Bacterial strains used as probiotics, compositions thereof, deposited strains, and methods for identifying probiotic bacterial strains

Abstract

The present invention relates to the use of a bacterial strain or a combination of bacterial strains in the preparation of a probiotic for fish, said bacterial strains being selected from the following: a Chryseobacterium massiliae strain identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020 and a Flavobacterium sp. strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020. The probiotic use includes preventing or minimizing Flavobacterium columnare infection in fish. The present invention also relates to the deposited bacterial strains, or a probiotic composition or kit comprising the same, and a method for identifying a probiotic bacterial strain resistant to pathogen infection.

Description

Technical Field

[0001] The present invention relates to the field of probiotics, and in particular to the use of bacterial strains as active ingredients as probiotics or for designing probiotic compositions suitable for administration to fish. Thus, the present invention relates to the treatment of such fish with such active ingredients to have a prophylactic and / or beneficial effect on their health. The active ingredients of the present invention particularly show benefits against challenges of infection by the bacterium Flavobacterium columnare. The present invention also relates to methods for identifying probiotic bacterial strains. Background Art

[0002] The animal resident microbiota forms complex and long-term associations that perform important community-level functions essential for host development and physiology [1, 2]. The microbiota ecosystem also provides protection against exogenous pathogens through a combination of inhibiting the colonization and growth of pathogens and / or stimulating the host immune system [3-7]. From the perspective of the microbial community composition, changes or reductions in the diversity of the resident microbiota, a phenomenon commonly referred to as dysbiosis, are often associated with increased susceptibility to infection caused by substantial reductions or changes in the abundance of key members of the microbial community [7, 8]. These observations early supported the view that the addition or promotion of individual protective or community-protective bacteria (such as probiotics) could minimize dysbiosis of the microbiota or directly prevent infection to restore host health [9-11].

[0003] Although the efficacy of probiotics has been demonstrated in animals and humans, little is known about their mechanisms of action, and diversity surveys or low-throughput experimental models provide limited information on the contribution of bacterial species to community function [1, 6, 12-14]. In addition, the characterization of bacterial strains that improve colonization resistance remains hampered by the complexity of the host commensal ecosystem. Recently, zebrafish have emerged as a powerful tool for studying microbe-microbe and host-microbe interactions [15-20]. Zebrafish can be easily raised axenically or gnotobiotically in association with specific bacterial species [15, 21]. In addition, the zebrafish bacterial community is increasingly well characterized, and a large number of phylogenetically diverse zebrafish gut bacteria can be cultured, making this model system amenable to direct manipulation of the microbiota and assessment of the impact of probiotics on host anti-infectivity [22-25]. Several studies have used zebrafish to evaluate the impact of exogenously added potential probiotics on host anti-infectivity [23-25]. However, although various lactic acid bacteria (Lactobacilli spp., Bacillus spp.) have been shown to improve infection outcomes against many zebrafish pathogens (such as Aeromonas hydrophila, A. veronii, Streptococcus agalactiae, and Vibrio parahaemolyticus) [26-30], the reported protective effects are often partial, reflecting the difficulty in identifying fully protective exogenous probiotics.

[0004] The inventors used axenic and conventional zebrafish larvae to explore the indigenous commensal microbiota to obtain bacterial species that defend against Flavobacterium columnare, a Bacteroidetes pathogen that affects wild and farmed fish species (including carp, channel catfish, goldfish, eels, salmon, and tilapia) [31, 32]. They identified two anti-infective scenarios that prevent death caused by F. columnare, mediated by the Bacteroidetes bacterium Chryseobacterium massiliae or by a consortium of nine other non-protective bacterial species that form a protective community. Their results constitute a powerful approach for exploring the host microbiota and identifying key members that mediate colonization resistance, providing insights into how to design microbial communities to defend against pathogens in aquaculture and other environments.

[0005] In fact, although wild fish stocks are approaching biologically unsustainable limits, fish aquaculture is a rapidly growing industry that supplies more than half of the fish consumed for human food

[101] . However, intensive aquaculture has promoted pathogen outbreaks, and high mortality rates in aquaculture facilities represent an important bottleneck in fish production [102-104]. These health problems mainly affect immature larvae and juveniles that are immunologically naive, and vaccinating them is impractical [105, 106], which has prompted the prophylactic and therapeutic use of antibiotics and chemical disinfectants to prevent fish diseases [107-109]. However, the widespread use of antibiotics and chemical disinfectants is associated with safety risks for the end consumer, environmental pollution, and the spread of antibiotic resistance, which has raised widespread human health concerns [104, 110]. In this context, the use of probiotics to improve fish health and protect disease-susceptible juveniles appears to be an economically and ecologically sound alternative strategy to antibiotic treatment [111-113].

[0006] Probiotics are live microorganisms that confer health benefits to the host through mechanisms that include growth promotion, immune stimulation, or direct inhibition of pathogenic microorganisms [114-116]. Given the important protective role of the host microbiota against pathogenic microorganisms (a process known as colonization resistance) [117-119], the fish-associated microbiota is considered a source of probiotics of interest [120-122]. In fact, the gastrointestinal tract and fish mucus are the most common sources of potential fish probiotics [123, 124]. However, the selection of probiotics is usually empirical or hampered by the lack of reproducibility and repeatability of in vivo challenges, which are often carried out under relatively poor control conditions and with high inter-individual microbial composition [121, 125].

