under-matured / underdeveloped immune system, adults, including adults with or without a fully mature / underdeveloped immune system, or may be immunocompromised.

[0092] According to a preferred embodiment, the treated fish are included in a population of treated fish that includes larvae, or the treated animals are fish larvae.

[0093] In certain embodiments, the treated host is a fish larva.

[0094] According to a particular embodiment, the treated fish is a rainbow trout, is included in a population of treated rainbow trout that includes larvae, or is a rainbow trout larva.

[0095] Thus, in a particular embodiment, the fish (or some of the fish) is a rainbow trout in the larval stage of development.

[0096] According to certain embodiments, the treated fish are present in an aquaculture environment or a fish management environment.

[0097] As mentioned above, strains used within the scope of the present invention may be defined and further functionally defined by a single parameter or a series of parameters applied cumulatively, such as the ANI value and / or the so-called "16S" value.

[0098] According to certain embodiments, the bacterial strains, including when present in the combinations according to the invention, maintain the properties, in particular the biological properties, more particularly the probiotic properties, of the corresponding natural strains from which they are derived. The properties of said corresponding natural strains can be easily determined by the tests disclosed in the experimental section of this specification. Whether the bacterial strains of the invention maintain said properties can be easily determined by comparative or comparative tests or by full tests according to the description given herein.

[0099] According to a particular embodiment, the bacterial strain, including when present in the combination according to the present invention, is selected from bacterial strains comprising a 16s rDNA sequence having at least 97% sequence identity, in particular at least 98% or at least 99% sequence identity, with the 16s rDNA sequence present in the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020, or the Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020, respectively.

[0100] Additionally or alternatively, the bacterial strain, including those present in the combinations according to the present invention, may be selected from bacterial strains containing a 16s rDNA sequence having at least 97% sequence identity with a 16s rDNA sequence present in a reference sequence of a Chryseobacterium massilia or Flavobacterium species strain, respectively. Examples of reference sequences are described above. They may be Chryseobacterium massilia CCUG 51329 and Flavobacterium spartani ATCC BAA-2541, respectively. The ratio may be as described in any embodiment or paragraph described herein.

[0101] Additionally or alternatively, the bacterial strain, including when present in the combination according to the invention, is selected from bacterial strains comprising a 16s rDNA sequence having at least 97% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4, respectively, which correspond to the 16s rDNA sequences present in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, the boundaries of which can be readily determined by the skilled person by reference to the annotations found in the database entries and by general knowledge.

[0102] According to certain embodiments, the sequence identity using the so-called taxonomic classification tool of 16s rDNA sequences may be 98% or more, 99% or more, or even reach 100%.

[0103] This technique has been extensively described in the literature and is illustrated herein, so that the skilled person has knowledge on how to perform taxonomic classification of 16s rDNA sequences, in particular how to determine at least 97% sequence identity (see e.g.

[78] and the experimental section herein).

[0104] The taxonomic classification of 16s rDNA sequences is well documented in the literature. In particular, the percent identity can be conventionally calculated using local or preferably global sequence alignment algorithms and their available computer implementations. In the most preferred embodiment, the percent identity is calculated over the entire length of the compared 16s rDNA sequences. When the sequences in the set in question are similar and roughly the same size, a global alignment, which attempts to align all residues in all sequences, is most useful. The computer implementation of the algorithm used is usually accompanied by default parameters in the literature that can be used to run the algorithm. Those skilled in the art can easily adapt it taking into account the purpose or sequence comparison performed.

[0105] However, whichever algorithm is used, it is recognized that a value of 97% identity between 16s rDNA sequences is an appropriate cut-off value for distinguishing between two species, i.e., for classifying the genomes of strains sought to be classified within an existing species (annotated in the database) or to define a new species unknown to date.

[0106] According to a particular embodiment, the bacterial strain, including when present in the combination according to the invention, is selected from the group consisting of the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020 (SEQ ID NO: 2) and the Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020 (SEQ ID NO: 1).

[0107] Throughout this application, CNCM stands for Collection Nationale de Culture de Microorganismes (Institut Pasteur, 28 rue du Dr Roux, 75724 Paris CEDEX 15, France).

[0108] According to a particular embodiment, the present invention relates to a Chryseobacterium massilia bacterial strain for use as a probiotic, in particular the Chryseobacterium massilia bacterial strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020 (SEQ ID NO: 2).

[0109] The Chryseobacterium massilia bacterial strain, accession number I-5479, is also known as UGB 3610 in the CNCM. This bacterial strain was isolated from zebrafish. An example of a suitable growth medium is a combination of tryptone yeast extract salts and R2A agar (Reesona 2A agar). Recommended incubation conditions are 28°C, aerobic, and shaking at 250 rpm.

[0110] According to a particular embodiment, the present invention relates to bacterial strains of Flavobacterium sp. for use as probiotics, in particular the Flavobacterium sp. strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020 (SEQ ID NO: 1).

[0111] The Flavobacterium bacterial strain, accession number I-5481, is also known in the CNCM as UGB 4466. This bacterial strain was isolated from rainbow trout. An example of a suitable growth medium is R2A agar (Reesona 2A agar). Recommended incubation conditions are 20°C, aerobic, and shaking at 250 rpm (revolutions per minute).

[0112] According to a particular embodiment, the bacterial strain or combination of bacterial strains is selected from the group consisting of: a. a host that is a fish, especially a bony fish, more especially a rainbow trout, especially a larval form thereof; or b. A host comprising, consisting essentially of, or consisting of a homogenous or mixed population of fish, e.g., a population including fish at different stages of development or growth, e.g., rainbow trout and used as described herein, to be administered to

[0113] A "homogeneous" population of fish refers to a population of fish whose individuals are substantially related to or strictly related to the same species. According to certain embodiments, the "homogeneous" population may include fish at different stages of development or fish that can be considered to be at the same stage of development.

[0114] Conversely, a "mixed" population of fish refers to a fish population in which the individuals are related to different species, i.e., include different fish species. According to certain embodiments, the "homogeneous" population may include fish at different stages of development, or fish that can be considered to be at the same stage of development.

[0115] The developmental stages of the fish may be eggs, larvae, young fish, or adults with or without a mature immune system.

[0116] According to a particular embodiment, the developmental stage of the fish administered with the active ingredient of the present invention is the larval developmental stage (meaning that in the case of a fish population, the population includes at least partly or entirely larval fish). Essentially, the larval stage lasts from hatching to the juvenile stage, which is defined as the moment when the fish has all of its fin rays and begins to grow scales (scale arrangement). The key event for defining the end of the larval stage and the beginning of the juvenile stage is when the notochord associated with the caudal fin ventral to the spinal cord shows bending (becomes flexible). It should be noted that the larval stage can be further subdivided into the pre-flexion stage, the flexion stage, and the post-flexion stage, or into the yolk sac larvae stage and the transformation stage following yolk sac absorption, which ends the yolk sac larvae stage. While these regulations are not so strict as to prevent the administration of the active ingredients described herein to juvenile fish or populations of juvenile fish at other stages of development, including adults, the present invention represents a clear advancement in the art when it comes to treating larval fish, i.e., fish at a stage that cannot be vaccinated, since vaccination of larval fish is generally known to be inefficient or inappropriate.

[0117] In certain embodiments applicable throughout the specification, the combinations defined herein may include at least one additional bacterial strain of the native microbiota of the treated host species or host species within the treated host population.

[0118] Examples of some bacterial strain communities in the native microbiota of fish include: - For rainbow trout, Delftia acidovorans, Flavobacterium spp., Aeromonas rivipollensis, Pseudomonas helmanticensis, Pseudomonas baetica, Aeromonas hydrophila, Flavobacterium plurextorum, Acinetobacter spp., and Pseudomonas spp. - for zebrafish (Danio rerio throughout this specification): Chryseobacterium massilia, Aeromonas veronii, Pseudomonas mosselli, Stenotrophomas maltophilia, Aeromonas caviae, Pseudomonas peli, Pseudomonas sediminis, Phyllobacterium myrsinacearum, Pseudomonas nitroreducens.

[0119] According to another aspect, the bacterial strains of the present invention or combinations thereof, as probiotics or on their own, are further suitable for the following purposes (one or more of the purposes according to all possible combinations of the purposes in the list below): a. Preventing the occurrence of infection by the Flavobacterium columnare pathogen in the host fish species or in a population of treated host fish species; b. Preventing the occurrence of further infections with Flavobacterium columnare pathogens in the host fish species or treated population of the host fish species, or minimizing the effects of existing infections; c. increasing the resistance of the treated host fish species or population of treated host fish species to the Flavobacterium columnare pathogen; and / or d. Preventing or minimizing the spread (extent and impact) of subsequent infection by the Flavobacterium columnare pathogen in the host fish species or in the treated host fish population, or increasing the resistance of the treated host fish species or in the treated host fish population to the Flavobacterium columnare pathogen; and / or e. preventing or controlling disease in the treated host fish species or population of treated host fish species, particularly resulting from infection with the Flavobacterium columnare pathogen, and in particular preventing or minimizing the spread of disease within the treated host fish species population; and / or f. Extending the lifespan of or reducing mortality in the treated host species or populations of the treated host species.

[0120] The present invention is directed to the pathogen Flavobacterium columnare, which belongs to the genus Flavobacterium, as a "pathogen" that is constantly or periodically present or likely to be present in aquaculture environments. Information about the pathogen can be found, for example, in "Columnaris Disease in Fish: A Review With Emphasis on Bacterium-Host Interactions" (DOI: 10.1186 / 1297-9716-44-27, Declerq et al. Veterinary Research 2013, 44:27).

[0121] Infection of fish with Flavobacterium columnare is thought to occur in association with seasonal epidemics in aquaculture environments, and these epidemic-related diseases are also infectious diseases of interest within the context of the present invention.

[0122] In a particular embodiment, the present invention relates to the prevention of infection of fish with the pathogen Flavobacterium columnare, in which the administration of an active ingredient of the present invention as a prophylactic in a host not infected with the pathogen is contemplated.

[0123] As used herein, "infection" refers to the invasion and proliferation of a pathogen into the body of a host. It is understood that an infection may or may not cause symptoms, being asymptomatic or clinically evident, respectively. An infection may remain localized or may spread through blood or lymphatic vessels and become systemic (spread throughout the body).

[0124] The present invention also relates to increasing the resistance of the fish under consideration to the Flavobacterium columnare pathogen, whether the fish under consideration is already infected or at risk of infection with the Flavobacterium columnare pathogen, and whether the fish of interest is asymptomatic or symptomatic to infection with Flavobacterium columnare.

[0125] In another aspect, the present invention also relates to the prevention and / or suppression of diseases that are caused in particular by infection with the Flavobacterium columnare pathogen (depending on the cause-effect relationship), i.e. diseases whose initial cause is the presence of infection with this pathogen, or diseases that are known to result from or are known to be strongly associated with a prior infection of the host with this pathogen.

[0126] Thus, according to a particular embodiment, the bacterial strains of the present invention or combinations thereof are used against diseases caused by (or induced by or caused by) Flavobacterium columnare bacteria, in particular columnaris disease.

[0127] The best-known disease resulting directly from infection with Flavobacterium columnare is columnaris disease (also referred to in the literature as columnariosis disease).

[0128] According to a specific embodiment, the present invention aims to alleviate fish diseases, particularly columnaris disease (also referred to in the literature as columnariosis), in rainbow trout. In this respect, and according to a specific embodiment, since the disease is columnaris disease, the present invention aims to alleviate columnaris disease in fish infected with Flavobacterium columnare. If the fish is rainbow trout, the present invention aims to alleviate columnaris disease in rainbow trout.

[0129] Since it is understood that the active ingredients of the present invention have a beneficial effect on the host organism, minimizing or ameliorating comorbidity events due to pathogen infection at the level of a single individual and / or at the level of the host population as a whole, "prolonging the lifespan of the treated host species or a population of treated host species" means, conversely, "reducing the mortality of the treated host species or a population of treated host species." Those skilled in the art can easily assess the reduction in mortality by observing the treated host species or a population of treated host species. Those skilled in the art can also easily assess the increase in lifespan in light of the prior occurrence of events known to be detrimental to the health of the treated fish, or by statistical evaluation taking into account individual observations made on untreated fish, or similar information that can be deduced or derived from the general knowledge of those skilled in the art.

[0130] "Extended lifespan" or "reduced mortality" may also be achieved by reducing, mitigating, treating, managing or suppressing diseases at the level of the entire fish population, diseases being described above.

[0131] According to a particular embodiment, the present invention aims at reducing fish diseases, in particular columnaris disease, in rainbow trout in the context of aquaculture research and management.

[0132] According to particular embodiments, the bacterial strains or combinations thereof are administered to bony fish or populations comprising bony fish, more particularly to rainbow trout or populations comprising rainbow trout, or to an aquaculture or fish management environment. In addition to trout, a list of fish species described in this paragraph is provided in the table above. Specific fish species are also described above.

[0133] The aquaculture environment may be a natural environment or tank of fresh or salt water with flow-through or recirculating water supply management.

[0134] According to specific embodiments, the bacterial strains or combinations thereof are used to prevent or mitigate fish disease in rainbow trout in an aquaculture environment or fish management environment upon detection of a pathogen in the aquaculture environment, in particular a pathogen as defined in any embodiment herein, in particular the fish disease in rainbow trout being columnaris disease.

[0135] According to certain embodiments, the bacterial strains or combinations thereof, including their mutants, are introduced into the fish parasitic microflora by adding the bacterial strains or combinations thereof in the form of live bacteria directly to the fish breeding water.

[0136] Possible routes and / or modes of administration to the host to be treated can be deduced depending on the respective embodiments detailed herein above and / or the formulation of the active ingredient to be delivered. The active ingredients of the present invention may be delivered in individual form, with or without additional ingredients, or in a mixture of active ingredients. - the active ingredient may be administered as, but not limited to, a capsule, a liquid, a powder, powdered beads, a tablet, etc.; and / or The active ingredient may be administered in any form or type of formulation as described herein, together with the feed (e.g. with so-called fish meal and / or fish oil) or in a food product. The present invention therefore also relates to a kit comprising, consisting essentially of, or consisting of, an active ingredient of the invention according to any embodiment described herein, at least one food product, in particular a fish food product, and, where appropriate, a leaflet of instructions for administration with a view to achieving the effects described herein.

[0137] Thus, the active ingredients of the present invention may be administered directly to the host's environment, for example by adding them to the water in which fish, including larvae, live and grow, or may be administered directly to the host's body by any route, particularly by bringing the bacterial strain (liquid or food) into contact with the gastrointestinal tract (GIT) of the fish.

[0138] The invention is generally described herein using the term "for use" where applicable. Using synonymous terms, the invention also relates to a method of administering a probiotic active ingredient, including isomers, as defined in any of the embodiments described herein to a host, particularly fish, in need thereof. Thus, all instances of "for use" can be translated as a method of administering an active ingredient, including variants described herein, to a host for the purposes described herein, depending on any embodiment described.

[0139] The present invention provides a method for treating a host species, or a population of treated host species, preferably fish, administered with an active ingredient of the invention according to any of the embodiments described herein for administration to the host, wherein: a. Prevent the occurrence of further infections with Flavobacterium columnare pathogens or minimize the effects of current infections with Flavobacterium columnare pathogens; and / or b. Increased resistance to the pathogen Flavobacterium columnare, and / or c. Prevent or minimize the spread (extent and impact) of subsequent infection by Flavobacterium columnare pathogens or increase resistance to Flavobacterium columnare pathogens; and / or d. Preventing or controlling disease caused by infection, particularly by the Flavobacterium columnare pathogen, and in particular preventing or minimizing the spread of disease within the treated host species population; and / or e. Extending the lifespan of the treated host species or population of treated host species or reducing mortality of the treated host species or population of treated host species, preferably fish. It also relates to a method for

[0140] The present invention also relates to a bacterial strain selected from the group consisting of the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020 (SEQ ID NO: 2) and the Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020 (SEQ ID NO: 1), or mutants thereof in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated.

[0141] When a complete or long genome sequence is considered, by reference to the SEQ ID NOs of the present disclosure as well as the sequences obtainable by sequencing the genome of the deposited strain as described herein, it can be understood that the claimed invention should not be susceptible to errors resulting from the sequencing process. Exemplary protocols and sequencing methods are provided herein and are well known to those skilled in the art who, by common sense, will readily be able to determine sequencing errors.

[0142] According to a particular embodiment, the bacterial strain is an isolated bacterial strain.

[0143] According to certain embodiments, the bacterial strain is an isolated live bacterium. According to certain embodiments, the bacterial strain is an isolated lyophilized bacterial strain that still maintains its rehydrating and / or resuscitating and / or reactivating properties, if applicable.

[0144] According to another aspect, the present invention provides a method for manufacturing a method of manufacturing a semiconductor device comprising: (i) a Chryseobacterium massilia strain that has an average nucleotide identity (ANI) value of at least 95% with the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020 (also disclosed in SEQ ID NO: 2), but that differs from said strain in particular by nucleotide deletions or substitutions achieved by non-native sequence modifications, resulting in the deletion or substitution of at least one (i.e. one, two, three or more) nucleotide with respect to the naturally deposited strain, so that the resulting strain does not have the characteristics of the corresponding naturally occurring strain, in particular the characteristics of the corresponding genetically identical strain, and maintains the characteristics detailed herein for the strain I-5479; (ii) a Flavobacterium species strain having an average nucleotide identity (ANI) value of at least 95% or more with the Flavobacterium strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020 (also disclosed in SEQ ID NO: 1), but differing therefrom in particular by nucleotide deletions or substitutions achieved by non-native sequence modifications, resulting in the deletion or substitution of at least one (i.e., one, two, or more) nucleotide with respect to the naturally deposited strain, so that the resulting strain does not have the characteristics of the corresponding naturally occurring strain, in particular the corresponding genetically identical strain characteristics, and maintains the characteristics detailed herein for the strain I-5481; or (iii) a variant of the Chryseobacterium massilia strain identified under accession number I-5479 deposited at the CNCM on January 24, 2020 (also disclosed in SEQ ID NO: 2) having an average nucleotide identity (ANI) value of at least 95% or more, or a variant of the Flavobacterium species strain identified under accession number I-5481 deposited at the CNCM on January 24, 2020 (also disclosed in SEQ ID NO: 1) having an average nucleotide identity (ANI) value of at least 95% or more, or a variant of any one of the above strains (i) or (ii) according to all described embodiments, in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated. Also includes.