[0007] To circumvent the experimental difficulties associated with evidence-based identification of fish probiotics, the use of axenic or fully controlled gnotobiotic hosts is a promising strategy [126, 127], and several economically important fish species have been successfully reared under axenic conditions, including Atlantic cod (Gadus morhua L.)

[128] , Atlantic halibut (Hippoglossus hippoglossus)

[129] , European sea bass (Dicentrarchus labrax)

[125] , or turbot (Scophthalmus maximus)

[130] (for relevant reviews, see [131, 132]). Other studies have used axenic or gnotobiotic-reared laboratory zebrafish (Danio rerio) to evaluate the effects of adding exogenous probiotics on host infections [133-136]. However, in most cases, the probiotics tested are exogenous to the host and are short-term microbiota inhabitants that only provide partial protection against the tested pathogens.

[0008] Therefore, the inventors investigated the potential of members of the microbiota of rainbow trout (Oncorhynchus mykiss) to defend against infection by Flavobacterium columnare, a fish pathogen that causes significant losses in aquaculture fish species, especially catfish and salmon

[137] . Using a new protocol for rearing juvenile trout under axenic conditions, they showed that axenic but not conventional juvenile trout were highly susceptible to infection by F. columnare. They then used recolonization of axenic trout to identify bacterial species from the microbiota of trout (Flavobacterium sp.) or zebrafish (Chryseobacterioum massiliae), which fully restored the defense against F. columnare infection. Their findings indicate that their new gnotobiotic trout model enables the discovery of the microbiota of teleost fish to rationally identify fish probiotics that can help prevent columnaris disease in rainbow trout and other fish, either alone or in combination, in the context of aquaculture research and livestock farming.

[0009] Accordingly, the present invention relies on the experiments described herein to propose new means and tools for solving the above problems. In particular, the present invention relates to providing relevant probiotic materials for protecting fish against pathogens and their harmful effects. Summary of the Invention

[0010] Accordingly, the present invention relates to a bacterial strain or a combination of bacterial strains for use as a probiotic in a fish or a fish population, wherein at least one bacterial strain of the bacterial strain or the combination is selected from the following: a Chryseobacterioum massiliae strain; a Chryseobacterioum massiliae strain in which one or more virulence factor-encoding genes and / or antibiotic resistance genes are deleted or inactivated; a Flavobacterium sp. strain, the genome of which has at least 95% average nucleotide identity (ANI) with SEQ ID NO: 1 or the Flavobacterium sp. strain has at least 95% ANI with the Flavobacterium sp. strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020; and a Flavobacterium sp. strain, the genome of which has at least 95% average nucleotide identity (ANI) with SEQ ID NO: 1 or the Flavobacterium sp. strain has at least 95% ANI with the Flavobacterium sp. strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020, in which one or more virulence factor-encoding genes and / or antibiotic resistance genes are deleted or inactivated.

[0011] Address of the CNCM: Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France, Collection Nationale de Cultures de Micro-organismes (CNCM), France.

[0012] SEQ ID NO: 1 is the complete genome sequence of a specific Flavobacterium species strain described herein, which was sequenced according to the experiments described herein and is also available in the ENA (European Nucleotide Archive) database under the primary accession number ERS4574862 (version 1) and the secondary accession number SAMEA6847264 (Tax ID 2730889, scientific name Flavobacterium sp. UGB 4466).

[0013] In this document, the 16S rRNA gene sequence of the specific Flavobacterium species strain shown in SEQ ID NO: 1 is disclosed in SEQ ID NO: 3.

[0014] In this document, the expressions "16S rRNA" and "16S rDNA" are used interchangeably.

[0015] It should be understood that the term "fish" (singular) or "fish" (plural) (the latter generally referring to a school of fish) mentioned herein is intended to be interchangeable throughout this specification, unless otherwise specified by the context or in the general sense.

[0016] The value of average nucleotide identity (ANI) can be readily determined by a person skilled in the art using common general knowledge and available tools, which are described in detail in the literature. ANI between two genomes (especially prokaryotic genomes) is generally known as a taxonomic method for classification that emerged in the genomics era. Prior to this, DNA-DNA hybridization (DDH) had been used as the standard for prokaryotic species delineation at the genomic level for nearly 50 years. Other methods are also available, and some of them are also described in detail in this application (including in the experimental section), such as 16S rRNA gene sequence similarity, recA gene sequence similarity, or rplC gene sequence similarity. In addition to the guidance provided herein, this specification also provides references for implementing the 16S rRNA gene sequence similarity method, which can be used as guidance for a person skilled in the art.