[0145] It should be understood that the present invention also encompasses strains that are close in structure to naturally occurring strains and that maintain the characteristics detailed herein for the deposited strains described herein by reference to their CNCM accession numbers or complete genome sequences, but that have been modified or altered to differ from any naturally occurring strain. The variants described herein are included within the scope of this embodiment.

[0146] However, the present invention also relates to food products, in particular solid or liquid fish food products, or mixtures thereof, comprising the bacterial strain of the present invention according to any embodiment, including the mutants.

[0147] The present invention also relates to an encapsulated bacterial strain of the invention according to any embodiment, including mutants, or mixtures thereof.

[0148] The present invention also relates to the use of the bacterial strain of the invention as described in any embodiment herein, including mutants, or mixtures thereof, or the encapsulated bacterial strain of the invention as described in any embodiment herein, including mutants, or mixtures thereof, or the composition of the invention as described herein as an additive (to a food product), in particular a probiotic additive to a food product. The food product may be a dry (solid) or liquid product.

[0149] In certain embodiments, the bacterial strains of the present invention, including mutants, are administered in or together with food-based products. The carriers or delivery vehicles described herein are adapted accordingly according to conventional practices in the art. The bacterial strains may be encapsulated when present in or administered together with food products.

[0150] One skilled in the art can readily determine the dosage, as conventionally practiced in the art.

[0151] According to certain embodiments, the bacterial strains containing the mutants or combinations thereof are used to produce a final liquid dose of the active ingredient of 5×10 4 cfu / mL to 5 × 10 6 cfu / mL or 5 x 10 8 cfu / mL, especially 5 × 10 5The formulations are administered to a host in need thereof in a dosage or formulation that may be a dose of cfu / mL. The present invention extends to dry / solid dosages, which may be liquid dosages equivalent to the dosages listed above. Dosages applied in dry form may be expressed in grams instead of mL. This range and specific values ​​are likely to provide complete protection of the host, particularly a fish host, from subsequent infection with the Flavobacterium columnare pathogen. In this regard, the level of protection achieved can be demonstrated by in vivo challenge infection using gnotobiotic zebrafish or rainbow trout as animal models, examples of which are described herein or in the literature. While Flavobacterium columnare killed germ-free (GF) larval fish within 48 hours, fish fed with the Chryseobacterium massilia or Flavobacterium species strains of the present invention were shown to be more resistant to infection, with survival rates up to 90% compared to experiments in which the protective bacterial strains of the present invention were not administered to the model, as shown in the examples described herein. Thus, "complete" protection can be assessed by an in vivo challenge test using gnotobiotic zebrafish or rainbow trout as animal models, demonstrating that the survival rate of the model can be increased by up to 90% after administration of the subject strains of the present invention. A description of the performance of this test can be found in the experimental section.

[0152] It is noted that the dosages that provide "total" protection require a concentration of the active ingredient, which is characteristic of the technical modifications made to the administered product.

[0153] The present invention also relates to a probiotic composition comprising, consisting essentially of, or consisting of at least one bacterial strain selected from the group consisting of a Chryseobacterium massilia strain and a Flavobacterium species strain having an average nucleotide identity (ANI) value of at least 95% with the Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020, in any combination thereof, in particular at least two or three different bacterial strains of said group, as well as an acceptable carrier or delivery vehicle and optionally adjuvant components.

[0154] The bacterial strains that may be present in the compositions of the invention are, in view of their intended administration, the same as those described herein, in particular those mentioned above, including in particular all the described variants and embodiments.

[0155] In particular, the probiotic compositions of the present invention may encompass variants of naturally occurring strains, as defined herein, particularly modified strains, which have been altered to differ from the naturally occurring strains and which maintain the properties detailed herein for the deposited strains described herein by reference to their accession numbers in the CNCM.

[0156] Probiotic compositions of the invention also include compositions comprising, consisting essentially of, or consisting of any of the strains described herein or mixtures thereof in dosages that provide "total" protection of the host as defined herein.

[0157] Acceptable carriers or delivery vehicles refer to any substance, agent, or ingredient that can be safely administered to a host to deliver active ingredients to said host so that they can perform their function, i.e., probiotic function. They may be water or other liquids, or other acceptable substances, agents, or ingredients. They may be pharmaceutical or veterinary carriers or delivery vehicles. They may also be food. More details regarding acceptable carriers or delivery vehicles can be found herein above, and these sections are also referenced in terms of the compositions described herein. When food is used as a vehicle for the probiotic bacteria or compositions of the present invention, it allows the probiotics to reach the intestines of fish.

[0158] Also described are probiotic compositions comprising the Flavobacterium species strain of the invention (characterized by reference to the deposited strain - see herein) and further comprising the Delftia acidovorans strain identified by accession number I-5480 deposited at the CNCM on January 24, 2020 (sequence findable in the ENA (European Nucleotide Archive) database under primary accession number ERS4574863 (version 1) and secondary accession), namely Aeromonas livipolensis, Pseudomonas hermannticensis, Pseudomonas baetica, Aeromonas hydrophila, Flavobacterium purexturum, Acinetobacter species, Pseudomonas species.

[0159] In this respect, said bacterium may be isolated from the microflora of trout, in particular rainbow trout.

[0160] According to another embodiment described herein, the composition comprises a Chryseobacterium massilia strain and further comprises the following bacteria: at least one Aeromonas veronii strain, in particular two different Aeromonas veronii strains, Pseudomonas mosseri, Stenotrophomonas maltophilia, Aeromonas caviae, Pseudomonas peri, Pseudomonas sediminis, Phyllobacterium myrsinacearum, Pseudomonas nitroreducens.

[0161] In this regard, the bacteria may be isolated from the zebrafish microbiota.

[0162] According to a particular embodiment, at least one bacterial strain of the probiotic composition of the invention is derived from the group consisting of the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020, and the Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020, and mutants thereof in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated.

[0163] According to a particular embodiment, at least one bacterial strain of the probiotic composition of the present invention is selected from the group consisting of a Chryseobacterium massilia strain comprising, or consisting essentially of, or consisting of the genome of SEQ ID NO: 2, and a Flavobacterium species strain comprising, or consisting essentially of, or consisting of the genome of SEQ ID NO: 1, and mutants in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated.

[0164] Any one of the embodiments described herein, particularly those probiotic compositions of the present invention described in the preceding paragraphs relating to probiotic compositions, can be used for the purposes detailed in any of the embodiments described herein.

[0165] The present invention also relates to methods of producing a composition, formulation, food product (liquid or dry), or kit comprising the active ingredients described herein according to any embodiment disclosed herein.

[0166] The present invention also relates to the use of any of the active ingredients described herein, including variants, according to any embodiment disclosed herein, to make or manufacture a composition, formulation, food product (liquid or dry) or kit suitable for administration to a host, particularly fish, in need thereof, which composition, formulation, food product (liquid or dry) or kit may be used for probiotic or prophylactic purposes or against a disease in a host, according to any embodiment described herein.

[0167] The present invention also relates to the use of any of the active ingredients described herein, including variants, according to any embodiment disclosed herein, to make or manufacture a composition, formulation, food product (liquid or dry) or kit suitable for administration to a host, particularly fish, in need thereof.

[0168] The present invention also relates to the use of any of the active ingredients described herein, including variants, according to any embodiment disclosed herein, for administration to a host in need thereof, particularly fish. The administration may be for probiotic or prophylactic purposes, according to any embodiment described herein, or for the prevention of disease in the host.

[0169] The present invention provides a method for identifying bacterial strains that are probiotic against pathogen infections, comprising the steps of: a) providing a germ-free model trout susceptible to infection with at least one defined pathogen that does not infect standard trout in a manner that adversely affects their health; b) determining the microflora, in particular the cultivable microflora, of the typical trout of step a) and optionally identifying the most representative bacterial strains of said microflora, and optionally isolating (so that they can be identified) and then identifying said most representative bacterial strains of said microflora; c) if identified, a step of re-standardizing the germ-free mass model by inoculating the germ-free mass with the microflora determined in step b) or the most representative bacterial strain of said microflora identified in step b), followed by contacting the germ-free mass model with at least one determined pathogen of step a); d) determining the probiotic effect of the microbiota or its most representative bacterial strains inoculated into the re-standardized germ-free mass model; The present invention also relates to a method comprising:

[0170] Regarding step a), the germ-free trout model may be a trout larva. According to a particular embodiment, the germ-free trout model is a rainbow trout. This document details how the inventors first obtained this novel model.

[0171] According to more specific embodiments, step a) may alternatively comprise identifying a pathogen that infects, and in particular kills, the germ-free trout model but does not infect, and in particular does not kill, the standard trout.

[0172] The fact that the pathogen provided kills the germ-free trout model used allows for an increased level of rigor in the method for identifying bacterial strains. Indeed, by this standard, the method exhibits a greater hit rate (i.e., it can be referred to as a "high-throughput" method). It is understood that the observation of symptoms less severe than death in fish does not negate the successful performance of the method (i.e., the observation of symptoms of infection in fish, and possibly relief in step (c)), albeit with a lower efficiency (low-throughput, but still a hit rate).

[0173] Specific examples detailing how steps a) to d) can be achieved are detailed in the experimental section herein. The method for identifying bacterial strains described herein is applicable to many different pathogens (in step a)) and many different microbiota (which may depend in part on the environment), depending on the experimental environment. Those skilled in the art can easily determine the experimental environment, and an explanation is provided in the experimental section below.

[0174] Renormalization means inoculating a host lacking said microbiota with the corresponding microbiota. Comprehensive examples and explanations that can be derived from them are given herein.

[0175] The determination in step d) can be made by observing the biological effect obtained after carrying out step c) on the re-normalized mass model. Comprehensive examples and explanations that can be derived therefrom are provided herein.

[0176] As used herein, the term "comprising," which is synonymous with "including" or "containing," is open-ended and does not exclude additional, unrecited elements, components, or method steps, whereas the term "consisting of" is a closed term that excludes any additional elements, steps, or components not specified.

[0177] The term "essentially consisting of" is a partially open term that does not exclude additional, unrecited elements, steps, or ingredients, as long as these additional elements, steps, or ingredients do not materially affect the basic and novel characteristics of the application.

[0178] Thus, the term "comprising" (or "comprise(s)") includes the term "consisting of" (or "consist(s) of"), as well as the term "essentially consisting of" ("essentially consist(s) of"). Thus, the term "comprising" (or "comprise(s)") in this application more specifically encompasses the term "consisting of" ("consist(s) of"), as well as the term "essentially consisting of" ("essentially consist(s) of").

[0179] For convenience to the reader of this application, the description has been divided into various paragraphs or sections. Such division should not be construed as meaning that the content of one paragraph or section is discontinuous with the content of another paragraph or section. To the contrary, this specification encompasses all possible combinations of the various sections, paragraphs, and sentences.

[0180] The relevant disclosures of all references cited herein are each specifically incorporated by reference.

[0181] These and other features of the present invention will become apparent from a reading of the examples and drawings which illustrate the experiments carried out by the inventors, as a complement to the features and definitions set out in this specification. The following examples are given for illustrative purposes, but are not intended to be limiting on the described invention. [Brief explanation of the drawings]

[0182] [Figure 1]Flavobacterium columnare kills germ-free, nonstandard zebrafish. Six-day-old germ-free or standard zebrafish larvae were exposed to different doses of Flavobacterium columnare ALG by tank immersion and then transferred to sterile water 3 hours later. The mean survival rate is indicated by a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistics correspond to unpaired, nonparametric Mann-Whitney tests comparing all conditions to uninfected germ-free (left) or standard (right). **** indicates p < 0.0001; absence of * indicates non-significant. The blue (gray in black and white) midline corresponds to unexposed larvae, and the red (light gray in black and white) midline corresponds to larvae exposed to Flavobacterium columnare. [Figure 2A] Selection of Flavobacterium columnare strains that are lethal to germ-free zebrafish. Survival of germ-free zebrafish larvae exposed to a collection of 28 strains of Flavobacterium columnare. [Figure 2B] Survival rates of germ-free and control zebrafish larvae exposed to the seven most virulent Flavobacterium columnare strains. Zebrafish larvae were infected at 6 dpf by immersion for 3 hours with 5.102 cfu / mL. The mean survival rate is indicated by a thick horizontal line, along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistics correspond to unpaired nonparametric Mann-Whitney tests comparing all conditions to uninfected germ-free. **** indicates p < 0.0001, *** indicates p < 0.001, ** indicates p < 0.005, * indicates p < 0.0005; no asterisk indicates non-significant. The blue / gray midline corresponds to uninfected larvae, and the red (light gray in the black and white version) midline corresponds to infected larvae. [Figure 3]Standard larvae from four different zebrafish facilities were also protected from Flavobacterium columnarum infection. A-B lineage zebrafish eggs were collected from four different zebrafish facilities: Facility 1, the Hôpital Robert Debre Institute (Paris, France); Facilities 2 and 3, two laboratories at the University of Paris 6 (Paris, France); and Facility 4, a private Amagen facility in Gif-sur-Yvette (France). Six-day-old germ-free or standard zebrafish larvae from each facility were exposed to Flavobacterium columnarum ALG by tank immersion and transferred to sterile water after 3 hours. The mean survival rate is shown as a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were collected. The blue (gray in the black and white version) midline corresponds to uninfected larvae, and the red (light gray in the black and white version) line corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 postinfection. Statistical values ​​shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, and the absence of * indicates no significance. [Figure 4] Restandardization of germ-free zebrafish larvae confers protection from Flavobacterium columnare infection. Resistance of germ-free and standard zebrafish larvae, reared in contact with fish facility tank water or mashed non-germ-free eggs, to Flavobacterium columnare ALG on day 0 (sterilization day) or 4 dpf (hatching day). Flavobacterium columnare ALG intake = 5 x 105 cfu / mL. Mean survival rates are indicated by thick horizontal lines with standard deviations. Number of zebrafish larvae per condition was n = 12. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistics correspond to unpaired nonparametric Mann-Whitney tests comparing all conditions to uninfected germ-free. **** indicates p < 0.0001; absence of * indicates non-significant. The blue (gray in the black and white version) midline corresponds to uninfected larvae, and the red (light gray in the black and white version) midline corresponds to infected larvae. [Figure 5]A mix of 10 culturable bacteria comprising the core zebrafish larval microbiota confers protection against Flavobacterium columnarum infection. Ten culturable strains identified as the core canonical microbiota were added to 4-dpf larvae at an equivalent concentration of 5 x 10 cfu / mL prior to challenge at 6 dpf. The mean survival rate is indicated by a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistics shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p < 0.0001; absence of * indicates non-significant. The blue (gray in black and white) midline corresponds to uninfected larvae, and the red (light gray in black and white) midline corresponds to infected larvae. [Figure 6] Dynamics of bacterial population establishment in re-standardized zebrafish larvae. The relative abundance of the 10 species constituting the core protective zebrafish microbiota in Mix 10 was identified at different time points using 16S rDNA gene amplicons of a pool of 10 larvae. The left line shows the relative abundance of the microbiota species in standard zebrafish larvae at day of hatching (4 dpf). For the re-standardized Mix 10 population, germ-free larvae fed Tetrahymena were incubated with an equal ratio of the 10 species (5 x 10 cfu / mL each) comprising the core 4 dpf microbiota. [Figure 7]Effect of antibiotic treatment on zebrafish survival. Germ-free and standard zebrafish larvae were treated with antibiotics for 16 hours at 4 dpf. The antibiotics were then washed out, and the larvae were infected with Flavobacterium columnarum ALG. Different concentrations of penicillin / streptomycin or kanamycin were used to identify non-toxic antibiotic treatments that cause microbiota dysbiosis. Penicillin / streptomycin dose 1 was 250 μg / mL, dose 2 was 15.6 μg / mL, and kanamycin doses 1, 200 μg / mL, dose 2 was 50 μg / mL, and dose 3 was 25 μg / mL. The mean survival rate is shown as a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. The blue (gray in black and white) midline corresponds to uninfected larvae, and the red (light gray in black and white) midline corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. Statistics shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates no significance. [Figure 8-1] Figure 8A shows the response of zebrafish larvae to Flavobacterium columnarum ALG infection after antibiotic dysbiosis. The figure shows the timing and treatment of the experiment. [Figure 8-2]Figure 8B shows 24 hours after antibiotic treatment, allowing for the restoration of protection in kanamycin-treated zebrafish larvae; the figure indicates the timing and treatment. The mean survival rate is shown as a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were counted. The blue (gray in the black and white version) midline corresponds to uninfected larvae, and the red (light gray in the black and white version) midline corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistics shown correspond to the unpaired nonparametric Mann-Whitney test. **** indicates p < 0.0001; absence of * indicates non-significant. [Figure 8-3] Figure 8C shows the population recovery profile with streptomycin / penicillin treatment. Figure 8D shows the population recovery profile with kanamycin treatment. Pools of 10 larvae were taken for 16S rDNA sequencing. [Figure 9] Protection of zebrafish from Flavobacterium columnare, restandardized with individual or mixed bacterial strains isolated from zebrafish larvae A. Figure 9A shows the determination of the level of protection conferred by each of the 10 bacterial species comprising the core protective zebrafish microbiota. Bacteria were individually added to the water on the day of hatching (dose: 5 x 10 cfu / mL). Figure 9B shows the level of protection conferred by different doses of Chryseobacterium massilia and Mix 9. Mix 9 conferred protection only at the highest dose. The mean survival rate is shown as a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were included. The blue (gray in black and white) midline corresponds to uninfected larvae, and the red (light gray in black and white) midline corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistical values ​​shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, and the absence of * indicates no significance. [Figure 10A]The eight-species combination of Protective Mix 9 does not protect against Flavobacterium columnare infection. Survival of zebrafish larvae against Flavobacterium columnare ALG infection at 6 dpf after re-standardization with different possible mixes containing the eight species of the Mix 9 consortium. Mix 10 = Re-ConvMix10, Mix 9 = Re-ConvMix9, Mix 8 = Re-ConvMix8. [Figure 10B] Table showing the different combinations used for renormalization at 4 dpf. [Figure 10C] Survival of zebrafish larvae against Flavobacterium columnare ALG infection at 6 dpf after testing re-standardization of non-protective Mix 8a with eight different combinations, doubling the amount of one of the eight species in each combination (shown in yellow in D). [Figure 10D] Table showing the different combinations used for renormalization at 4 dpf. Mix 10 = Re-ConvMix10, Mix 9 = Re-ConvMix9, Mix 8 = Re-ConvMix8. A and C: The mean survival rate is shown as a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. The blue (gray in black and white) midline corresponds to uninfected larvae, and the red (light gray in black and white) midline corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized 10 days after infection. Statistical values ​​shown correspond to the unpaired nonparametric Mann-Whitney test. **** indicates p < 0.0001; absence of * indicates non-significant. [Figure 11A]Zebrafish immune response to Flavobacterium columnarum infection. qRT-PCR analysis of host gene expression 18 hours after exposure to Flavobacterium columnarum in larvae renormalized with the indicated bacteria or bacterial mix. Each dot corresponds to an individual larva. Expression of Il10 (A), il1b (B), and il22 (C) in wild-type AB zebrafish. D: Comparison of survival rates between myd88- / - zebrafish and background-matched myd88+ / + zebrafish after renormalization and exposure to Flavobacterium columnarum ALG. The mean survival rate is indicated by a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 postinfection. The blue (gray in black and white) line corresponds to uninfected larvae, and the red (light gray in black and white) line corresponds to infected larvae. Statistical values ​​shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates not significant. [Figure 11B]qRT-PCR analysis of host gene expression 18 hours after exposure to Flavobacterium columnarum in larvae renormalized with the indicated bacteria or bacterial mix. Each dot corresponds to an individual larva. Expression of Il10 (A), il1b (B), and il22 (C) in wild-type AB zebrafish. D: Comparison of survival rates between myd88- / - zebrafish and background-matched myd88+ / + zebrafish after renormalization and exposure to Flavobacterium columnarum ALG. The mean survival rate is indicated by a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. Larval mortality was monitored daily, and surviving fish were euthanized 10 days after infection. The blue (gray in black and white) line corresponds to uninfected larvae, and the red (light gray in black and white) line corresponds to infected larvae. Statistics shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates no significance. [Figure 11C]qRT-PCR analysis of host gene expression 18 hours after exposure to Flavobacterium columnarum in larvae renormalized with the indicated bacteria or bacterial mix. Each dot corresponds to an individual larva. Expression of Il10 (A), il1b (B), and il22 (C) in wild-type AB zebrafish. D: Comparison of survival rates between myd88- / - zebrafish and background-matched myd88+ / + zebrafish after renormalization and exposure to Flavobacterium columnarum ALG. The mean survival rate is indicated by a thick horizontal line along with the standard deviation. For each condition, n = 12 zebrafish larvae were used. Larval mortality was monitored daily, and surviving fish were euthanized 10 days after infection. The blue (gray in black and white) line corresponds to uninfected larvae, and the red (light gray in black and white) line corresponds to infected larvae. Statistics shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates no significance. [Figure 11D] The blue (gray in black and white) line corresponds to uninfected larvae, and the red (light gray in black and white) line corresponds to infected larvae. Statistical values ​​shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates no significance. [Figure 12]Expression of i / lb in WT and myd88- / - mutant zebrafish renormalized with various bacteria or bacterial mixes as indicated. qRT-PCR analysis of i / lb gene expression was performed 18 hours after exposure to Flavobacterium columnarum ALG. Each dot corresponds to an individual larva. The blue (gray in black and white) midline corresponds to uninfected larvae, and the red (light gray in black and white) midline corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistics shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates non-significant. The mean survival rate is indicated by a thick horizontal line. The blue (gray in black and white) line corresponds to uninfected larvae, and the red (light gray in black and white) line corresponds to infected zebrafish. [Figure 13] The intestine of an infected germ-free zebrafish shows severe damage. Germ-free standard and re-standardized zebrafish larvae. Re-standardized zebrafish were inoculated with Mix 9 or Chryseobacterium massilia at 4 dpf. Figure 13A shows a representative intestinal photograph from an uninfected larva. Fish were fixed for histological analysis or electron microscopy at 7 dpf. Left: Giemsa staining (L = intestinal lumen). Right: Transmission electron microscopy. Figure 13B shows a representative intestinal photograph from an infected larva exposed to Flavobacterium columnare ALG at 7 dpf. Toluidine blue staining of Epon-embedded zebrafish larvae for light microscopy (left) or transmission electron microscopy at 7 dpf (right). [Figure 14]Flavobacterium columnare-infected larvae require food intake. Prior to Flavobacterium columnare ALG infection, germ-free zebrafish larvae were fed either germ-free Tetrahymena thermophila (sterile + tetras) or germ-free fish food powder (sterile + powder), or not. Fed larvae were susceptible to Flavobacterium columnare ALG infection, whereas unfed germ-free larvae survived infection with the fish pathogen. The mean survival rate is shown as a thick horizontal line with the standard deviation. For each condition, n = 12 zebrafish larvae were used. The blue (gray in black and white) midline indicates the survival rate of infected and unfed zebrafish larvae. The red (light grey in black and white) midline corresponds to uninfected larvae, and the red (light grey in black and white) midline corresponds to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. Statistical values ​​shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates no significance. [Figure 15]Pre-exposure to Chryseobacterium protects larval and adult zebrafish from Flavobacterium columnarum infection. Figure 15A shows that zebrafish larvae were inoculated with 5 × 10 cfu / mL of Chryseobacterium massilia for 48 hours at 4 dpf before infection with a virulent Flavobacterium columnarum strain at 6 dpf. Figure 15B shows the survival rate of adult zebrafish pre-exposed to Chryseobacterium massilia (2 × 10 cfu / mL for 48 hours) before exposure to Flavobacterium columnarum ALG (5 × 10 cfu / mL for 1 hour). The mean survival rate is indicated by a thick horizontal line along with the standard deviation. For each condition, n = 12 larval or adult zebrafish were collected. Zebrafish mortality was monitored daily, and surviving fish were euthanized 10 days post-infection. The blue (gray in black and white) line corresponds to uninfected larvae, and the red (light gray in black and white) line corresponds to infected zebrafish. Statistical values ​​shown correspond to unpaired nonparametric Mann-Whitney tests. **** indicates p<0.0001, *** indicates p<0.001, ** indicates p<0.005, * indicates p<0.05, and no * indicates no significance. [Figure 16] Protocol used in this study for rearing and infecting or restandardizing germ-free trout larvae. [Figure 17] Protocol used in this study for rearing and infecting or restandardizing germ-free trout larvae. After fertilization, eggs were sterilized (-5 dph) and maintained in sterile autoclaved mineral water at 16°C in Petri dishes until hatching. Once hatched, rainbow trout larvae were transferred to cell culture flasks with vented caps and maintained there throughout the entire experiment. Larvae were fed sterile powdered food every two days from 21 dph until the end of the experiment. Water was changed 30 minutes after feeding the animals. To test the protective effect of potential probiotic strains, larvae were restandardized at 22 dpf with one or several substrate bacteria diluted in water. Pathogenic bacteria were added to the water at 24 dph for 24 hours, and then the larvae were washed with fresh sterile water. [Figure 18] Growth performance of rainbow trout larvae reared under germ-free and standard conditions. Body size (A) and weight (B) of standard and germ-free fish were measured at 35 dph (n=6). [Figure 19] Anatomical comparison of standard and germ-free rainbow trout larvae. Whole mounts were analyzed by optical projection tomography after clearing fish based on the immunolabeling-enabled 3D imaging of solvent organ clearing method (iDISCO+). Lateral views of standard (A, B, C, and D) and germ-free (E, F, G, and H) rainbow trout larvae imaged at 21 dph. Brain (A and F), spleen (B and G), intestine (C and H), gills (D and I), and pronephros (E and J). Images represent two different fish per condition. [Figure 20] Comparison of the intestinal anatomy of standard and germ-free rainbow trout larvae. Whole mounts were analyzed by optical projection tomography after clearing the fish based on the immunolabeling-enabled 3D imaging of solvent organ clearing method (iDISCO+). Lateral views of standard (A and B) and germ-free (C and D) rainbow trout larvae imaged at 21 dph. The midgut (A and C) and posterior intestine (B and D). Images represent two different fish per condition. [Figure 21] Survival rates of germ-free and standard rainbow trout larvae infected with different pathogens. A) Survival rates of germ-free larvae exposed to Flavobacterium phychrophilum strain THCO2-90, Flavobacterium columnare strain Fc7, L. garvieae, Vibrio anguillarum, and Y. ruckeri strain JIP27 / 88 in a tank. B) Flavobacterium columnare strain Fc7 kills germ-free rainbow trout but not standard rainbow trout. The mean and SD plots show the average number of days post-infection at which infected fish died. N = 12 larvae per condition. All surviving fish were euthanized 10 days after infection. Asterisks indicate significant differences from the uninfected population (****p<0.0001). [Figure 22]Survival of re-standardized trout larvae after infection with Flavobacterium columnarum Fc7. Figure 22A shows that germ-free trout larvae exposed to water used for rearing standard fish at 21 dph exhibit similar survival rates after infection with Flavobacterium columnarum as standard trout larvae. Figure 22B shows that 11 species identified from the microbiota of standard fish were added to rainbow trout larvae at 22 dph, followed by infection with Flavobacterium at 24 dph. This bacterial mixture is able to protect the re-standardized larvae from infection. N = 12 larvae per condition. All surviving fish were euthanized 10 days after infection (****p<0.0001). [Figure 23] Protection of germ-free trout larvae from Flavobacterium columnare infection by individual species isolated from the standardized rainbow trout microbiota. Figure 23A shows that 11 species isolated from the standardized fish microbiota (Table 1) were individually added to rainbow trout larvae at 22 dph, followed by infection with Flavobacterium columnare Fc7 at 24 dph. Of the 11 different strains, only Flavobacterium species strain 4466 protected the restandardized larvae from infection. Figure 23B shows that Mix 11 and Mix 10 (a mix of all identified strains except Flavobacterium species strain 4466) were added to rainbow trout larvae at 22 dph, followed by infection with Flavobacterium columnare at 24 dph. Mix 11 protected the restandardized larvae from infection, whereas Mix 10 did not. n = 10 larvae for each condition. All surviving fish were euthanized 10 days after infection. Figure 23C shows the CFU / mL recovered from isolated intestines from germ-free fish exposed to Flavobacterium columnare Fc7, Flavobacterium species strain 4463, or both 24 hours after infection (****p<0.0001). [Figure 24]Chryseobacterium species (Chryseobacterium massilia) completely protect trout from Flavobacterium columnarum infection. Survival of axenic trout larvae exposed to Chryseobacterium species 48 hours before infection with Flavobacterium columnarum strains Fc7, IA-S-4, Ms-Fc-4, and ALG-00-530. Flavobacterium columnarum kills axenic trout but not trout pre-standardized with Chryseobacterium species. The mean and SD plots show the average number of days post-infection at which infected fish died. N = 12 larvae per condition. All surviving fish were euthanized 10 days after infection. Asterisks indicate significant differences from the uninfected population (****p<0.0001, **p<0.01). [Figure 25] Survival rates of germ-free and standard rainbow trout larvae infected with different fish pathogens. Kaplan-Meier graph of survival rates of germ-free larvae after bath exposure with Flavobacterium psychrophilum strain THCO2-90, Flavobacterium columnare strain Fc7, Lactococcus garvieae strain JIP28 / 99, Vibrio anguillarum strain 1669, and Yersinia luchenii strain JIP27 / 88. Mean and SD plots show the average survival rate of fish over 10 days after exposure to the different pathogenic microorganisms. n = 10 larvae per condition. All surviving fish were euthanized 10 days after infection. Asterisks indicate significant differences from the uninfected population (**p<0.01, ***p<0.001, ****p<0.0001). [Figure 26]Survival of re-standardized trout larvae after infection with Flavobacterium columnare Fc7. Kaplan-Meier graph of the survival of germ-free larvae after exposure to Flavobacterium columnare strain Fc7 in a tank. Figure 26A shows that Flavobacterium columnare strain Fc7 kills germ-free rainbow trout but not standard rainbow trout. Figure 26B shows that germ-free trout larvae exposed to water used for rearing standard fish at 22 dph exhibit a similar survival rate after infection with Flavobacterium columnare as standard trout larvae. Figure 26C shows that 11 strains identified from the microbiota of standard fish were added to rainbow trout larvae at 22 dph, followed by infection with Flavobacterium columnare at 24 dph. This bacterial mixture is capable of protecting the re-standardized larvae from infection. Mean and SD plots show the average survival rate of fish over 10 days after exposure to different pathogenic microorganisms. For each condition, n = 10 larvae. All surviving fish were euthanized 10 days after infection. Asterisks indicate significant differences from the uninfected population (****p<0.0001). [Figure 27] Histological comparison of the posterior intestine of infected and uninfected germ-free and standard rainbow trout larvae. Figure 27A shows representative images of the intestine of uninfected germ-free and standard trout larvae. Figure 27B shows representative images of the intestine of infected germ-free and standard larvae exposed to Flavobacterium columnare strain Fc7. Fish were fixed for histological analysis 1 day post-infection (dpi). Figure 27C shows the average number of goblet cells per microvilli in the posterior intestine. Lines indicate the mean ± SD per cilium in the same area of ​​the posterior intestine of three fish per condition. Paraffin-embedded rainbow trout larvae were stained with a combination of Alcian blue and PAS for light microscopy. Images and quantification data represent three different fish per condition. [Figure 28]Representative images of the in vitro growth inhibitory activity of Flavobacterium sp. strain 4466 against different virulent Flavobacterium / columnae strains. Figure 28A shows no growth inhibition of Flavobacterium columnare Fc7 after the addition of 5 μl of Flavobacterium sp. culture supernatant. Figure 28B shows the ring of growth inhibition of Flavobacterium columnare FC7 surrounding a Flavobacterium sp. colony on a Flavobacterium columnare strain Fc7 layer. Figure 28C shows the ring of growth inhibition of Flavobacterium columnare ALG-00-530, IA-S-4, and Ms-Fc-4. The agar layering technique was performed by spreading a Flavobacterium columnare bacterial suspension in soft agar solution onto TYES agar and then depositing 5 μl of an overnight culture of Flavobacterium sp. strain 4466. Incubation was carried out for 24 hours at 28° C. The experiment was performed in triplicate. [Figure 29] Phylogenetic tree showing the relationship of Flavobacterium species strain 4466 to the 15 closest Flavobacterium species based on ANI analysis. The tree was constructed with RAxML (version 8.2.8) using the 400 most conserved proteins for each strain's proteome. Bootstrap support values ​​are indicated at the connection points. [Figure 30] To test whether protective Flavobacterium species isolated from the standard rainbow trout microbiota could protect rainbow trout, germ-free fish larvae were re-standardized with Flavobacterium species 48 h before exposure to four virulent Flavobacterium columnare strains (Fc7, ALG-00-530, IA-S-4, and Ms-Fc-4) belonging to genomovars I and II and isolated from different geographical origins and host fish species. Flavobacterium species strain 4466 conferred protection to rainbow trout larvae against all Flavobacterium columnare strains. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0183] result Flavobacterium columnare kills axenic but non-standard zebrafish To investigate microbiota-based resistance to infection in zebrafish, we compared the susceptibility of germ-free and standard zebrafish larvae to Flavobacterium columnare, an important fish pathogen previously shown to infect and kill adult zebrafish. Using bath immersion, 6-day-postfertilization (6 dpf) germ-free and standard zebrafish larvae were exposed to a collection of 28 Flavobacterium columnare strains belonging to four different genomovars for 3 h at 5.10 μg / mL. 5 Daily monitoring showed that 16 of 28 Flavobacterium columnare strains killed the axenic larvae in less than 48 hours (Figure 2A), whereas the standard larvae survived exposure to all virulent Flavobacterium columnare strains tested (Figure 2B). The highly virulent strain ALG-00-530 (hereafter referred to as Flavobacterium columnare) ALG ), sterile death was rapid (1 day) and dose-dependent, and standard zebrafish died at the highest dose (10 7 The results showed that all but 1000 cfu / mL survived the infection (Figure 1). Similar survival of infected standard larvae was obtained for AB strain zebrafish eggs obtained from four different zebrafish facilities (Figure 3), suggesting that the standard zebrafish microbiota may confer protection against Flavobacterium columnare infection.

[0184] Ten culturable bacterial strains are sufficient to protect against Flavobacterium columnare infection In our rearing conditions, a standard larval microbiota is obtained after hatching from the microorganisms present in the egg chorion and in the water of the fish facility. The microorganisms associated with standard eggs are Flavobacterium columnarum. ALGTo test the hypothesis that re-Conv eggs provide protection against Flavobacterium columnarum, sterilized eggs were exposed to either fish facility tank water or mashed, unsterilized standard eggs aged 0 or 4 dpf (before or after hatching, respectively). In both cases, these re-Conv zebrafish, like standard zebrafish, were able to protect against Flavobacterium columnarum. ALG To confirm the composition of the canonical zebrafish microbiota, a clone library was generated and cloned using Flavobacterium columnarum. ALG We sequenced the 16S rDNA gene components of homogenous pools of 10 standard zebrafish larvae at 4, 6, and 11 dpf, sampled over a 3-month period from different larval batches, exposed or not to IL-16. We identified 13 dominant OTUs, 10 of which were identified in all experiments (Table 1). Furthermore, deep sequencing of the V3-V4 region of the 16S rDNA of gDNA collected from larvae from the four other zebrafish facilities mentioned above revealed that most of these 10 OTUs were also detected in standard larvae (Table 2).