[0017] The methods for performing average nucleotide identity (ANI) and the software tools for calculating ANI values are also readily available to a person skilled in the art: they can be freely accessed via the Internet in particular. Examples can be found in https: / / www.ezbiocloud.net / tools / aniThe literature provides details of the relevant available tools. In particular, the percentage identity can generally be calculated by local (preferably global) sequence alignment algorithms and their available computerized implementations. In a most preferred embodiment, the percentage identity is calculated over the entire length of the comparison sequences, which can be the entire genome of the comparison strain. When a set of query sequences are similar and approximately equal in size, a global alignment that attempts to align every residue in each sequence is most useful. The computerized implementations of the algorithms used are generally associated with the default parameters in the literature, which can be used to run the algorithms. Those skilled in the art can easily adjust them according to their purposes or the sequence alignments performed.

[0018] However, regardless of the algorithm used, an ANI value of 95% is recognized as a suitable cut-off for differentiating two species, i.e., for classifying the genome of a strain sought to be classified among existing species (as annotated in a database) or for defining a new species hitherto unknown.

[0019] As described herein, the identification of the specific strains of the present invention is carried out by whole-genome-based identification and is performed by the TrueBac ID system (v1.92, DB: 20190603) [https: / / www.truebacid.com / ]

[66] . The main parts of the TrueBac ID - Genome system include: (1) a proprietary reference database called the TrueBac database, which is designed to store the latest nomenclature, 16S rRNA gene, and genomic sequences of type / reference strains; and (2) an optimized bioinformatics pipeline that uses average nucleotide identity (ANI) to provide the identification of query genomic sequences. The algorithmic identification scheme using WGS works as follows: First, a pool of phylogenetically most closely related taxa is identified by retrieving three genes extracted from whole-genome assemblies - 16S rRNA, recA, and rplC. Then, for gene-based retrieval, the Mash tool (https: / / github.com / marbl / mash) is used for additional rapid whole-genome-based retrieval. Then, the top-hits of the above four retrievals are pooled, and the ANI is calculated using the MUMmer tool. When possible or when the 16S rDNA gene sequence similarity > 99%, the species-level identification is carried out based on the algorithm cut-off of 95% for ANI (for accurate species identification, thus enabling the identification of new species of Flavobacterium strains).

[0020] Therefore, those skilled in the art can easily determine the ANI value using the MUMmer tool described above and herein.

[0021] According to one specific embodiment, the genome of the Chryseobacterium massiliae strain described herein or used in the present invention, as an alternative or supplement to any other feature described herein, has an average nucleotide identity (ANI) value of at least more than 95%, preferably more than 96%, with SEQ ID NO: 2.

[0022] SEQ ID NO: 2 is the complete genome sequence of a specific Chryseobacterium massiliae strain described herein, which was sequenced according to the experiments described herein and is also available in the ENA (European Nucleotide Archive) database under the primary accession number ERS4385998 (version 1) and the secondary accession number SAMEA6623857 (Tax ID 204089, scientific name Candidatus Chryseobacterium massiliae).

[0023] In one specific embodiment, the strain is a variant of the above strain, in which more than one virulence factor-encoding gene and / or antibiotic resistance gene is deleted or inactivated.

[0024] In the present text, the 16S rRNA gene sequence of the specific Chryseobacterium massiliae strain shown in SEQ ID NO: 2 is disclosed in SEQ ID NO: 4.

[0025] The following table provides the closest hit taxonomic units of the specific strains (Flavobacterium species and Chryseobacterium massiliae strains described herein) described herein, respectively.

[0026]

[0027] It can be seen therefrom that the Flavobacterium species strain of the present invention (also referred to as Flavobacterium species 4466 herein) has an average nucleotide identity (ANI) of 94.65% with the Flavobacterium spartansii ATCC BAA-2541 strain, and its sequence is available in the GenBank database under the accession number MUHG01000041.1 (version 1) - https: / / www.ncbi.nlm.nih.gov / nuccore / MUHG01000041.1. Therefore, the strain whose sequence is disclosed in SEQ ID NO: 1 constitutes a novel Flavobacterium species that has never been described to date.

[0028] Therefore, in a specific embodiment, the present invention relates to or utilizes a Flavobacterium species strain that has an average nucleotide identity (ANI) value of at least more than 95% with the Flavobacterium spartansii ATCC BAA-2541 bacterial strain as a reference sequence. As described herein, such strains can be used as probiotics for fish or fish populations.

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

[0030] In this article, the 16S rRNA gene sequence of the Flavobacterium spartansii ATCC BAA-2541 strain is disclosed in SEQ ID NO: 5.

[0031] Therefore, in a specific embodiment, the present invention relates to or utilizes a Flavobacterium species strain, the genomic 16S rDNA sequence of which has at least 98% sequence identity with SEQ ID NO: 5 as a reference sequence.

[0032] According to a specific embodiment, the present invention relates to or utilizes a Flavobacterium species strain, the genome of which has an average nucleotide identity (ANI) value of at least 95% or more with the Flavobacterium spartansii ATCC BAA-2541 strain as a reference sequence, and the 16S rDNA sequence contained therein has at least 98% sequence identity with SEQ ID NO: 5 as a reference sequence.

[0033] In a specific embodiment, the strain is a variant of any of the above strains, in which one or more virulence factor-encoding genes and / or antibiotic resistance genes are deleted or inactivated.