[0185] [Table 4A]

[0186] [Table 4B]

[0187] [Table 4C]

[0188] To isolate culturable zebrafish microbiome bacteria, dilutions of homogenized pools of 6 and 11 dpf larvae were fixed onto various growth media, and 10 distinct bacterial morphotypes were identified. Using 16S-based analysis followed by whole-genome sequencing, 10 bacteria corresponding to 10 strains of nine different species were identified that were also consistently detected using the culture-free approach (Table 1). 4 dpf germ-free zebrafish were then cultured with a mix of all 10 identified culturable bacterial species (hereafter referred to as Mix 10), with 5.10 each. 5 Flavobacterium columnarum at 6 dpf, re-standardized at a concentration of cfu / mL. ALG We monitored zebrafish survival after exposure to Mix 10 and re-standardized zebrafish (Re-Conv Mix10 ) exhibited strong levels of protection against all identified highly virulent Flavobacterium columnare strains (Figure 5). These results demonstrated that Mix 10 constitutes a core protective bacterial population that provides complete protection for zebrafish larvae against Flavobacterium columnare infection.

[0189] [Table 5]

[0190] Population dynamics under antibiotic dysbiosis reveal major contributors to resistance to Flavobacterium columnare infection Flavobacterium columnare ALG To further analyze the determinants of Mix 10 protection against infection, 4 dpf larvae were treated with 10 species (5.10 each). 5Equal ratio mixtures of bacteria (cfu / mL) were inoculated into the zebrafish and their establishment was monitored over an 8-hour period. First, we verified that the overall bacterial composition of the larvae was not significantly different from that of the excised gut (p=0.99). We then sampled pools of 10 larvae immediately after renormalization (t0), 20 minutes, 2 hours, 4 hours, and 8 hours later, and tracked the relative abundance of bacteria using 16S rDNA sequencing. At t0, all species were present in the zebrafish at greater than 4% abundance, with the exception of Aeromonas veronii 1 (0.2%) and 2 (not detected) (Figure 6). Aeromonas caviae was the most abundant species detected, followed by Stenotrophomonas maltophilia and Chryseobacterium massilia, which together accounted for 67.5% of the population (Figure 6). The relative abundance of species was relatively stable for most species during the process of population establishment, likely reflecting their initial colonization ability, and the evenness of species was comparable at t0 (E = 0.84) and t8h (E = 0.85). However, the evenness of species in the standard and Re-Conv Mix10 Both larvae are Flavobacterium columnarum. ALG While protected against infection, the overall structure of the reconstituted Mix10 population differed from that of the standard population at 4 dpf (Figure 6). To verify the resilience of the sensitivity and protection afforded by the Mix10 cell population, we performed Re-Conv Mix10 Zebrafish were subjected to non-toxic antibiotic treatment at 4 dpf using either a 250 μg / mL penicillin / streptomycin combination (all members of the Mix 10 cells are sensitive to penicillin / streptomycin) or 50 μg / mL kanamycin (affects all members of the Mix 10 bacteria except Chryseobacterium massilia, Phyllobacterium myrsinacearum, and Stenotrophomonas maltophilia) (Figure 7). At 5 dpf, after 16 h of contact, the antibiotic was washed off and the zebrafish were immediately inoculated with Flavobacterium columnarum. ALG Both antibiotic treatments significantly reduced the number of Re-Conv Mix10 Flavobacterium columnare ALGThis resulted in a complete loss of protection against infection (Figure 8A). The same antibiotic treatment was then used, but only at 6 dpf, with a 24-hour recovery period after antibiotic washout at 5 dpf (Figure 8B). Mix10 The larvae continued to show protection comparable to that of infected germ-free larvae, whereas the kanamycin-treated Re-Conv Mix10 Zebrafish showed recovery of protection after a 24-hour recovery period and survived comparable to untreated standard fish (Figure 8B). Sampling and 16S analysis during the recovery experiment at different time points showed that the evenness of the bacterial population decreased similarly after antibiotic administration for both treatments (E = 0.85 for the control at 4 dpf, E = 0.72 at t0 for kanamycin, and E = 0.7 at t0 for penicillin / streptomycin) and continued to decrease during recovery (E = 0.6 and 0.64 after a 24-hour recovery period for kanamycin and penicillin / streptomycin treatments, respectively). Notably, Chryseobacterium massilia remained similarly detectable immediately after both antibiotic treatments, whereas penicillin / streptomycin treatment led to a significant decrease in its relative abundance (Figure 8C). In contrast, the relative abundance of Chryseobacterium massilia reversed 6 h after the end of kanamycin treatment and became the dominant member of the reconstituted microbiota after a 24-h recovery period (Figure 8D), suggesting that the protective effect observed in kanamycin-treated larvae was likely due to the recovery of Chryseobacterium massilia.

[0191] Resistance to Flavobacterium columnare infection is conferred by both individual and population-level protection Flavobacterium columnare ALG To examine the potential key role of Chryseobacterium massilia in protecting against infection, 4-dpf germ-free zebrafish were exposed to Chryseobacterium massilia alone, which increased the risk of infection by 5.10%. 2We found that individual protection was provided at doses as low as 5.10 cfu / mL (Figure 9). Interestingly, none of the other nine species that comprised Mix 10 were individually protective (Figure 9A), but their equi-ratio combination (designated Mix 9) conferred protection to zebrafish, except at 5.10 4 Doses below cfu / mL did not confer protection, and the reproducibility was not as good as that of Chryseobacterium massilia (Figure 9B). Mix9 To determine whether zebrafish protected against Flavobacterium columnare infection, we tested all nine eight-species combinations (Mix 8) as well as multiple combinations of seven, six, four, or three species, but no protection was observed (Figure 10 and Table 3). We then tested whether the lack of protection of Mix 8 compared to Mix 9 could be due to a density effect resulting from doubling the concentration of any of the species in the nonprotective Mix 8a (Figure 10B), but no protection was observed. Thus, these results suggested that microbiota-based protection against Flavobacterium columnare infection relies on either a Chryseobacterium massilia-dependent membership effect or a population-dependent effect mediated by the Mix 9 community.

[0192] [Table 6A]

[0193] [Table 6B]

[0194] [Table 6C]

[0195] [Table 6D]

[0196] [Table 6E]

[0197] Inflammatory and anti-inflammatory cytokine production is mediated by Flavobacterium columnarum ALG Does not contribute to microbiota-mediated protection against infection To verify the contribution of the innate immune response of larval zebrafish to resistance to Flavobacterium columnarum infection, we used qRT-PCR to measure cytokine mRNA expression in germ-free and standard zebrafish, as well as the expression of Chryseobacterium massilia (re-Conv Cm ), Mix 10 (re-Conv Mix10 Cytokine mRNA expression in larvae restandardized with Mix 4 (Aeromonas caviae, both Aeromonas veronii species, Pseudomonas mosseri) as a non-protective control (Table 3) or with or without exposure to Flavobacterium columnarum ALG was also measured. Genes encoding IL1β (pro-inflammatory), IL22 (pro-intestinal repair), and IL10 (anti-inflammatory) were examined. While some variation in il10 expression was observed in uninfected restandardized larvae, this did not correlate with protection. Furthermore, il10 expression was not modulated by infection under any of the conditions tested (Figure 11A). In contrast, Flavobacterium columnarum ALG ALG In germ-free zebrafish exposed to IL1b, a strong induction of IL1b and IL22 was observed (Fig. 11B-C). However, this induction was not observed in the protected canonical form, Re-Conv. Cm , Re-Conv Mix10 or non-protective Re-Conv Mix4These findings suggest that the presence of gut microbiota significantly (down-)modulates the inflammatory response induced by Flavobacterium columnare infection. However, this effect does not correlate with protection, suggesting that inflammatory modulation is not the primary mechanism of microbiota-induced resistance. Given that Myd88 is a major adaptor downstream of IL-1 and Toll-like receptor signaling, we also generated germ-free and typical larvae from myd88 null zebrafish mutants

[35] and showed that the inflammatory response induced by Flavobacterium columnare infection was not observed in larvae. ALG Remarkably, despite the lack of innate immunity, the virus was able to survive in either the standard or Re-Conv Mix10 Myd88 mutants, which are germ-free but not germ-free, survived Flavobacterium columnare infection similarly to wild-type zebrafish (Figure S11D). Furthermore, il10 induction by Flavobacterium columnare infection was observed only in germ-free larvae and was myd88-independent (Figure S12). Therefore, these results are inconsistent with a prominent role for cytokines in the microbiota-mediated protection against Flavobacterium columnare infection through modulation of pathogen-induced inflammatory responses.

[0198] Chryseobacterium massilia and Mix 9 protect zebrafish from intestinal damage after Flavobacterium columnare infection Flavobacterium columnare ALG Histological analysis of axenic larvae fixed 24 hours after exposure to Flavobacterium columnare revealed extensive intestinal damage (Figure 13A) before any significant signs were observed in other potential target organs such as the gills or skin. ALG To verify the requirement for intestinal access in the infection process, we modified our standard germ-free fish husbandry protocol to involve feeding live, germ-free Tetrahymena thermophila. We found that if left unfed, germ-free zebrafish could infect Flavobacterium columnarum. ALG While they did not die after contact, feeding them either Tetrahymena thermophila or other food sources such as sterilized fish meal resulted in the death of Flavobacterium columnare.ALG We found that susceptibility to infection was restored (Fig. 14), suggesting that feeding and ingestion are necessary for successful infection.

[0199] Histological sections consistently demonstrated severe intestinal damage, with blebbing and vacuole formation within the microvilli, in germ-free larvae infected with Flavobacterium columnare (Figure 13). In contrast, zebrafish preincubated with Chryseobacterium massilia or a mixed 9 consortium at 4 dpf and then exposed to Flavobacterium columnare at 6 dpf showed no differences compared to uninfected or standard-infected larvae (Figure 13), confirming complete protection against Flavobacterium columnare at the intestinal level.

[0200] Chryseobacterium massilia protects larval and adult zebrafish against virulent Flavobacterium columnare strains The clear protection afforded by Chryseobacterium massilia against Flavobacterium columnare infection led us to examine whether exogenous addition of this bacterium could improve microbiota-based protection against this widespread fish pathogen. We first showed that zebrafish larvae colonized with Chryseobacterium massilia were completely protected against all virulent Flavobacterium columnare strains identified in this study (Figure 15A). To examine whether Chryseobacterium massilia could also protect adult zebrafish from Flavobacterium columnare infection, we treated 3- to 4-month-old standard adult zebrafish with a high dose (5 × 10 6 cfu / mL) Flavobacterium columnarum ALGZebrafish were treated with Chryseobacterium massilia for 48 hours before challenge with Flavobacterium columnaris. Mortality monitoring revealed that pretreatment with Chryseobacterium massilia significantly increased the survival rate of adult zebrafish after infection with Flavobacterium columnaris ALG compared with untreated standard fish (p = 0.0084) (Figure 15B). Taken together, these results demonstrate that Chryseobacterium massilia is a putative broad-spectrum probiotic that protects zebrafish against columnaris disease caused by Flavobacterium columnaris.

[0201] Generation of germ-free rainbow trout larvae To investigate the potential protection offered by endogenous or exogenous bacteria against pathogens attacking a microbiologically controlled rainbow trout host (Oncorhynchus mykis), we first aimed to generate germ-free trout larvae. To this end, we exposed newly fertilized eggs to the aforementioned antibiotic and antifungal mixture for 5 hours

[0136] , followed by 15 minutes of exposure to 0.005% bleach, followed by 10 minutes of treatment with Romeiod, an iodophor disinfectant solution. The germ-free eggs were then placed in an antibiotic-supplemented aqueous solution at 16°C and maintained under sterile conditions in a Class II hood. 50 μl of rearing water was then sampled and culture-based and 16S-based PCR tests were performed to assess the germ-free status of the treated eggs 24 hours after treatment (Figure 16).

[0202] The germ-free eggs hatched naturally the same number of days after fertilization (dpf) as the untreated standard eggs, indicating that our sterilization protocol did not affect egg viability. Conversely, we determined that egg sterilization had a positive effect on hatching efficiency, which was 72±5.54% for the treated eggs and 48.6±6.2% for the standard eggs. After hatching, up to 12 larvae were placed in 75cm jars containing only fresh, sterile water without antibiotics. 3The fish were then transferred to cell culture flasks with vented caps (Figure 17). The water in the flasks was changed every 48 hours, and the germ-free and standard fish relied on their own yolk reserves until 21 days post-hatching (dph), after which they were fed gamma-sterilized fishmeal 30 minutes before water changes every 48 hours (Figure 17). For germ-free fish, 50 μl of fish water was collected before each water change and one larval sample was collected weekly for culture-based and 16S-based PCR sterility testing until the end of the experiment (35 dph).

[0203] Germ-free trout at 35 dph show normal development and growth compared to standard larvae. To verify the results of rearing germ-free larvae under sterile conditions, the growth performance of standard and germ-free larvae reared from the same egg batch was compared. No significant differences were observed in standard body length and weight at 35 dph, 2.33 ± 0.20 cm vs. 2.16 ± 0.11 cm and 0.72 ± 0.21 g vs. 0.64 ± 0.19 g for standard and germ-free larvae, respectively (Figure 18).

[0204] Consistently, anatomical comparison of standard and germ-free trout by optical projection tomography

[0138] revealed no anatomical differences in organ development between standard and germ-free fish at 21 dph, even for organs in direct contact with the fish microbiota, such as the gills (Figures 19D and 19I) and intestine (Figures 19C and 19H, 20), or organs potentially influenced by the gut microbiota, such as the brain (Figures 19A and 19F), spleen (Figures 19B and 19G), or pronephros (Figures 19E and 19J).

[0139] These results suggest that the natural microbiota does not significantly affect the development and growth of rainbow trout at this stage in their life cycle.

[0205] Identification of a fish pathogen that causes death in sterile but non-standard trout larvae To identify pathogens capable of infecting germ-free rainbow trout larvae via natural infection routes, several bacterial trout pathogens were tested, including Flavobacterium psychrophilum strain THC-O2 / 90, Flavobacterium columnare strain Fc7, Lactococcus garvieae, Vibrio anguillarum strain 1669, and Yersinia ruckeri strain JIP 27 / 88. At 24 dph, germ-free rainbow trout larvae were incubated with 10 test pathogens. 7 The fish were exposed to water containing 100 CFU / mL for 24 hours. The fish were then washed three times by replacing 90% of the infection tank with fresh, sterile water and then maintained at 16°C under sterile conditions. Of all the pathogens tested, Flavobacterium columnare strain Fc7 was the most virulent, leading to high and reproducible mortality in germ-free trout within 48 hours of exposure (Figures 21A and 25). In contrast to the susceptibility of germ-free trout to Flavobacterium columnare, standard larvae reared from non-sterile eggs resisted infection under all test conditions (Figures 21B and 26). Consistently, histological analysis performed at 25 dph (24 hours post-infection) showed that infected germ-free fish exhibited dissection of the gill epithelium and severe damage to the intestinal region. In contrast, standard rainbow trout larvae infected with Flavobacterium columnare showed no difference from uninfected germ-free or standard larvae. More precisely, histological analysis performed on germ-free and standard larvae at 25 dph (24 h post-infection) did not reveal any signs of intestinal damage (Figure 27). However, we observed an increase in goblet cell numbers when comparing infected and uninfected germ-free larvae, whereas standard-infected larvae did not show the opposite phenotype when compared to uninfected standard larvae (Figure 27).

[0206] Standard rainbow trout microbiota protects against Flavobacterium columnare infection Based on the high susceptibility of germ-free but nonstandard rainbow trout to Flavobacterium columnare Fc7, we hypothesized that the observed resistance to infection was conferred by the standard microbiota. To test this, germ-free rainbow trout larvae were exposed to water used to rear standard fish at 21 dph, 1 week before challenge with Flavobacterium columnare Fc7. The restandardized rainbow trout larvae tolerated Flavobacterium columnare equally well as standard larvae, whereas those reared under sterile conditions died rapidly within the first 24 hours post-infection (Figure 22A), suggesting that the standard rainbow trout microbiota conferred resistance to Flavobacterium columnare Fc7 infection. To identify culturable species within the standard trout microbiota, growth medium dilutions of material extracted from three standard rainbow trout larvae at 35 dph were plated on various agar plates. Independent 16S-based determination of bacterial diversity colonizing the tested media identified 11 different bacterial strains, which were individually isolated and stored (Table 4).

[0207] [Table 7]

[0208] To examine whether these 11 culturable strains could contribute to the protection against Flavobacterium columnare infection observed in standard trout, germ-free rainbow trout larvae at 22 dph were inoculated with a mix of all 11 bacterial strains (hereafter referred to as Mix 11) at 5 × 10 5 These re-standardized trout were exposed to Flavobacterium columnare strain Fc7 and survival was monitored. Mix11 Larvae survived as well as control fish (Figure 22B). These results demonstrate that the presence of 11 bacterial strains isolated from the rainbow trout microbiota reproduced the complete protection against Flavobacterium columnare infection observed in control fish.

[0209] Resistance to Flavobacterium columnare infection is conferred by one member of the trout microbiota To determine whether some individual members of the Protective Mix 11 may play a key role in infection resistance, 22 dph germ-free mass was cultured at 5.10 5 Single-restandardized fish were challenged with 11 isolated bacterial strains at concentrations of CFU / ml, followed by Flavobacterium columnarum Fc7. We found that only Flavobacterium species strain 4466 restored standard-level protection, while the other 10 strains, whether added individually (Figure 23A) or as a mix (Mix 10 shown in Figure 23B), showed no protection. To assess colonization of the gut by Flavobacterium species strain 4466 and / or Flavobacterium columnarum Fc7, intestines from single-restandardized fish 24 h postinfection were fixed on TYES agar after excision under sterile conditions. Interestingly, both Flavobacterium sp. strain 4466 and Flavobacterium columnare Fc7 were able to successfully colonize the intestines of rainbow trout after a single contact (Figure 23C). However, we were only able to detect Flavobacterium sp. after infection of trout with Flavobacterium columnare Fc7 (Figure 23C), suggesting potential competition between both bacterial species. Consistently, cell-free spent supernatant of Flavobacterium sp. strain 4466 showed no inhibitory effect on Flavobacterium columnare Fc7 in layering assays (Figure 28A). However, Flavobacterium sp. strain 4466 colony growth inhibited the growth of Flavobacterium columnare Fc7 (Figure 28B) and all Flavobacterium columnare strains tested (Figure 28C), suggesting potential contact-dependent inhibition. The present inventors have demonstrated that the T6SS of the type 6 secretion system (T6SS), a contact-dependent antagonistic system found only in the Bacteroidetes phylum, iiiidentified a cluster of 12 genes potentially associated with this phenotype in the Flavobacterium species strain 4466 genome (tssB, tssC, tssD, tssE, tssF, tssG, tssH, tssI, tssK, tssN, tssP, and tssQ) characteristic of the Flavobacterium species

[32] . To improve the taxonomic identification of protective Flavobacteria isolated from trout larvae microbiota, we performed whole-genome sequencing followed by average nucleotide identity (ANI) analysis. We determined that despite similarity to Flavobacterium spartansii (94.65%) and Flavobacterium tractatora (94.62%), these values ​​were lower than the 95% ANI required to identify the two organisms as the same species

[0204] . Furthermore, full-length 16S rRNA and recA gene comparisons also showed high similarity to Flavobacterium spartansii and Flavobacterium tracta, although the values ​​obtained were below the 99% similarity threshold required to consider the two organisms to belong to the same species (see the table on pages 5-6 of this specification). Similarly, a maximum likelihood-based phylogenetic tree (Figure 29) generated from the sequences of 15 bacterial strains from the genus Flavobacterium revealed that the sequences of Flavobacterium sp. strain 4466 clustered with those of Flavobacterium spartansii and Flavobacterium tracta, but Flavobacterium sp. strain 4466 could not be identified to the species level.

[0210] Endogenous Flavobacterium species strain 4466 protects germ-free rainbow trout against infection with various Flavobacterium columnare strains. To test whether protective Flavobacterium species isolated from the standard rainbow trout microbiota could protect rainbow trout, germ-free fish larvae were re-standardized with Flavobacterium species 48 h before exposure to four virulent Flavobacterium columnare strains (Fc7, ALG-00-530, IA-S-4, and Ms-Fc-4) belonging to genomovars I and II and isolated from different geographical origins and host fish species. Flavobacterium species strain 4466 conferred protection to rainbow trout larvae against all Flavobacterium columnare strains (Figure 30). Therefore, Flavobacterium species strains identified from trout mix 11 are putative probiotics that may protect trout and other fish from columnaris disease.

[0211] Use of germ-free mass to identify exogenous probiotics against Flavobacterium columnare infection Our results demonstrated that germ-free trout can be used as a gnotobiotic model to identify bacteria that protect against Flavobacterium columnarum infection. To determine whether this controlled gnotobiotic approach could be used to identify probiotics outside the range of bacteria present in the trout microbiota, we pre-exposed 22-dph germ-free rainbow trout larvae to Chryseobacterium massilia, a bacterium previously shown to protect larval and adult zebrafish from infection with Flavobacterium columnarum [Stressmann et al.]. 10 6After 48 hours of balneation with cfu / mL Chryseobacterium massilia, trout larvae were infected with four Flavobacterium columnare strains, Fc7, ALG-00-530, IA-S-4, and Ms-Fc-4, belonging to genomovars I and II and isolated from different geographical origins and hosts (Table 2). As previously observed in a zebrafish pure culture model, Chryseobacterium massilia also protected rainbow trout larvae against Flavobacterium columnare infection (Figure 24). Taken together, these results demonstrate that germ-free rainbow trout may enable the rational identification of bacterial species with probiotic potential against highly virulent Flavobacterium pathogens, whether endogenous to trout or not.

[0212] Bacterial taxonomic identification of protective microorganisms To taxonomically identify the protective Chryseobacterium and Flavobacterium strains isolated from the zebrafish and trout larval microbiota, we performed whole-genome sequencing followed by average nucleotide identity (ANI) analysis. The morphotype corresponding to Chryseobacterium was identified at the species level as Chryseobacterium massilia, with a genome-wide similarity of 95.85% (see the table on pages 5–7 of this specification). Regarding Flavobacterium strain 4466, we determined that despite its similarity to Flavobacterium spartansii (94.65%) and Flavobacterium tracta (94.62%), these values ​​were lower than the 95% ANI required to identify the two organisms as the same species

[0204] . Furthermore, full-length 16S rRNA and recA gene comparisons also showed high similarity with Flavobacterium spartansii and Flavobacterium tracta, although the values ​​obtained were also below the 99% similarity threshold required to consider the two organisms to belong to the same species (see the table on pages 5–7 of this specification).

[0213] Antibacterial gene prediction Antimicrobial resistance (AMR) genes were found in the entire sequenced genome of each strain using AMRFinderPlus. This tool is based on the following: Feldgarden, M., Brover, V., Haft, DH, Prasad, AB, Slotta, DJ, Tolstoy, I., Tyson, GH, Zhao, S., Hsu, C.-H., McDermott, PF, Tadesse, DA, Morales, C., Simmons, M., Tillman, G., Wasilenko, J., Folster, JP, Klimke, W., 2019. Validating the NCBI AMRFinder Tool and Resistance Gene Database Using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of NARMS Isolates. Antimicrob. Agents Chemother. 63 no. 11 (November 1, 2019): e00483-19 The implementation thereof is described in https: / / doi.org / 10.1128 / AAC.00483-19, which can be referenced by those skilled in the art.

[0214] No evidence of AMR genes was detected in the genome of Chryseobacterium species. The chromosome of Flavobacterium species 4466 revealed genes encoding resistance to carbapenems, licosamides, streptogramins, pleuromutilins, and fluoroquinolone antibiotics (Table 6).

[0215] [Table 8]

[0216] Toxicity gene prediction The isolated strain Chryseobacterium species contained five putative virulence factors, including several proteins involved in capsule biosynthesis, the heat shock protein HtpB subunit, the KatA catalase, and the ClpP protease proteolytic subunit (Table 7).

[0217] Table 7. Identification of virulence genes for Chryseobacterium massilia. Using the virulence factor database, virulence-coding genes were predicted using either protein annotation or nucleotide sequence from the whole genome of Chryseobacterium massilia.

[0218] In the case of Flavobacterium sp. 4466, genes encoding capsule, sialic acid synthase, type IV and type VI secretion system effectors and catalase were found as possible virulence factors (Table 8).

[0219] [Table 9]

[0220] Essay Numerous studies have focused on the effect of microbial diversity on higher-level bacterial community characteristics. In this study, we documented a novel, population-level protective effect of the resident microbiota against lethal infection. More specifically, using restandardization of otherwise germ-free zebrafish, we demonstrated that a series of 10 culturable bacterial strains belonging to nine different species from the standard laboratory zebrafish microbiota provided a conventional level of protection against infection with a wide range of highly virulent Flavobacterium columnare strains. With the exception of Chryseobacterium massilia (Bacteroidetes), this protective community was dominated by Proteobacteria, including Pseudomonas and Aeromonas, as well as bacteria commonly found in aquatic environments. Despite the relative tolerance of the zebrafish larval microbiota to environmental and sample-to-sample variability

[36] , we demonstrated that these 10 bacteria were also identified as dominant in four separate zebrafish facilities, suggesting the existence of a core microbiota with important functional properties.

[0221] Using germ-free and gnotobiotic zebrafish larvae exposed to controlled combinations of bacterial species, we were able to demonstrate a very robust species-specific protective effect in larvae associated with Chryseobacterium massilia alone. We also identified population-level protection conferred by combinations of nine other species that comprise a protective zebrafish larval microbiota that, when presented individually, would otherwise not be able to protect against Flavobacterium columnare. However, this protection was poorly reproducible (full protection was seen in only 50% of the tests performed) and was significantly less than the 5.10% observed in the case of Chryseobacterium massilia. 2 cfu / mL, at least 5.10 4 cfu / mL inoculum was required. Thus, these results suggest the existence of two distinct microbiota-based protection scenarios against infection by Flavobacterium columnare: a membership effect mediated by Chryseobacterium massilia, and a threshold effect mediated by the Mix9 consortium.

[0222] Neither of these two protective mechanisms against Flavobacterium columnare infection appears to rely on microbiota-based immunomodulation. However, we cannot exclude the possibility that some members of the protective Mix 10 individually induce pro- or anti-inflammatory responses that are hidden by the presence of the mixed microbiota. While further research is required to identify the mechanisms involved in population-level Mix 9 protection, renormalization and dysbiosis and recovery experiments demonstrated the key role of Chryseobacterium massilia in resistance to Flavobacterium columnare. This protection is likely conferred by several mechanisms, including nutrient depletion or competition, adhesion inhibition, release of inhibitory metabolites, and stimulation of host immune defenses [6, 12, 37]. Because Mix 9 and Chryseobacterium massilia showed differences in the minimum cell density required for protection, it is possible that in the latter case, Flavobacterium columnare infection induces a relatively density-independent protective mechanism in Chryseobacterium massilia through direct, antagonistic field competition. Indeed, both Flavobacterium columnare and Chryseobacterium massilia belong to the phylum Bacteroidetes, and in addition to direct resource competition, several mechanisms of field competition have been shown to occur between phylogenetically close Bacteroidetes species, including toxin production [38, 39] or toxin injection dependent on the type VI secretion system

[40] . Experiments are currently underway to identify non-protective Chryseobacterium massilia mutants and further analyze their protective mechanisms. Interestingly, infected larvae restandardized with either Chryseobacterium massilia or Mix 9 did not show signs of intestinal damage seen in axenic larvae, suggesting that both Chryseobacterium massilia and Mix 9 confer equivalent intestinal resistance to Flavobacterium columnare infection. While microbial colonization contributes to intestinal maturation and stimulates the production of epithelial passive defenses such as mucus [41, 42], lack of intestinal maturation is unlikely to contribute to Flavobacterium columnare-induced mortality, as monocolonized larvae or larvae restandardized with the nonprotective mix died as rapidly as axenic larvae.

[0223] Although several studies have observed the long-term assembly and development of the zebrafish microbiota from larvae to sexually mature adults, little is known about the initial colonization of larvae after hatching [43, 44]. Neutral (stochastic) and deterministic (host niche-based) processes [45-47] often lead to microbial communities represented by a limited number of species with high abundances and highly diverse populations with low abundances. In our experiments, the inoculum of the 10 mixed species corresponded to an equigeometric bacterial mix and therefore started from an assumed completely homogeneous state (E = 1) [48, 49]. In our study, the homogeneity was also relatively high (0.84) and remained very similar up to 8 h, suggesting that most of the 10 species were able to colonize the larvae. From the perspective of population competition, loss of diversity is often associated with decreased resistance to colonization, but it remains unclear whether this increased vulnerability is due to the loss of certain key members of the microbial community and / or changes in their dominance [7, 8]. We exposed established bacterial communities to different antibiotic perturbations and examined their resistance to infection following direct challenge with Flavobacterium columnare (to examine the sensitivity of the core microbiota to the perturbation) or subsequent recovery (to examine its resilience) [11, 50]. Antibiotics are known to alter the composition and relative abundance of the microbiota depending on their therapeutic range [12, 51]. We observed that the penicillin / streptomycin treatment, which was expected to affect most core species, reduced the abundance of all species except for two (Aeromonas veronii and Phyllobacterium myrsinacearum), which became relatively dominant during the recovery period but failed to provide protection against Flavobacterium columnarum. In the case of the kanamycin treatment, colonization resistance was fully restored toward the end of the recovery period, suggesting resilience that may be due to species that quickly recovered to pre-perturbation levels due to rapid growth, physiological plasticity, or mutation

[52] . Interestingly, evenness also decreased during the recovery period for both treatments, even taking into account potential biases associated with the use of 16S rDNA as a proxy index to determine relative abundance [53, 54]. However, phylum abundances shifted from >98% for Proteobacteria to 48% for Proteobacteria and 52% for Bacteroidetes after penicillin / streptomycin treatment. Furthermore, Chryseobacterium massilia was detected rarely (<1%) in typical larvae, suggesting that Chryseobacterium massilia may have a disproportionate effect on the population, or that population-level protection provided by the other nine bacterial species also contributed to the protection of typical larvae against Flavobacterium columnare infection.

[0224] Despite the phenotypic uniformity of symptoms associated with columnaris disease caused by Flavobacterium columnare in both cold- and warm-water fish, Flavobacterium columnare strains exhibit high genetic diversity, making standardization of animal models of infection difficult [34, 55, 56]. Our studies have shown that germ-free zebrafish larvae are highly susceptible to infection with a variety of genomovars isolated from different hosts, demonstrating their robustness as an animal model for studying the pathogenicity of Flavobacterium columnare. Flavobacterium columnare infection causes significant losses in aquaculture, but there is no consensus regarding the molecular basis of Flavobacterium columnare virulence. Secreted enzymes acting on connective tissue, such as chondroitin AC lyase [33, 57-59] and collagenase

[60] , have been proposed as possible virulence factors

[31] . Recently, a Flavobacterium columnare mutant in the type 9 secretion system (T9SS) was shown to be nonpathogenic in adult zebrafish, suggesting that proteins secreted by the T9SS may be key to virulence

[33] . The colonization process of Flavobacterium columnare also remains largely uncharacterized

[31] . In salmonids, the gills are the primary site of infection, but skin, fins, and tails are also frequently damaged, and septicemia can occur in severe cases

[57] . In salmon, low-virulence strains have been associated with gross tissue damage in multiple organs, while high-virulence strains cause death before such damage is observed

[31] . We were unable to identify clear sites of Flavobacterium columnare infection in zebrafish larvae by histology, likely due to the very low infectious dose, with less than 100 cfu recovered from infected, moribund larvae. However, multiple lines of evidence suggest that the intestine is the primary target of Flavobacterium columnarum infection in our model: (i) unfed germ-free larvae survived the contact, (ii) histological analysis showed severe gut disruption just a few hours after infection in germ-free larvae, and (iii) the primary function of IL-22 is to promote gut repair, suggesting that il22 is induced in germ-free larvae exposed to Flavobacterium columnarum.

[61] This induction appears to be the result of pathogen-mediated damage, as it was not observed in either standard or atypical larvae. The very rapid mortality of the larvae, likely due to this severe intestinal damage, likely explains why little damage was observed in other target organs common to columnaris disease, such as the gills, skin, and fins.

[0225] Numerous bacterial diseases affect aquaculture and cannot be controlled by vaccination, either because they affect juvenile fish or because no effective vaccines are available (e.g., for Flavobacterium). In these cases, the use of antibiotics remains the only option, potentially resulting in the spread of antibiotic resistance. There is an urgent need to develop alternative treatments

[11] . In the case of Flavobacterium columnare infection, both high genetic variability and a wide host range are important limiting factors for identifying effective probiotics against this broad-spectrum pathogen. This study demonstrated that Chryseobacterium massilia not only provided complete and robust protection against all virulent Flavobacterium columnare genomovars tested but also significantly enhanced the survival of adult standard zebrafish exposed to this pathogen, making it a promising probiotic candidate for preventing columnaris disease. While further research is needed to elucidate the protective potential of Chryseobacterium massilia in other teleosts, the endogenous nature of Chryseobacterium massilia suggests that it can establish itself as a long-term resident organism in the microbiota of larval and adult zebrafish, which could be an advantageous attribute when pursuing probiotic adaptation to target fish species.

[62] While short-term resident probiotics may limit unintended consequences for microbial populations and host strains, the use of endogenous resident organisms may stably regulate populations and provide long-term protection against recurrent disease outbreaks in fish.

[63]

[0226] In conclusion, the use of a simple and tractable fish model to mine the indigenous fish microbiota as a source of protection against fish pathogens further highlights the power of the zebrafish model for analyzing microbiota function. Our study contributes to expanding knowledge about microbiota-mediated colonization and resistance to infection against important fish pathogens. Future studies will determine the potential of endogenous bacteria as aquaculture probiotics to improve the health and production of other teleost fish.

[0227] The use of probiotics to improve fish growth performance and health and limit the use of chemicals and antibiotic treatments is currently a common approach to curbing disease outbreaks in the fish farming industry [123, 140, 141]. However, the identification and characterization of protective bacteria is hindered by experimental variability associated with treatment and challenge infections conducted in open environmental conditions using poorly controlled standard fish species. Therefore, the development of robust and reproducible gnotobiotic models is beneficial for the development of fish probiotics [125, 131]. Here, we established a novel, germ-free gnotobiotic model in rainbow trout, enabling controlled studies of probiotic-based protection against infections caused by various fish bacterial pathogens.

[0228] The resistance of rainbow trout eggs to effective sterilization protocols eliminated surface-associated microbial populations, allowing us to routinely rear germ-free larvae at 16°C for up to 35 days after incubation without constant antibiotic exposure. Our protocol is therefore comparable to gnotobiotic protocols used in zebrafish [136, 142], cod larvae

[0128] , and stickleback (Gasterosteus aculeatus)

[0143] , which do not rely on continuous antibiotic administration, thereby avoiding potential long-term effects on fish development.