[0034] In a specific embodiment, the Flavobacterium species strain of the present invention or as described in any embodiment herein retains the characteristics (especially the biological characteristics described herein) of the Flavobacterium species strain identified by accession number No. I-5481 deposited at the National Collection of Cultures and Microorganisms (CNCM) in France on January 24, 2020, and / or retains the characteristics (especially the biological characteristics described herein) of the Flavobacterium species strain shown in SEQ ID NO: 1. Particularly preferred refers to probiotic characteristics.

[0035]

[0036] Thus, it can be seen that the Chryseobacterium massiliense strain of the present invention has an average nucleotide identity (ANI) of 95.85% with the Chryseobacterium massiliense CCUG 51329 bacterial strain, and the sequence of the Chryseobacterium massiliense CCUG 51329 bacterial strain can be obtained in the GenBank database under accession number ASM338553 (version 1) - https: / / www.ncbi.nlm.nih.gov / assembly / GCF_003385535.1 / .

[0037] Accordingly, in a specific embodiment, the present invention relates to or utilizes a Chryseobacterium massiliense strain, and the genome of this Chryseobacterium massiliense strain has an average nucleotide identity (ANI) value of at least 96% or more with the Chryseobacterium massiliense CCUG 51329 bacterial strain used as a reference sequence. As described herein, such strains can be used as probiotics for fish or fish populations.

[0038] The Chryseobacterium massiliense strain described herein also has a 99.86% 16S rRNA gene sequence similarity with the Chryseobacterium massiliense CCUG 51329 bacterial strain.

[0039] The 16S rRNA gene sequence of the Chryseobacterium massiliense CCUG 51329 bacterial strain is publicly available herein as SEQ ID NO: 6.

[0040] According to a specific embodiment, the present invention relates to or utilizes a Chryseobacterium massiliense strain, and the genome of this Chryseobacterium massiliense strain has an average nucleotide identity (ANI) value of at least 96% or more with the Chryseobacterium massiliense CCUG 51329 bacterial strain used as a reference sequence, and the 16S rDNA sequence it contains has a sequence identity of at least 99.9% with SEQ ID NO: 6 used as a reference sequence.

[0041] Alternatively, the present invention relates to or utilizes a Chryseobacterium massiliense strain, and the genome of this Chryseobacterium massiliense strain has an average nucleotide identity (ANI) value of at least 97% or more with the Chryseobacterium massiliense CCUG 51329 bacterial strain used as a reference sequence.

[0042] In a specific embodiment, the strain is a variant of any of the above strains, in which one or more virulence factor encoding genes and / or antibiotic resistance genes are deleted or inactivated.

[0043] In a specific embodiment, the present invention or the Chryseobacterium massiliense strain (including variants) described in any embodiment herein retains the characteristics (especially the biological characteristics described herein) of the Chryseobacterium massiliense strain identified by the accession number No. I-5479 deposited at the National Collection of Cultures and Microorganisms (CNCM) on January 24, 2020, and / or retains the characteristics (especially the biological characteristics described herein) of the Chryseobacterium massiliense strain shown in SEQ ID NO: 2. In particular, it refers to probiotic characteristics.

[0044] According to a specific embodiment, the ANI value of the Flavobacterium species strain used in the present invention and the Flavobacterium species strain identified by accession number No. I-5481 deposited at the French National Collection of Cultures and Microorganisms (CNCM) on January 24, 2020 is as disclosed in any embodiment herein, and / or can reach more than 96%, more than 97%, more than 98%, more than 99%, or 100%.

[0045] Other methods for defining Flavobacterium species other than the ANI value covered by the present invention are described in detail herein. In the context of the present invention, those skilled in the art will understand that all these methods can easily define a suitable subgroup of Flavobacterium species strains. Table 1 below shows examples of relevant data during the process of verifying the taxonomic identity of prokaryotic genomes, although the classification methods are different.

[0046] As defined herein, the strains used in the present invention can be defined by a series of parameters, such as an ANI value of more than 95% and a so-called "16S" value of more than 97%, 98% or 99%. According to all the values and embodiments described herein, these combinations are covered herein by using the wording "and / or" to combine the parameters.

[0047] According to a specific embodiment, the Chryseobacterium massiliense strain used in the present invention is a Chryseobacterium massiliense strain having an average nucleotide identity (ANI) value of at least more than 95% with the Chryseobacterium massiliense strain identified by accession number No. I-5479 deposited at the French National Collection of Cultures and Microorganisms (CNCM) on January 24, 2020, and / or a Chryseobacterium massiliense strain having an average nucleotide identity (ANI) value of at least more than 95%, preferably more than 96%, with SEQ ID NO: 2.

[0048] When it comes to the fact that more than one virulence factor-encoding gene or antibiotic resistance gene is deleted or inactivated in the strain under consideration (which is a feature that can be related to any embodiment described herein), it has been observed that those skilled in the art can easily select more than one deleted or inactivated gene and (if necessary) engineer the resulting strain according to the guidance described herein and his general knowledge of attenuating or inactivating resistance genes in bacterial strains.

[0049] According to one embodiment, the bacterial strains of the present invention according to any embodiment described herein are non-toxic to healthy fish. The bacterial strains can be naturally non-toxic or can be made non-toxic by genetic attenuation and / or chemical attenuation.