[0131] Furthermore, after hatching, fish larvae were immediately transferred to cell culture flasks with vented caps. Rearing fish in flasks has several drawbacks that prevent long-term experiments, such as a lack of aeration or automated water exchange.

[0131] These limitations limit this model to being an effective method for short-term experiments. The relatively short duration of experiments conducted to investigate infection resistance in trout larvae, while effective in controlling germ-free conditions, poses the drawback of working with larvae with a less complex microbiota. Pure culture and gnotobiotic conditions are more artificial than traditional larval rearing conditions for both farmed and wild fish.

[0128] However, even if the addition of bacterial strains in pure culture cannot necessarily substitute for the effects of the natural host-associated microbiota, this model is an excellent tool that can be used to study the effects of specific bacterial additives without any microbial interference.

[0229] Rearing under germ-free conditions does not significantly affect the development and growth of rainbow trout larvae at 21 dph. Similar results were reported for germ-free stickleback larvae at 14 dph

[0143] . Germ-free reared sea bass (D. labrax L.) larvae were reported to grow faster and have more developed intestines than standard-reared larvae

[0144] . This discrepancy may be due to the fact that in our study and in the germ-free stickleback, anatomical analysis was performed before the first feeding, whereas the germ-free sea bass were fed externally. Fish initially obtain nutrients by absorbing internal egg yolk until the intestinal tract is open from the mouth to the outlet. We cannot exclude the possibility that differences in total body weight, as well as the structure and organ size, may occur between the intestines of germ-free and standard fish at later developmental stages or after the fish first feed.

[0230] Salmonids, including rainbow trout, are commercially important species, and their production in intensive aquaculture facilities is associated with increased susceptibility to diseases caused by viruses, bacteria, fungi, and parasites

[0145] . In this study, we tested the susceptibility of axenic and standard trout larvae to key salmonid freshwater pathogens. This led to the identification of Flavobacterium columnare as a hypervirulent species that is lethal to axenic larvae. Flavobacterium columnare is the causative agent of columnaris disease, which affects a variety of aquaculture fish species [137, 146] and is an emerging problem for larval and juvenile rainbow trout [147, 148]. In contrast to the high susceptibility of germ-free trout, standard larvae reared from non-sterile eggs were completely resistant to Flavobacterium columnare infection, indicating that the symbiotic microbiota harbored by standard trout larvae plays an essential role in protecting against Flavobacterium columnare.

[0231] Germ-free conditions cannot be compared with those prevailing in nature or those used in fish farming.

[0205] However, our results showed that germ-free rainbow trout larvae are highly susceptible to infection with Flavobacterium columnare, the causative agent of columnaris disease, which affects many aquacultured fish species. [206, 207] Our histological analysis comparing germ-free and standard larvae infected or uninfected with Flavobacterium columnare Fc7 did not reveal any major signs of lesional inflammation, but we observed that the number of goblet cells per crypt increased in infected germ-free larvae and decreased in standard larvae. A healthy intestine is determined by biological markers such as the number of goblet cells, which secrete mucus with bactericidal properties.

[0208] Interestingly, a significant decrease in goblet cell number was also observed in uninfected germ-free larvae compared with standard larvae, as previously reported in zebrafish.

[0209] The absence of stimulatory microorganisms in germ-free larvae likely led to a dysregulated acute immune response after Flavobacterium columnarum infection. These results suggest that the microbiota influences cell differentiation (or maturation) in the trout intestinal epithelium, potentially affecting some aspect of protection against Flavobacterium columnarum infection.

[0232] Various studies have demonstrated that a more diverse gut community provides greater protection to the host [149-151], which provides the basis for the paradoxical negative effects on fish health associated with the extensive use of antibiotics in aquaculture, which consequently reduces microbiota diversity and favors the colonization of opportunistic pathogens

[0152] .

[0233] This advocates for practices that enrich fish microbial communities to minimize pathogen introduction in aquaculture.

[0122] Meanwhile, our results demonstrate that resistance to infection can be achieved with the relatively low-complexity culturable microbiota identified in standard trout larvae. Although we identified only 11 distinct bacterial species from the standard rainbow trout larval microbiota, these species nonetheless confer complete protection to restandardized germ-free trout. While examples of infection resistance conferred by controlled bacterial consortia in gnotobiotic hosts often rely on the structure of the microbiota rather than on individual members of the microbiota, [153-156] we demonstrated that the protection observed for the bacterial consortium composed of the identified 11 microorganisms is primarily due to the presence of Flavobacterium sp. strain 4466. However, we cannot exclude the possibility that the presence of other bacterial species may be required for more efficient transfer or stability of protective members in the trout microbiota during subsequent developmental stages.

[0234] Over the past 30 years, the fish farming industry has made considerable efforts to identify probiotic microorganisms for rainbow trout, including Gram-positive and Gram-negative bacteria and yeasts.

[0157] However, the lack of reproducibility of many in vivo experiments, the high interindividual and seasonal variability in trout microbiota composition, and the limited colonization ability of exogenous microorganisms make it nearly impossible to definitively demonstrate probiotic properties. [158-160] The identification of Flavobacterium species, an indigenous member of the trout larval microbiota that protects against Flavobacterium columnare infection, suggested that this bacterium could be used as a probiotic to prevent infection. Although there is still no clear evidence that indigenous probiotics exert superior potency against the target host compared with exogenous probiotics,

[0161] the use of beneficial indigenous bacteria isolated from aquatic organisms is gaining recognition for pathogen control within the aquaculture industry.

[0162]

[0235] The high genetic variability and wide range of hosts of F. columnare are important limiting factors for identifying effective probiotics against this broad pathogen. Several probiotic candidates isolated from the host provided partial protection against F. columnare infection in other native fish species, such as walleye (Sander vitreous) and brook char (Salvelinus fontinalis) [163, 164]. However, depending on the fish family used, high variability in protection was observed among probiotic strains used against F. columnare-challenged brook char. The authors suggested that the genetic background of each family governs the efficacy of probiotics against pathogens, based on the fact that microbiota composition is directly influenced by the host genotype

[0163] . The use of germ-free or gnotobiotic animal models, as proposed in this study, should reduce this variability, allowing for a more accurate evaluation of candidate probiotic strains. According to the different criteria defined for the selection of bacterial probiotics [157, 161], our study suggests that Flavobacterium species can be considered as promising endogenous probiotics, whose potential in aquaculture needs further validation at different stages of the trout life cycle.

[0236] More precisely, the inventors specifically demonstrated the ability of Flavobacterium sp. strain 4466, isolated from standard trout larvae, to prevent Flavobacterium columnarum infection. Furthermore, this bacterium, but not its supernatant, inhibits Flavobacterium columnarum growth in vitro, suggesting a direct interaction between Flavobacterium sp. strain 4466 and Flavobacterium columnarum. Interestingly, Flavobacterium sp. strain 4466 utilizes a T6SS, a molecular mechanism unique to the Bacteroidetes phylum, to deliver antibacterial effector proteins upon contact with target cells. iiiThe complete subtype of Flavobacterium is encoded by the T6SS gene

[0211] . Members of the genus Flavobacterium are ubiquitous inhabitants of the microbiota of freshwater and marine fish, and both commensal and pathogenic Flavobacterium species often share the same ecological niche [212-214]. The T6SS of Flavobacterium species strain 4466 iii It is currently being investigated whether contact-killing strains of F. columnare contribute to colonization by inhibiting the potency of F. columnare Fc7. However, other mechanisms, such as pathogen exclusion in the event of nutrient competition or direct competition for attachment to host tissues, cannot be ruled out. This process has been suggested for infected zebrafish, where highly adherent probiotic strains colonized efficiently and extended the lifespan [215, 216, 217].

[0237] Interestingly, our germ-free rainbow trout larvae model enabled us to demonstrate the protective effect of Chryseobacterium massilia, a potentially probiotic bacterium isolated from standard zebrafish [Stressman], against various strains of Flavobacterium columnare from different hosts and geographical origins. These results support Chryseobacterium massilia as a potential probiotic for preventing columnaris disease in other teleost fish species, apart from its original host, zebrafish. Furthermore, this germ-free fish model offers extensive potential for the study of potential endogenous and exogenous probiotic strains against infection.

[0238] In conclusion, experimental conditions that reduce microbiota variability allow germ-free rainbow trout larvae to be challenged under gnotobiotic conditions, leading to a clear analysis of protective phenotypes against fish pathogens. This approach also facilitates the study of host-pathogen interactions under controlled conditions to better understand the virulence mechanisms used by fish pathogens. Overall, this model may contribute to the mitigation of fish diseases in rainbow trout from the perspective of aquaculture research and management.

[0239] Materials and Methods Bacterial Strains and Growth Conditions. The bacterial strains used in this study are listed in Table 1. Flavobacterium columnare strains (Table 5) were grown at 28°C in tryptone yeast extract salts (TYES) broth [0.4% (w / v) tryptone, 0.04% yeast extract, 0.05% (w / v) MgSO4 7H2O, 0.02% (w / v) CaCl2 2H2O, 0.05% (w / v) d-glucose, pH 7.2]. Flavobacterium columnare was assigned to four genomovar groups using 16S rDNA restriction fragment length polymorphism analysis, including genomovar I, I / II, II, and III

[64] . All 10 mixed 10 microbiota species were grown in Luria-Bertani (LB) medium at 28°C.

[0240] [Table 10A]

[0241] [Table 10B]

[0242] Ethical Statement All animal experiments described in this study were performed at the Institut Pasteur (larvae) or INRA Jouy-en-Josas (adults) in accordance with the European Union guidelines for the treatment of laboratory animals (http: / / ec.europa.eu / environment / chemicals / lab_animals / home_en.htm) and were approved by the relevant international Animal Health and Care Committees.

[0243] General handling of zebrafish. Wild-type AB fish or myd88 null mutant ( myd88 hu3568 / hu3568

[35] , kindly provided by A.H. Meijer (Leiden University, The Netherlands) were reared in our facility. Eggs were collected a few hours after oviposition, washed, and sorted under a dissecting scope to remove feces and unfertilized eggs. All the following procedures were carried out in a laminar flow microbiology cabinet using disposable plastic containers. A 25 cm 3 Fish were placed in sterile cell culture flasks with vented caps (0-6 dpf, 15 fish per flask) or in 24-well microtiter plates (6-15 dpf, one fish per 2 mL well) containing autoclaved mineral water (Volvic) and maintained at 28°C. Starting at 4 dpf, fish were fed sterile Tetrahymena thermophila parasites three times a week

[23] . Germ-free zebrafish were generated after sterilization of the egg chorion, protecting the otherwise sterile eggs, and then subjected to antibiotic and chemical treatments (see below). Meanwhile, standard larvae (with a facility-specific microbiota) were raised directly from unsterilized eggs, and the germ-free larvae were subsequently handled in exactly the same manner.

[0244] Sterilization of zebrafish eggs. Egg sterilization was performed using the procedure described above with some modifications

[23] . Freshly fertilized zebrafish eggs were first bleached (0.003%) for 5 min, washed in sterile water with gentle agitation for 3 min, and then placed in a 75 cm container containing 100 mL of heat-sterilized Volvic mineral water supplemented with methylene blue solution (0.3 μg / mL). 3Eggs were placed in 100-egg vented culture flasks with caps and maintained overnight. Eggs were then transferred to 50-mL Falcon tubes (100 eggs per tube) and treated with an antibiotic mixture (500 μL of penicillin G:streptomycin, 10,000 U / mL:10 mg / mL, GIBCO #P4333), 200 μL of filtered kanamycin sulfate (100 mg / mL, SERVA Electrophoresis #26899), and an antimycotic (50 μL of amphotericin B solution, Sigma-Aldrich (250 μg / mL), #A2942) for 2 hours at 28°C with agitation. Eggs were then washed three times in water with gentle agitation, bleached (0.003%) for 15 minutes, and resuspended by inversion every 3 minutes. The eggs were again washed three times in water and incubated for 10 min with 0.01% Romeiod (COFA, Cooperative Francaise de l'Aquaculture). Finally, the eggs were washed three times in water and placed in a 25 ml flask containing 20 mL of water. 3 The eggs were transferred to a vented-cap culture flask. After sterilization, 30–35 eggs were transferred to each flask. At 4 dpf, the eggs were transferred to new flasks and then re-standardized to 10–15 eggs per flask. Sterility was monitored at several locations throughout the experiment by dropping 50 μL of water from each flask onto LB, TYES, and YPD agar plates. All plates were incubated at 28°C under aerobic conditions. The plates were left for at least 3 days to allow for the proliferation of slow-growing organisms. Spot checks for bacterial contamination were also performed by PCR amplification of water samples using a 16S rDNA gene printer, according to the procedure detailed below. If a particular flask was contaminated, the fish from that flask were excluded from the experiment.

[0245] Germ-free zebrafish rearing procedures. After hatching, fish were fed sterile Tetrahymena thermophila three times a week starting at 4 dpf. (i) Tetrahymena thermophila stock. Axenic strains of Tetrahymena thermophila were stocked in 20 mL of PPYE (0.25% proteose peptone BD Bacterium #211684, 0.25% yeast extract BD Bacterium #212750) supplemented with penicillin G (10 units / mL) and streptomycin (10 μg / mL) and maintained at 28°C. The medium was inoculated with 100 μL of the preceding Tetrahymena thermophila stock. After 1 week of growth, samples were taken and tested for sterility on LB, TYES, and YPD plates and then returned to stock. (ii) Growth. Tetrahymena thermophila were stocked in MYE broth (1% milk powder, 1% yeast extract) inoculated from the stock suspension at a 1:50 ratio and incubated at 28°C. After 24 h of growth, the Tetrahymena were transferred to Falcon tubes and washed three times in 50 mL of autoclaved Volvic water (4400 rpm, 25°C, 3 min each). Finally, the Tetrahymena thermophila were resuspended in water and added to culture flasks (500 μL to 20 mL) or 24-well plates (50 μL per well). The sterility of the Tetrahymena thermophila was tested by plating and 16S rDNA PCR as described in the previous section. (iii) Fine Powder Feeding. When indicated, the animals were fed a pre-gamma-sterilized fine powder diet (ZM-000 Fish Feed, ZM Ltd) appropriate for the early initial feeding oral size every 48 h

[65] .

[0246] Restandardization of germ-free zebrafish. At 4 dpf, immediately after hatching, zebrafish larvae were restandardized with a single bacterial population or a mixture of bacteria. Ten bacterial species constituting the core protective microbiota were grown at 28°C in the appropriate medium (TYES or LB). The bacteria were then pelleted, washed twice with sterile water, and all were cultured at the same cell density (OD 600 =1 or 5.10 7 (i) Re-standardization by individual species. Bacteria were resuspended and added to the culture flasks containing the germ-free fish at a final concentration of 5.10 5(ii) Re-standardization with bacterial mixtures. To prepare Mix 10, Mix 9, Mix 8, and all other mixes used, equimolar mixtures were prepared with each bacterial species at an initial concentration of 5.10 7 The bacterial suspension was added to a final concentration of 5 × 10 cfu / mL in a culture flask containing germ-free fish. 5 cfu / mL.

[0247] Challenge infection. Flavobacterium columnare strains (Table 5) were grown overnight at 28°C in TYES broth. 2 mL of culture was then pelleted (10,000 rpm for 5 min) and washed once in sterile water. Germ-free zebrafish were challenged at 6 dpf for 3 h with a bacterial dose of 5.10 2 ~5.10 7 Fish were exposed to the test pathogens by immersion in culture flasks at concentrations ranging from 0.1 cfu / mL. Fish were then transferred to individual wells of a 24-well plate containing 2 mL of water and 50 μL of freshly prepared, sterile Tetrahymena thermophila. Mortality was monitored daily as described

[23] , and 54 ± 9 cfu of Flavobacterium columnare were recovered from infected larvae. All zebrafish experiments were terminated on day 9 post-infection, when zebrafish were euthanized with tricaine (MS-222, Sigma-Aldrich #E10521). Each experiment was repeated at least three times, with 10–15 larvae per condition and experiment.

[0248] Collection of eggs from other zebrafish facilities Standard zebrafish eggs were collected in 50 mL Falcon tubes from the following facilities: Facility 1: Nadia Soussi-Yanicostas facility at the Hôpital Robert Debre in Paris; Facility 2: Jussieu A2, University Paris 6; Facility 3: Jussieu-C8 (UMR7622), University Paris 6; Facility 4: Private AMAGEN facility, Gif sur Yvette. Larvae were handled according to the same housing conditions, sterilization procedures, and infection procedures used in the Pasteur Institute facilities.

[0249] Determination of fish bacterial load using cfu counting. Zebrafish were euthanized with 0.3 mg / mL tricaine (MS-222, Sigma-Aldrich #E10521) for 10 minutes. They were then washed in three separate baths of sterile PBS-0.1% Tween to remove loosely attached bacteria from the skin. Finally, the fish were transferred to a tube containing calibrated glass beads (acid-washed, 425 μm–600 μm, Sigma-Aldrich #G8772) and 500 μL of autoclaved PBS. The fish were homogenized for 45 seconds at maximum speed (6.5 m / s) using a FastPrep cell disruptor (BIO101 / FP120 QBioGene). Finally, serial dilutions of the collected suspension were spotted onto TYES agar plates and incubated at 28°C for 48 hours, after which cfu counts were performed.

[0250] Characterization of zebrafish bacterial content. Experiments were performed three times independently over a three-month period, with three separate batches of eggs collected from separate pairs of fish in different tanks. Larvae were reared as described above. Germ-free and standard larvae were collected at 4, 6, and 11 dpf per batch. Infected standard larvae were infected with Flavobacterium columnarum by immersion for 3 hours as described above. ALGFor each experimental group, triplicate pools of 10 larvae (one per experimental batch) were euthanized, washed, and lysed as described above. The lysates were divided into three aliquots: one for culture and subsequent 16S rDNA gene sequencing (A), one for 16S rDNA gene library generation and Sanger sequencing (B), and one for Illumina metabarcoding-based sequencing (C).