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

[0051] According to one embodiment, attenuation of a bacterial strain is achieved by partial or complete deletion of one or more genes, more specifically by partial or complete deletion of one or more genes involved in or responsible for the production of virulence factors in the bacterial strain. For each strain considered separately, such genes can be listed as in Table 7 of this specification (see the Results section).

[0052] More precisely, it has been shown that strains of the isolated Chryseobacterium sp. contain five predicted virulence factors, including some proteins involved in capsule biosynthesis, the HtpB subunit of the heat shock protein, KatA catalase, and the proteolytic subunit of the ClpP protease. According to a specific embodiment, one or more of the genes in the Chryseobacterium sp. of the present invention are partially or completely deleted among those it contains (such as can be determined according to the guidance and tools provided and described in the Results and Methods sections herein), especially among those having the above involvements.

[0053] It has been shown that the Flavobacterium sp. 4466 contains genes encoding capsule, sialic acid synthase, type IV and type VI secretion system effectors, and catalase as potential virulence factors. According to a specific embodiment, one or more of the genes in the Flavobacterium sp. 4466 strain of the present invention are partially or completely deleted among those it contains (such as can be determined according to the guidance and tools provided and described in the Results and Methods sections herein), especially among those having the above involvements.

[0054] For all these embodiments, exemplary representative genes are provided in Table 6, the content of which is referred to herein: any one or more of the genes precisely cited in this table can be deleted or partially deleted in the variant strains of the present invention.

[0055] This also applies to antibiotic resistance genes. According to one embodiment (which can be accumulated with other embodiments described in this application), the bacterial strain is engineered by partial or complete deletion of one or more genes of the bacterial strain, more specifically by partial or complete deletion of one or more genes involved in antibiotic resistance of the bacterial strain. For each strain considered separately, such genes can be listed in Table 6 of this specification (see the Results section).

[0056] Flavobacterium species 4466 has been shown to contain genes encoding resistance to carbapenems, lincosamides, streptogramins, pleuromutilins, and fluoroquinolones. According to one specific embodiment, the Flavobacterium species 4466 strain of the present invention has one or several of those genes (such as can be determined according to the guidance and tools provided and described in the Results and Methods section herein), especially those involved as above, partially or completely deleted.

[0057] In all cases, the partial deletion is to an extent sufficient to inactivate the gene function.

[0058] For the sake of description in this specification, a bacterial strain in which more than one virulence factor-encoding gene or antibiotic resistance gene is deleted or inactivated may also be referred to herein as a "variant". However, the variants of the present invention retain the functional characteristics of their parental strains, especially they retain their probiotic effects.

[0059] Unless otherwise specified by the context or in general meaning, when referring to a bacterial strain in this specification, its variants as defined above will be systematically included in the definition of the said bacterial strain or a combination or association of strains (wherein, according to all their possible combinations, the variants of one strain may be associated with other non-variant strains or with other variant strains).

[0060] According to one specific embodiment (applicable throughout the specification), in a single composition or separate compositions (comprising a mixture of different bacterial strains, or consisting essentially of a mixture of different bacterial strains, or consisting of a mixture of different bacterial strains), the bacterial strain (and its variants) is combined with an acceptable carrier or delivery vehicle and optional adjuvant components. According to one specific embodiment (applicable throughout the specification), the bacterial strains of interest in the present invention are used for the purposes described herein, or for the compositions described herein, provided that they retain their probiotic properties, especially with respect to / against any applications described herein, especially against the pathogen infections and their consequences described herein, especially when considering Flavobacterium columnare, and / or with respect to / against the fish or fish populations to be treated described in any embodiment of this specification.

[0061] According to one specific embodiment, when only one bacterial strain is used, the bacterial strain is administered to a host in need thereof without an acceptable carrier or delivery vehicle (or adjuvant components, when relevant), or in a composition according to any embodiment defined above and described in this specification.

[0062] According to one specific embodiment, when a combination of different bacterial strains is used, the bacterial strains are administered to a host in need thereof in the following manner:

[0063] i. As a single bacterial strain in the absence of an acceptable carrier or delivery vehicle (or adjuvant component, when relevant); or

[0064] ii. As a mixture of different bacterial strains in a single composition; or

[0065] iii. In different compositions, each composition comprising at least one bacterial strain, or consisting essentially of at least one bacterial strain, or consisting of at least one bacterial strain; or

[0066] iv. As a combination of at least one individualized (selected) bacterial strain and different compositions, each composition comprising at least one bacterial strain, or consisting essentially of at least one bacterial strain, or consisting of at least one bacterial strain.

[0067] An "individualized" or "selected" bacterial strain refers to a bacterial strain in the absence of an acceptable carrier or delivery vehicle (or adjuvant component, when relevant).

[0068] Those skilled in the art can readily understand that the bacterial strains of the present invention, having the desired protective properties as described herein, can be in direct contact with fish or a fish population as probiotics. For example, the bacterial strains of the present invention can be directly added to the rearing water of the fish.

[0069] Pharmaceutical products can also be designed. Examples of drugs for delivering probiotics are: capsules, liquids, powder microparticles, tablets... In a specific embodiment, the bacterial strains of the present invention are administered in a liquid formulation. In a specific embodiment, the bacterial strains of the present invention are administered in a powder formulation.

[0070] Alternatively, as is known in the art, the bacterial strains of the present invention can be conveniently provided to fish together with food or incorporated in food to achieve their probiotic effect: the bacterial strains of the present invention can be provided to fish together with fish food (i.e., co-provided: provided simultaneously but not mixed with the fish food before feeding the fish), or incorporated in a fish food product. In a specific embodiment, the bacterial strains of the present invention are encapsulated.

[0071] The carrier or delivery vehicle mentioned herein is adjusted accordingly according to the conventional practice in the art.

[0072] According to a specific embodiment, the probiotic effect is achieved when the bacterial strains of the present invention are delivered to the gastrointestinal tract (GIT) of the fish to be treated. This application demonstrates the colonization of the gastrointestinal tract of the treated fish.

[0073] According to a specific embodiment, the probiotic effect is achieved when the bacterial strain is alive (live bacteria), or at least in a viable state, or capable of being rehydrated and / or revived and / or resuscitated to be alive when in contact with the intended host.

[0074] According to one specific embodiment (which applies throughout the specification), the bacterial strain of the present invention is a live bacterium.

[0075] Nevertheless, according to one specific embodiment, the bacterial strain of the present invention is lyophilized. Lyophilized commercial preparations have the advantages of storage and transportation.

[0076] In one specific embodiment, the bacterial strain of the present invention is encapsulated. Encapsulation of probiotics is also a convenient means to improve the stability of probiotics, facilitate the handling and storage of probiotic cultures, and protect bacteria from harmful conditions (such as oxygen exposure, freezing temperatures, or acidic environments) during production, storage, and gastrointestinal transit (when relevant). It can also be applied to directly administer bacteria to the rearing water of fish. Thus, "encapsulated" means that the bacteria have undergone an encapsulation process, which is defined as encapsulating cells (especially live cells) in a shell material to provide protection against adverse environmental conditions and may (but is not mandatory) allow their controlled release under intestinal conditions. Several methods for encapsulating probiotics are known in the art, such as spray drying, extrusion, emulsion or phase separation, freeze drying, ion gelation, etc. This list is not restrictive. Probiotic encapsulation technology (PET) generally allows the fixation of microorganisms in semi-permeable and / or biocompatible materials. The literature readily provides examples and guidance to those skilled in the art. For example, see: 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 N.V., Journal of Applied Microbiology 2015, 119, 917 - 935, doi.org / 10.1111 / jam.12886. Thus, the carriers or delivery vehicles mentioned herein include those that allow the encapsulation of the bacterial strain of the present invention.

[0077] Conversely, the bacterial strain or combination of bacterial strains of the present invention can be present in a composition, wherein the composition consists essentially of or consists of at least one bacterial strain, an acceptable carrier or delivery vehicle, and optionally an adjuvant component.

[0078] According to all embodiments described herein, such compositions may allow the bacterial strains or combinations of bacterial strains of the present invention to meet formulation requirements as formulations suitable for the intended purpose, together with the selected carrier or delivery vehicle. The formulations may include: liquid or powder formulations, capsules, powder microparticles, tablets, encapsulated bacterial strains as described above, appropriately formulated (e.g., in a liquid).

[0079] It should be understood that a person skilled in the art can easily determine the suitable carrier or delivery vehicle present in such "compositions" or "formulations", and detailed information is provided above and herein.

[0080] According to a specific embodiment (applicable throughout the specification), when using a combination of different bacterial strains, the bacterial strains are administered to a host in need thereof simultaneously or in any order, either individually or sequentially in any order.

[0081] According to a specific embodiment (applicable throughout the specification), the bacterial strains of the present invention are isolated bacterial strains.

[0082] "Probiotic" means that a bacterial strain or combination of bacterial strains has the ability to exert a beneficial effect on the organism to which they are administered, preferably an effect beneficial to the health condition. Probiotics are organisms that, when administered to a host, confer health benefits to the host. The beneficial effect can be achieved through the interaction of the bacterial strain or combination of bacterial strains in the context of the present invention with the microbiota of the host to which they are administered. Thus, the "probiotic" characteristic used as an adjective to define the bacterial strains, active ingredients, compositions or liquid products described herein means that they have the intended function included in the definition of "probiotic".

[0083] In a specific aspect, it has been demonstrated herein the colonization of the bacterial strains of the present invention in the gastrointestinal tract, as well as the beneficial effects of protecting the host and increasing survival after administration of the bacterial strains of the present invention.

[0084] According to a specific embodiment, "probiotic" or "probiotic effect" refers to the ability to colonize the gastrointestinal tract of a host administered with probiotics.

[0085] According to another specific embodiment (which may be a supplement to other features including the above features), "probiotic" or "probiotic effect" refers to the ability to protect a host administered with probiotics from the harmful effects on health conditions caused by infection with other pathogens (such as the pathogen Flavobacterium columnare). In this regard, the harmful effects on health conditions caused by Flavobacterium columnare infection are described in detail herein. Any reversal, even a partial reversal effect, of the harmful effects on the host can be considered to have a probiotic effect or constitute a probiotic effect. Therefore, a person skilled in the art can determine whether a probiotic effect is achieved by observing the beneficial effects on health conditions obtained as compared with non-treatment.