[0251] A) Bacterial culture and subsequent 16S rDNA gene-based identification The lysates were serially diluted and immediately plated on R2A, TYES, LB, MacConkey's, BHI, BCYE, TCBS, and TSB agar plates and incubated at 28°C for 24–72 hours. For each agar plate, the colony morphotype was documented, and colonies were picked and streaked onto duplicate agar plates. To identify individual morphotypes, individual colonies were picked from each agar plate for each identified morphotype, vortexed in 200 μL of DNA-free water, and boiled at 90°C for 20 minutes. Five μL of this bacterial suspension was used as a template for colony PCR to amplify the 16S rDNA gene with universal primer pairs for domain bacteria 8f (5'-AGA GTT TGA TCC TGG CTC AG-3' (SEQ ID NO: 7)) and 1492r (5'-GGT TAC CTT GTT ACG ACT T-3' (SEQ ID NO: 8)). Each primer was used at a final concentration of 0.2 μM in a 50 μL reaction. PCR cycling conditions included an initial denaturation at 94°C for 2 minutes, followed by 32 cycles of denaturation at 94°C for 1 minute, baking at 56°C for 1 minute, and extension at 72°C for 2 minutes, with a final extension step at 72°C for 10 minutes. 16S rDNA gene PCR products were verified on a 1% agarose gel and purified with a Qiaquick® PCR purification kit. Two PCR products per morphotype were sent for sequencing (Eurofins, Ebersberg, Germany). 16S rDNA sequences were manually proofread, and low-quality sequences were excluded from the analysis. Primer sequences were trimmed, and sequences were compared with GenBank (NCBI) and BLAST, and with the Ribosomal Database Project and SeqMatch. A 95% similarity cutoff was used for genus assignment and a 98% cutoff for operational taxonomic unit assignment.

[0252] B) 16S rDNA gene clone library generation Total DNA was extracted from the lysate using the Mobio PowerLyzer® Ultraclean® Kit according to the manufacturer's instructions. Axenic larvae and DNA-free water were also extracted as control samples. The extracted genomic DNA was verified by Tris-acetate-EDTA-agarose gel electrophoresis (1%), stained with GelRed, and quantified by directly applying 2.5 μL to a NanoDrop® ND-1000 spectrophotometer. The 16S rDNA gene was amplified by PCR using primers 8f and 1492r, and the product was checked and purified as described in Section A. Here, we added 25–50 μg of DNA as template to a 50 μL reaction. Clone libraries were generated using the pGEM®-T Easy Vector System (Promega) according to the manufacturer's instructions. The presence of cloned inserts was confirmed by colony PCR in conjunction with the vector primers gemsp6 (5'-GCT GCG ACT TCA CTA GTG AT-3' (SEQ ID NO: 9)) and gemt7 (5'-GTG GCA GCG GGA ATT CGA T-3' (SEQ ID NO: 10)). Clones with the correct size insert were purified as described above and sent for sequencing (Eurofins, Ebersberg, Germany). Blanks using DNA-free water were run as controls for all procedures. The coverage of the clone library was calculated according to the following formula: [1 - (n1 / N2)] x 100, where n1 represents the number of singletons detected in the clone library and N2 represents the number of clones generated for this sample. Clone libraries were generated to achieve a minimum coverage of 95%, yielding at least 48 clones per sample. Sequence analysis and identification were performed as described in Section A.

[0253] C) Illumina sequencing of the 16S rDNA gene To identify 16S rDNA gene diversity in fish collected from our facility and four other zebrafish facilities, fish were raised as described above. Germ-free fish were sterilized as described above, and non-infected germ-free and standard fish were collected at 6 and 11 dpf. Flavobacterium columnarum was used as described above. ALG Infection with Flavobacterium columnare was performed by immersion in a tank for 3 hours, followed by transfer to clean water. Infected standard fish were collected at 6 dpf (6 hours after infection) and at 11 dpf, similar to uninfected fish. Infected germ-free larvae were collected only at 6 dpf (6 hours after infection), but by 11 dpf, all larvae had died from infection. Triplicate pools of 10 larvae were euthanized, washed, and lysed as described above. Total DNA was extracted using the Mobio PowerLyzer® Ultraclean® kit as described above, quantified on a NanoDrop® ND-1000 spectrophotometer, and sent to IMGM Laboratories GmbH (Germany) for Illumina sequencing. Primers Bakt_341F (5′-CCTACGGGNGGCWGCAG-3′ (SEQ ID NO: 11)) and Bakt_805R (5′-GACTACHVGGGTATCTAATCC-3′ (SEQ ID NO: 12)) were used for amplification in conjunction with amplification of variable regions 3 and 4 of the 16S gene [ 63 ].

[0254] Each amplicon was purified using solid-phase reversible immobilization (SPRI) paramagnetic particle-based technology (AMPure XP beads, Beckman Coulter) at a bead-to-DNA ratio of 0.7:1 (v / v) according to the manufacturer's instructions. Amplicons were normalized using the Sequal-Prep kit (Life Technologies), resulting in each sample containing approximately 1 ng / μl of DNA. Samples, positive controls, and negative controls were generated in a single library. The quality of the purified amplicon library was checked using a High Sensitivity DNA LabChip kit on a 2100 Bioanalyzer system (both from Agilent Technologies). For cluster generation and sequencing, the MiSeq® Reagent Kit 500-Cycle Nano v2 (Illumina Inc.) was used. Prior to sequencing, cluster generation by two-dimensional bridge amplification was performed, followed by bidirectional sequencing, resulting in 2 × 250 bp paired-end (PE) reads.

[0255] MiSeq® Reporter 2.5.1.3 software was used for primary data analysis (signal processing, demultiplexing, adapter trimming). CLC Genomics Workbench 8.5.1 (Qiagen) was used for read splicing, quality trimming, and QC reporting, and OTU definition was performed within the Bacterial Genomics module of the CLC plug-in.

[0256] Comparison of the whole body and intestinal bacterial contents of larvae Mix 10 was re-standardized and cultured for 3 hours at 6 dpf. ALGLarvae infected with B. tumefaciens were euthanized and washed. DNA was extracted from a pool of 10 whole larvae or a pool of 10 intestinal tracts excised with sterile surgical forceps, and Illumina 16S rDNA gene sequencing was performed on them. Samples of germ-free larvae and excised germ-free intestines were collected as controls. No statistically significant differences were observed between whole-body and intestinal bacterial contents of fish (p = 0.99). Therefore, whole larvae were used in experiments to monitor bacterial establishment and recovery.

[0257] Whole genome sequencing Chromosomal DNA from 10 species comprising the core of the zebrafish larval microbiota was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) with RNase treatment. DNA quality and quantity were assessed on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA sequencing libraries were created using the Nextera DNA Library Preparation Kit (Illumina Inc.), and library quality was checked on a Bioanalyzer 2100 (Agilent Technologies) using a High-Sensitivity DNA LabChip Kit. Sequencing clusters were generated using the MiSeq Reagent Kit v2 500 Cycle (Illumina Inc.) according to the manufacturer's instructions. DNA sequencing was performed at the Helmholtz Center for Infection Research by bidirectional sequencing, generating 2 × 250-bp paired-end (PE) reads. A range of 1,108,578 to 2,914,480 reads were generated per sample, with a median of 1,528,402. Reads were quality selected and trimmed, adapters were filtered out using fastq-mcf (Aronesty, 2011 #29), and the genome was assembled using SPAdes 2.5.1 (Bankevich, 2012 #123).

[0258] Bacterial species identification. Whole-genome-based bacterial species identification was performed using the TrueBac ID system (v1.92, DB:20190603) [https: / / www.truebacid.com / ;

[66] . Species-level identification was performed based on an algorithm cutoff set at 95% ANI, where possible, or if 16S rDNA gene sequence similarity was >99%.

[0259] Monitoring bacterial dynamics Three independent experiments were conducted over a 6-week period, with eggs collected from separate pairs of fish from different tanks to monitor establishment and recovery. Larvae were reared, sterilized, and infected as described above, with the only difference being that eggs were grown at 75 cm to accommodate the larger number of larvae required. 3 Culture flasks with vented caps (filled with 50 mL of sterile Volvic) were used because in each experiment, larvae intended for time-series Illumina sequencing were sequentially removed from the experiment where larval survival was monitored. Animals were pooled (10 larvae per time point / condition), euthanized as described above, washed, lysed, and stored at -20°C until the end of survival monitoring and until all triplicate pools had been collected.

[0260] A) Establishing a group To track the establishment of the 10 core species in the larvae, the germ-free larvae were reconstituted with an equal ratio of Mix 10 as described above. Mix10 Larval samples were collected at 4 dpf immediately after addition of the 10 core species, and then at 20 min, 2 h, 4 h, and 8 h. Samples of sterile standard larvae and inoculum were also collected as controls.

[0261] B) Induction of dysbiosis Different doses of kanamycin (dose 1 was 200 μg / mL, dose 2 was 50 μg / mL, and dose 3 was 25 μg / mL) and a penicillin / streptomycin antibiotic mixture (dose 1 was 250 μg / mL, and dose 2 was 15.6 μg / mL) were administered in the reconstituted mice. Mix10Zebrafish larvae at 4 dpf were tested to identify antibiotic treatments that, when added to flask water, were non-toxic to the larvae but caused dysbiosis. Sixteen hours after treatment, the antibiotics were extensively washed away with sterile water, and the larvae were incubated with Flavobacterium columnarum. ALG As a result of challenge with 250 μg / mL penicillin / streptomycin and 50 μg / mL kanamycin, all larvae died and colonization resistance was successfully abolished with the best results in all replicates where antibiotic treatments were used, e.g., 250 μg / mL penicillin / streptomycin and 50 μg / mL kanamycin.

[0262] C) Population recovery B) After 8 hours of incubation, 4 dpf recon Mix10 Larvae were treated with 250 μg / mL penicillin / streptomycin and 50 μg / mL kanamycin for 16 hours. The antibiotics were washed out extensively, and the larvae were then left to recover in sterile water for 24 hours to assess the resilience of the bacterial population. Samples (pools of 10 larvae) were taken at 3, 6, 12, 18, and 24 hours during the recovery period and sent for 16S rDNA Illumina sequencing. At 6 dpf, the larvae were then incubated with Flavobacterium columnarum. ALG Mice were challenged with 1000kJ / ml for 3 hours and monitored daily for survival for 10 days post-infection. All time series samples were sequenced by IMGM Laboratories GmbH as described above.

[0263] Statistical analysis of metataxonomic data. 16S RNA analysis was performed using SHAMAN (Volant, 2019 #125). Library adapters, primer sequences, and base pairs occurring at the 5' and 3' ends with a Phred quality score below 20 were trimmed using Alientrimmer (v0.4.0). Reads with positive matches to the zebrafish genome (mm10) were excluded. Filtered high-quality reads were merged into amplicons using Flash (v1.2.11). The resulting amplicons were clustered into operational taxonomic units (OTUs) using VSEARCH (v2.3.4) [Rognes, T., Flouri, T., Nichols, B., Quince, C., & Mahe, F. (2016). VSEARCH: a versatile open source tool for metagenomics. PeerJ, 4, e2584.] This process includes several steps for dereplication, singleton removal, and chimera detection. Clustering was performed at a 97% sequence identity threshold, resulting in 459 OTUs. OTU taxonomic annotation was performed against the SILVA SSU (v132) database (Quast, 2012 #126), which was completed using VSEARCH and included 16S sequences from 10 bacterial populations, and filtered according to their identity with the reference (Yarza, 2014 #127). Annotations were retained if the identity between the OTU sequence and the reference sequence was 78.5% or greater for taxonomic class, 82% or greater for order, 86.5% or greater for family, 94.5% or greater for genus, and 98% or greater for species. In this study, 73.2% of the OTUs were annotated, and of those, 91.69% were annotated at the genus level.

[0264] Input amplicons were then aligned against the set OTUs, and VSEARCH global alignment was used to generate an OTU contingency table listing the number of amplicons associated with each OTU. The matrix of OTU count data was normalized for library size at the OTU level using weighted non-null count normalization. Normalized counts were then summed within genera. Subsequently, the DESeq2 R package was used to analyze the data. 95 A generalized linear model (GLM) implemented in was applied to detect differences in genus abundance between groups. We defined a GLM including treatment (condition) and time (variable) as main effects and the interaction between treatment and time. The resulting P values ​​were adjusted according to the procedure of Benjamini and Hochberg.

[0265] The structural analysis can be reproduced in SHAMAN by loading the count tables describing the taxonomic results together with the target and contrast files available on Figshare (https: / / doi.org / 10.6084 / m9.figshare.11417082.v2).

[0266] Determining cytokine levels Total RNA from individual zebrafish larvae was extracted 18 hours after pathogen exposure (12 hours after washing) using the RNeasy kit (Qiagen). Oligo(dT17)-primed reverse transcription was performed using M-MLV H reverse transcriptase (Promega). Quantitative PCR was performed using Takyon SYBR Green PCR Mastermix (Eurogentec) on a StepOne thermocycler (Applied Biosystems). Primers for ef1a (a housekeeping gene used to normalize cDNA amounts), il1b, il10, and il22 were described in [Rendueles 2012]. Data were analyzed using the ΔΔCt method. Four larvae per condition were analyzed. Zebrafish genes and proteins mentioned in the text are as follows: ef1a NM_131263, il1b BC098597, il22 NM_001020792, il10 NM_001020785, myd88 NM_212814.

[0267] Histological comparison of germ-free, standard, and restandardized fish infected or not with Flavobacterium columnare Fish were collected 24 hours post-infection (7 dpf), fixed in Trump's fixative (4% methanol-free formaldehyde, 1% glutaraldehyde in 0.1 M PBS, pH 7.2)

[67] for 24 hours at 4°C, and sent to the PIBiSA microscopy facility at the Faculte de Médecine de Tours (France) (https: / / microscopies.med.univ-tours.fr / ), where whole fixed animals were processed and embedded in Epon. Semi-thin sections (1 μm) were cut using an X-ultramicrotome and then stained with toluidine blue for light microscopy and imaging, or processed for transmission electron microscopy.

[0268] Pretreatment of adult zebrafish with Chryseobacterium massilia Zebrafish strain AB was used. Fish were raised in dechlorinated, recirculating water at 28°C and then transferred to continuous-flow tanks at 3–4 months of age for infection experiments. Chryseobacterium massilia was grown in TYES broth at 150 rpm and 28°C to stationary phase. The bacterial cultures were washed twice in sterile water and the OD 600nm The Chryseobacterium massilia bacterial suspension was adjusted to a final concentration of 2.10 6 Adult fish were re-standardized by adding bacteria directly to the fish water (1 L) at 1000 cfu / mL. The water flow was stopped, removed, and then restarted to maintain contact with the fish for 24 hours. After water replacement, Chryseobacterium massilia was administered twice. The same volume of sterile water was added to the control group.

[0269] Challenge infection of adult zebrafish Immediately after restandardization of fish with Chryseobacterium massilia, Flavobacterium columnare infection was performed. This infection was performed with slight modifications as previously described by Li and coworkers [Li et al., 2017]. Briefly, Flavobacterium columnare strain ALG-0530 was grown in TYES broth at 150 rpm and 28°C to stationary phase. The bacterial culture was then diluted to a final concentration of 5.10 6 The bacteria were diluted at cfu / mL directly into 200 mL of aquarium water. The water flow was stopped, removed, and then restarted, allowing the bacteria to remain in contact with the fish for 1 hour. Sterile TYES broth was used as a control. Bacterial counts were confirmed at the start of the immersion challenge by plating serial dilutions of the water sample on TYES agar. The water was maintained at 28°C and continuously oxygenated during the immersion period. Groups consisted of 10 fish. Virulence was assessed based on fish mortality 10 days after infection.

[0270] Statistical methods. Statistical analysis was performed using the unpaired nonparametric Mann-Whitney test. Analysis was performed using Prism v8.2 (GraphPad Software). Evenness: The Shannon diversity index was calculated using the formula (HS = -Σ[P(ln(P)]), where P represents the relative abundance of species. Overall evenness was calculated by the Shannon index E = H S / H max The lower the interspecific evenness (and the presence of dominant species) in a population, the lower this index.

[0271] Handling of rainbow trout larvae Fertilized rainbow trout eggs were obtained from Aqualande Group, France. Upon arrival, the eggs were acclimated at 16°C before manipulation. All procedures were carried out in a laminar flow microbiology cabinet using single-use disposable plasticware. Eggs were placed in 145 x 20 mm Petri dishes and maintained in 75 mL of autoclaved, dechlorinated water until hatching. After hatching, fish were transferred to 250 mL vented cell culture flasks containing 100 mL of sterile water and maintained at 16°C. At 21 days post-hatching, the fish were fed irradiated powdered food. To avoid waste accumulation and oxygen limitation, half of the water was replaced every two days to maintain the health of the rainbow trout larvae.

[0272] Sterilization and rearing of germ-free rainbow trout Rainbow trout eggs were first transferred to sterile Petri dishes (140 mm, 150 eggs per dish) and washed twice with sterile methylene blue solution (0.05 mg / mL). Freshly fertilized eggs were then maintained in 75 mL of methylene blue solution and exposed to the antibiotic mixture described above (750 μL of penicillin G (10,000 U / mL) / streptomycin (10 mg / mL), 300 μL of filtered kanamycin sulfate (100 mg / mL), and 75 μL of the antifungal amphotericin B solution (250 μg / mL)) for 5 hours at room temperature with stirring. The eggs were then washed three times with fresh sterile water. They were then bleached (0.005%) for 15 minutes. The eggs were again washed three times with sterile water. They were then treated with Romeiod, an iodophor disinfectant, for 10 minutes. Finally, the eggs were washed three times and placed in 75 mL of sterile water supplemented with antibiotics and kept at 16°C until hatching. Eggs hatched naturally 5–7 days after treatment. Immediately after hatching, the fish were placed in a 75 cm 3 The eggs were transferred to a vented cap culture flask (12 larvae per flask). The hatching rate was determined by counting the number of larvae that hatched in the Petri dish and comparing it with the total number of eggs.

[0273] Sterility was monitored at various times throughout the experiment by dropping 50 μL of rearing water from each flask onto LB agar plates, YPD agar plates, and TYES agar plates, all incubated at 16°C under aerobic conditions. Fish larvae were also checked weekly for bacterial contamination. Randomly selected fish were killed by an overdose of filtered tricaine methanesulfonate solution (MS222, 300 mg / L). The whole fish were mechanically agitated in a Lysing matrix tube containing 1 mL of sterile water and 425–600 μm glass beads (Sigma). The aerobic conditions were monitored for 45 seconds, 6.0 ms, and 10 min using a FastPrep-24 instrument (XXX). -1Samples were homogenized using a 100-ml syringe pump. Serial dilutions of the homogenized solution were plated on TYES agar, YPD agar, and LB agar. If water samples or collected, euthanized, and homogenized fish showed any bacterial CFU in any of the various media used, these animals (or flasks) were excluded from the experiment. The absence of any contamination in the fish larvae was confirmed by PCR using primers specific to the 16S region of the chromosome (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 13), 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 14)).

[0274] Bacterial strains and growth conditions The bacterial strains used in this study are listed in Table 2. Flavobacterium columnare strains Fc7 and IA-S-4 and Chryseobacterium massilia were grown in tryptone yeast extract salts (TYES) broth [0.4% (w / v) tryptone, 0.04% yeast extract, 0.05% (w / v) MgSO4 7H2O, 0.02% (w / v) CaCl2 2H2O, 0.05% (w / v) D-glucose, pH 7.2] at 150 rpm and 18°C. Flavobacterium psychrophilum strains THCO2-90 and FRGDSA 1882 / 11 were grown in TYES broth at 150 rpm and 28°C. Yersinia ruckeri strain JIP 27 / 88 was grown in TYES broth at 150 rpm and 28°C. Cultures were grown in Luria-Bertani (LB) medium at 150 rpm and 28°C. Vibrio anguillarum strain 1669 was grown in tryptic soy broth (TSB) at 150 rpm and 28°C. Lactococcus garvieae was grown in brain heart infusion (BHI) medium at 150 rpm and 28°C. 15 g / L agar was added when necessary for solid media. Stock cultures were maintained at -80°C in their respective medium containing 20% ​​(vol / vol) glycerol.

[0275] Fish challenge infection Pathogens were grown in suitable media at various temperatures to advanced stationary phase. Each culture was then pelleted (10,000 rpm for 5 minutes) and washed once in sterile water. Bacteria were resuspended and diluted to a final concentration of 10 7 The pathogens were added to the culture flasks at a concentration of cfu / mL. After 24 hours of incubation at 16°C, the fish were washed three times with each water change. 10–12 larvae were used per condition and experiment. Bacterial counts were confirmed at the beginning and end of the immersion challenge by plating serial dilutions of water samples on media specific for each pathogen. Each experiment was repeated at least twice. Virulence was assessed according to fish mortality 10 days after infection.

[0276] Characterization of culturable standard rainbow trout microbiota To identify species constituting the culturable standard microbiota, three standard rainbow trout larvae were euthanized at 31 dpf by overdosing with MS222. These fish were homogenized according to the protocol described above, and serial dilutions of the homogenized suspension were plated on different media: TYES agar, LB agar, R2A agar, and TSA. The plates were incubated at 16°C for 48–72 h. After incubation, morphologically distinct colonies (based on morphology, size, color, texture, height, and margins) were isolated and maintained at −80°C in their respective media containing 15% (v / v) glycerol. Individual 16S base identification was performed by amplifying and sequencing the 16S chromosomal region using the universal oligonucleotides 27F and 1492R. The 16S rRNA gene sequences were then compared with those available in the EzBioCloud database

[0178] .

[0277] Re-standardization of germ-free rainbow trout microbiota Each isolated bacterial species was grown in the appropriate medium for 24 hours at 150 rpm and 28°C. The bacteria were then pelleted and washed twice with sterile water. They were diluted to a final concentration of 5 x 10 7 At 22 dph, 1 mL of each bacterial suspension was added to the flask (final concentration 5 × 10 5cfu / mL) and germ-free rainbow trout were re-standardized. When fish were re-standardized with bacterial consortia, after bacterial washing, all isolated species were collected in aqueous suspension at a concentration of 5 × 10 7 The bacterial suspension was mixed at 0.05 cfu / mL. This mixed bacterial suspension was then added to flasks containing germ-free rainbow trout as described. In all cases, fish were restandardized for 48 hours, followed by a challenge infection with Flavobacterium columnare. The bacterial suspension was added immediately after water change. Each experiment was repeated at least twice.

[0278] Histological verification Histological sections were used to compare microscopic lesions between germ-free and standard fish after Flavobacterium columnarum infection. Sacrificed animals were fixed in Trump's fixative (4% methanol-free formaldehyde, 1% glutaraldehyde in 0.1 M PBS, pH 7.2) for 24 hours at 4°C.

[0179] Fixed fish were whole-body processed and embedded in Epon. Semi-thin sections (1 μm) were cut using an ultramicrotome and stained with toluidine blue for light microscopic observation and image processing.

[0279] Three-dimensional imaging of transparent fish using optical projection tomography (iDISCO) For whole-body 3D imaging of cleared fish, fish were fixed in 4% formaldehyde in PBS and left overnight at 4°C. Fixed samples were washed with PBS. To clear the tissue, fish were first pre-decolorized by pre-immersion in 0.5X SSC twice for 1 hour at room temperature, followed by incubation in 0.5X SSC + 0.5% KOH + 3% H2O2 for 2 hours at room temperature. Decolorization was stopped by incubation in PBS twice for 15 minutes. Fish were then post-fixed in 2% formaldehyde in PBS for 2 hours at room temperature, followed by two 30-minute rinses in PBS. The decolorized fish were cleared using the iDISCO+ protocol [Renier, 2016] (Renier et al. 2016, PMID 27238021). Briefly, samples were gradually dehydrated in an increasing methanol series (20%, 40%, 60%, 80%, and 100% in HO twice, each time for 1 hour). The dehydrated samples were bleached by overnight incubation at 4°C in methanol + 5% HO, followed by two 1-hour incubations in 100% methanol. They were then successively incubated in 67% dichloromethane + 33% methanol for 3 hours, dichloromethane for 1 hour, and finally dibenzyl ether until the fish were completely transparent. Whole-body samples were acquired on a light-sheet ultramicroscope (LaVision Biotec, Bielofeld, Germany) using a 2x objective and a 0.63x zoom factor. Autofluorescence was obtained by illuminating both sides of the sample with a 488 nm laser. Z-stacks were acquired with a 2 μm z-step.

[0280] Whole genome sequencing. Chromosomal DNA of Flavobacterium species strain 4466 isolated from rainbow trout larval microbiota was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) with RNase treatment. DNA quality and quantity were assessed on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA sequencing libraries were generated using the Nextera DNA Library Preparation Kit (Illumina Inc.), and library quality was checked on a Bioanalyzer 2100 (Agilent Technologies) using a High-Sensitivity DNA LabChip Kit. Sequencing clusters were generated using the MiSeq Reagent Kit v2 500 Cycle (Illumina Inc.) according to the manufacturer's instructions. DNA sequencing was performed by bidirectional sequencing at the Institut Pasteur Microbiology Interactive Platform, generating 2 × 250 bp paired-end (PE) reads. Reads were quality selected, trimmed, and adapters were removed using fastq-mcf

[0218] , and the genome was assembled using SPAdes 3.9.1

[0219] .

[0281] Genome phylogenetic analysis. The proteomes of the 15 closest Flavobacterium strains identified by ANI analysis were retrieved from the NCBI RefSeq database (Table below).

[0282] [Table 11]

[0283] These sequences were analyzed along with the proteome of Flavobacterium sp. strain UGB 4466 using Phylophlan (version 0.43, March 2020). This method uses the 400 most conserved proteins to construct a maximum likelihood phylogenetic tree using RAxML (version 8.2.8). The maximum likelihood was bootstrapped with 1000 replicates.

[0284] Agar layering assay for detecting growth inhibition The growth inhibitory effect of Flavobacterium sp. strain 4466 was evaluated using an agar spot test. Briefly, 125 μl of an overnight culture of various strains of Flavobacterium columnare, adjusted to an OD of 1, was mixed with 5 mL of top agar medium (0.7% agar) and overlaid on a TYLES agar plate. 5 μl of an overnight culture of Flavobacterium columnare sp. 4466 was then dropped onto the target bacterial overlay. The plate was incubated at 28°C for 24 hours. Growth inhibition of Flavobacterium columnare was recorded by the observation of a distinct halo surrounding the Flavobacterium sp. strain colony. Sterile TYES culture medium was used as a mock, and the experiment was performed in triplicate.

[0285] Whole genome sequencing for taxonomic identification and prediction of antibiotic resistance and virulence factors based on whole genome sequence analysis Chromosomal DNA of Chryseobacterium sp., Delftia sp. strain 4465 (available in the ENA (European Nucleotide Archive) database under primary accession number ERS4574863 (version 1) and secondary accession number SAMEA6847265 (taxpayer ID number 80866, scientific name Delftia acidovorans)) and Flavobacterium sp. strain 4466 was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) with RNase treatment. DNA quality and quantity were assessed on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA sequencing libraries were generated using the Nextera DNA Library Preparation Kit (Illumina Inc.), and library quality was checked on a Bioanalyzer 2100 (Agilent Technologies) using the High Sensitivity DNA LabChip Kit. Sequencing clusters were generated using the MiSeq Reagent Kit with 500-cycle v2 chemistry (Illumina Inc.) according to the manufacturer's instructions. DNA sequencing was performed by bidirectional sequencing at the Institut Pasteur Microbiology Interactive Platform, generating 2 × 150 bp paired-end (PE) reads. Reads were quality-selected, trimmed, and adapters were removed using fastq-mcf

[0201] , and the genome was assembled using SPAdes 3.9.1

[0202] .

[0286] Whole genome analysis for taxonomic identification and prediction of antibiotic resistance and virulence factors based on whole genome sequence analysis Whole-genome analyses of Chryseobacterium sp., Delftia sp. strain 4465 (available in the ENA (European Nucleotide Archive) database under primary accession number ERS4574863 (version 1) and secondary accession number SAMEA6847265 (tax ID number 80866, scientific name Delftia acidovorans)), and Flavobacterium sp. strain 4466 were performed using the TrueBac ID system (v1.92, DB:20190603) (https: / / www.truebacid.com / )

[0203] . When 16S rDNA gene sequence similarity was greater than 99%, species-level identification was performed based on an algorithm cutoff set at 95% average nucleotide identity (ANI). Virulence factors were identified using the Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / ). Antimicrobial resistance (AMR) genes were discovered using AMRFinderPlus. This is a tool that identifies AMR genes using either protein annotation or nucleotide sequence via the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / pathogens / antimicrobialresistance / AMRFinder / ). (References) TIFF0007808045000021.tif207170TIFF0007808045000022.tif222170TIFF0007 808045000023.tif218170TIFF0007808045000024.tif218170TIFF0007808045000 025.tif218170TIFF0007808045000026.tif218170TIFF0007808045000027.tif218170TIFF0007808045000028.tif218170TIFF0007808045000029.tif119170(Deposit certificate) JPEG0007808045000030.jpg241170JPEG0007808045000031.jpg229170JPEG0007808045000032.jpg224170TIFF0007808045000033.tif132164JPEG0007808045000034.jpg233170JPEG0007808045000035.jpg238170JPEG0007808045000036.jpg238170TIFF0007808045000037.tif131167JPEG0007808045000038.jpg239170JPEG0007808045000039.jpg233170JPEG0007808045000040.jpg233170TIFF0007808045000041.tif132166

Claims

1. An agent for use as a probiotic in fish, comprising a bacterial strain or a combination of bacterial strains, wherein at least one bacterial strain of the bacterial strain or combination thereof is selected from the group consisting of: (i) the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020; (ii) a Chryseobacterium massilia strain having a genome which has an average nucleotide identity (ANI) of at least 95% with the Chryseobacterium massilia strain of (i) and which has a probiotic effect in fish; (iii) a Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020; and (iv) a Flavobacterium species strain having a genome which has an ANI of at least 95% with the Flavobacterium species strain of (iii) and which has a probiotic effect in fish.

2. a. if only one bacterial strain is used, said bacterial strain is administered to the fish in need thereof without an acceptable carrier or delivery vehicle or in a composition; or b. If a combination of different bacterial strains is used, the bacterial strains are administered to fish in need thereof: i. as individual bacterial strains without an acceptable carrier or delivery vehicle; or ii. as a mixture of different bacterial strains present in a single composition, or iii. in different compositions each comprising at least one bacterial strain; or iv. at least one individualized bacterial strain and a collection of different compositions each comprising at least one bacterial strain; is administered, 2. The method of claim 1, wherein when a combination of different bacterial strains is used, the bacterial strains are administered simultaneously or separately in any order, or sequentially in any order to a host in need thereof.

3. The bacterial strains are - respectively, whose genome has an average nucleotide identity (ANI) of at least 96% with SEQ ID NO: 2 or with the Chryseobacterium massilia strain identified under accession number I-5479 deposited at the CNCM on January 24, 2020, and / or whose genome has an average nucleotide identity (ANI) of at least 96% with SEQ ID NO: 1 or with the Flavobacterium species strain identified under accession number I-5481 deposited at the CNCM on January 24, 2020, and / or - comprising a 16s rDNA sequence that has at least 97% sequence identity with the 16s rDNA sequence present in a Chryseobacterium massilia strain, the genome of which comprises SEQ ID NO: 2, or the strain identified under accession number I-5479 deposited at the CNCM on 24 January 2020, or a Flavobacterium species strain, the genome of which comprises SEQ ID NO: 1, or the strain identified under accession number I-5481 deposited at the CNCM on 24 January 2020, respectively; and / or - derived from the group consisting of the Chryseobacterium massilia strain, whose genome comprises SEQ ID NO: 2, or the strain identified under accession number I-5479, deposited at the CNCM on 24 January 2020, and the Flavobacterium species strain, whose genome comprises SEQ ID NO: 1, or the strain identified under accession number I-5481, deposited at the CNCM on 24 January 2020; and / or - a mutant of any one of these strains in which one or more virulence factor-encoding genes and / or antibiotic resistance genes have been deleted or inactivated, 3. The agent according to claim 1 or 2, selected from bacterial strains.

4. At least one bacterial strain a. a host that is a bony fish; or b. A host comprising, consisting essentially of, or consisting of a homogeneous or mixed population of fish The agent according to any one of claims 1 to 3, which is administered to

5. 5. The agent according to claim 1, further comprising at least one other bacterial strain of the native microflora of the fish species to be treated.

6. An agent according to any one of claims 1 to 5, consisting essentially of or consisting of at least one bacterial strain and an acceptable carrier or delivery vehicle.

7. a. Prevent or minimize infection by Flavobacterium columnare in host fish or treated host fish populations, or increase resistance of treated host fish or treated host fish populations to Flavobacterium columnare; and / or b. Prevent or control disease in the treated fish species or treated host fish population; and / or c. Extending the lifespan of or reducing the mortality of treated host fish or treated host fish populations. The agent according to any one of claims 1 to 6 for use in treating atopic dermatitis.

8. 8. The agent according to any one of claims 1 to 7, for use against infection caused by the Flavobacterium columnare pathogen or a disease caused by infection by the Flavobacterium columnare pathogen.

9. 9. The agent according to any one of claims 1 to 8, wherein at least one bacterial strain is administered to a bony fish or a population comprising bony fish.

10. 10. The agent according to any one of claims 1 to 9 for preventing or mitigating fish diseases in rainbow trout in an aquaculture environment or fish management environment upon detection of a pathogen in the aquaculture environment or fish management environment.

11. 11. The agent according to any one of claims 1 to 10, wherein the bacterial strain or combination of bacterial strains is introduced into the fish environment or into the fish parasitic microbial population by administration of encapsulated bacterial strains or combinations thereof.

12. The bacterial strain or combination of bacterial strains is effective in delivering 5 x 10 bacterial strains to a host in need thereof. 4 cfu / mL or more 5×10 6 12. The agent according to any one of claims 1 to 11, administered at a dose of less than or equal to cfu / mL.

13. A probiotic composition or food product comprising at least one bacterial strain selected from the group consisting of: (i) the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020; (ii) a Chryseobacterium massilia strain having a genome with an average nucleotide identity (ANI) of at least 95% with the Chryseobacterium massilia strain of (i) and having a probiotic effect in fish; (iii) a Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020; and (iv) a Flavobacterium species strain having a genome with an ANI of at least 95% with the Flavobacterium species strain of (iii) and having a probiotic effect in fish.

14. 14. A probiotic composition or food product according to claim 13, wherein at least one bacterial strain is a bacterial strain as defined in claim 3.

15. 15. A probiotic composition or food product according to claim 13 or 14 for the use as defined in any one of claims 1 to 12.

16. A bacterial strain selected from the group consisting of: (i) the Chryseobacterium massilia strain identified by accession number I-5479 deposited at the CNCM on January 24, 2020; (ii) a Chryseobacterium massilia strain having a genome with an average nucleotide identity (ANI) of at least 95% with the Chryseobacterium massilia strain of (i) and having a probiotic effect in fish; (iii) a Flavobacterium species strain identified by accession number I-5481 deposited at the CNCM on January 24, 2020; and (iv) a Flavobacterium species strain having a genome with an ANI of at least 95% with the Flavobacterium species strain of (iii) and having a probiotic effect in fish.

17. 16. A kit comprising an agent according to any one of claims 1 to 12, or a probiotic composition or food product according to any one of claims 13 to 15.

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