[0086] According to another specific embodiment (which may be a supplement to other features including the above features), "probiotic" or "probiotic effect" refers to the ability to confer increased survival to a host or a group of hosts after administration of the bacterial strain of the present invention. The experimental part herein also provides guidance on how to evaluate whether increased survival is conferred to a host or a group of hosts after administration of the bacterial strain of the present invention. Therefore, a person skilled in the art can determine whether a probiotic effect is achieved according to this criterion by observing the increased survival effect on the host or the group of hosts obtained as compared with non-treatment. The data provided herein show that the present invention confers beneficial effects on the health of the hosts administered with the present invention, improving their survival, even after being attacked by pathogens.

[0087] According to the present invention, the host is a fish, particularly a teleost fish (i.e., a fish of the subclass Actinopterygii), preferably a fish that may be affected or is affected by columnaris disease.

[0088] In a specific embodiment, the fish is selected from one or several species listed in the following table: Those species are usually farmed fish.

[0089] Table 1

[0090]

[0091]

[0092]

[0093] According to one specific embodiment, the fish are selected from: eels (Anguilla sp.), salmon (Oncorhynchus sp. and Salmo sp.), tilapia (Oreochromis sp.), hybrid striped bass (Morone chrysops × M. saxatilis), walleye (Stitzostedion vitreum), channel catfish, sunfish (e.g., largemouth bass (Micropterus salmoides)), baitfish (Pimephales promelas), goldfish (Carassisu auratus), carp (Cyprinus carplo), and ornamental fish (tropical fish species, e.g., black molly (Poecilia sphenops)) and swordtail (Xiphophorus maculatus).

[0094] Such fish are generally considered to be target fish species for the pathogen Flavobacterium columnare.

[0095] According to one specific embodiment, the fish is rainbow trout (a synonym of Oncorhynchus mykiss throughout this specification).

[0096] According to one specific embodiment, the fish being treated is at any stage of its development. For example, it can be an egg, particularly a larva that does not have a fully mature / developed immune system, an adult, including an adult with or without a fully mature / developed immune system. The fish can be immunocompromised.

[0097] According to a preferred embodiment, the fish being treated is in a population of treated fish including larvae, or the animal being treated is a fish larva.

[0098] In one specific embodiment, the host being treated is a fish larva.

[0099] According to one specific embodiment, the fish being treated is rainbow trout and is in a population of treated rainbow trout including larvae, or is a rainbow trout larva.

[0100] Thus, in one specific embodiment, the fish (or some of the fish) is rainbow trout at the larval stage of development.

[0101] According to one specific embodiment, the fish being treated is present in an aquaculture environment or a fish rearing environment.

[0102] As described above, the strains used in the present invention can be defined by a single parameter or a series of parameters applied cumulatively, such as ANI values and / or the so-called "16S" values, and can be further defined by functional definitions.

[0103] According to a specific embodiment, bacterial strains (including the combinations according to the invention) retain the characteristics of their natural counterparts from which they are derived, in particular biological characteristics, and more specifically probiotic characteristics. The characteristics of the natural counterparts can be easily determined by the tests disclosed in the experimental part of the present specification. Whether the bacterial strains of the present invention retain said characteristics can be easily determined by comparison, or by a comparative assay, or by a whole assay following the guidance provided herein.

[0104] According to a specific embodiment, the bacterial strains (including the combinations according to the invention) are selected from the following bacterial strains: the 16S rDNA contained in the bacterial strain has at least 97% sequence identity, particularly at least 98% or at least 99% sequence identity, with the 16S rDNA sequence present in the Chryseobacterium massiliense strain identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020 or the Flavobacterium species strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020.

[0105] As an addition or alternative to the above, the bacterial strains (including the combinations according to the invention) are selected from the following bacterial strains: the 16S rDNA contained in the bacterial strain has at least 97% sequence identity with the 16S rDNA sequence present in the reference sequences of Chryseobacterium massiliense or Flavobacterium species strains. Examples of reference sequences are as described above: respectively, they can be Chryseobacterium massiliense CCUG 51329 and Flavobacterium spartansii ATCC BAA-2541, respectively. The percentage can be as described in any embodiment or paragraph herein.

[0106] As an addition or alternative to the above, the bacterial strains (including the combinations according to the invention) are selected from the following bacterial strains: the 16S rDNA contained in the bacterial strain has at least 97% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 4, respectively. These are sequences corresponding to the 16S rDNA sequences present in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Those skilled in the art can easily determine the boundaries of the 16S rDNA sequences by referring to the annotations in the database entries and common general knowledge.

[0107] According to a specific embodiment, the sequence identity using the so-called 16S rDNA sequence taxonomic classification tool can reach more than 98%, more than 99%, or 100%.

[0108] Those skilled in the art have knowledge of how to perform 16S rDNA sequence taxonomic classification, particularly of determining at least 97% sequence identity, as this technique is well documented in the literature and guidance is provided herein (for example, see

[78] discussed herein and the experimental part).

[0109] The literature provides detailed information on the particularities of the taxonomic classification of 16S rDNA sequences. In particular, the percentage of identity can generally be calculated by local, preferably global, sequence alignment algorithms and their feasible computerized implementations. In a most preferred embodiment, the percentage of identity is calculated over the entire length of the 16S rDNA sequences being compared. Global alignment (which attempts to align every residue in each sequence) is most useful when the sequences in the query set are similar and of roughly equal size. The computerized implementation of the algorithms used is generally associated with the default parameters in the literature, which can be used to run the algorithms. Those skilled in the art can easily adjust them according to their purpose or the sequence comparison being carried out.

[0110] However, regardless of the algorithm used, an identity value of 97% between 16S rDNA sequences is recognized as an appropriate cut-off for distinguishing two species, i.e., for classifying the genome of a strain to be classified among existing species (as annotated in the database) or for defining a new species hitherto unknown.

[0111] According to a specific embodiment, the bacterial strains (including the combinations according to the present invention) are selected from the following bacterial strains: the Chryseobacterium massiliense strain (SEQ ID NO: 2) identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020, and the Flavobacterium sp. strain (SEQ ID NO: 1) identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020.

[0112] Throughout the present application, CNCM represents the National Collection of Cultures and Microorganisms of France (Pasteur Institute, 25 rue du Dr Roux, 15th arrondissement of Paris, France, zip code F-75724).

[0113] According to a specific embodiment, the present invention relates to a Chryseobacterium massiliense bacterial strain, in particular the Chryseobacterium massiliense strain (SEQ ID NO: 2) identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020, for use as a probiotic.

[0114] The Chryseobacterium massiliense bacterial strain with accession number No. I-5479 is also known as the identification reference number UGB 3610 of the CNCM. This strain was isolated from zebrafish. An example of a suitable growth medium is a combination of tryptone yeast extract salts and R2A agar (Reasoner's 2A agar). The recommended incubation conditions are: 28 °C, aerobic, shaking at 250 rpm (revolutions per minute).

[0115] According to a specific embodiment, the present invention relates to a Flavobacterium species bacterial strain, in particular the Flavobacterium species strain (SEQ ID NO: 1) identified by the accession number No. I-5481, deposited at the CNCM on January 24, 2020, for use as a probiotic.

[0116] The Flavobacterium species bacterial strain with the accession number No. I-5481 is also known as the CNCM identification reference number UGB 4466. This strain was isolated from rainbow trout. An example of a suitable growth medium is R2A agar (Reasoner's 2A agar). The recommended incubation conditions are: 20 °C, aerobic, shaking at 250 rpm (revolutions per minute).

[0117] According to a specific embodiment, a bacterial strain or a combination of bacterial strains is used for the use as defined herein, such that at least one bacterial strain is administered to:

[0118] a. a host, which is a fish, in particular an osteichthyan, more particularly a rainbow trout, especially their larvae, or

[0119] b. a host that comprises a conspecific fish population or a mixed fish population, or consists essentially of a conspecific fish population or a mixed fish population, or consists of a conspecific fish population or a mixed fish population, such as a population comprising fish (e.g., rainbow trout) at different stages of their development or growth.

[0120] A "conspecific" fish population refers to a fish population in which the individuals are essentially of the same species or strictly of the same species. According to a specific aspect, such a "conspecific" population may comprise fish at different developmental stages or fish at apparently the same developmental stage.

[0121] Conversely, a "mixed" fish population refers to a fish population in which the individuals belong to different species, i.e., it comprises different fish species. According to a specific aspect, such a "mixed" population may also comprise fish at different developmental stages or fish at apparently the same developmental stage.

[0122] The developmental stages of the fish can be: eggs, larvae, juvenile fish, adults with or without a mature immune system.

[0123] According to a specific embodiment, the developmental stage of the fish to which the active ingredient of the invention is applied is the larval developmental stage (in the case of a school of fish, this means that the school at least partially contains fish larvae or is entirely fish larvae). Basically, the larval stage lasts from hatching to the juvenile stage and is defined as the moment when all fin rays appear on the fish and the scales begin to grow (scaly). The key event that defines the end of the larval stage and the beginning of the juvenile stage is the flexion (becoming flexible) of the notochord associated with the ventral caudal fin of the spinal cord. It is worth noting 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 larval stage and the transformation stage after the yolk sac is absorbed (the latter ends the yolk sac larval stage). Although these regulations are not strict (so that the active ingredients described herein cannot be applied to juveniles or fish at other developmental stages, including adults), it is observed that since fish vaccination is generally known to be ineffective or irrelevant at the larval stage, when fish at the larval stage (i.e., fish at a stage where vaccination is considered to fail) are treated, the present invention provides a significant advancement in the art.

[0124] In one specific aspect (applicable throughout the specification), a combination as defined herein may further comprise at least one other bacterial strain of the indigenous microbiota of the treated host species or of a host species in the treated host population.

[0125] Relevant examples of several bacterial strains from the inherent microbiota of fish can be:

[0126] -Associated with rainbow trout: Delftia acidovorans, Flavobacterium sp., Aeromonas rivipollensis, Pseudomonas helmanticensis, Aeromonas rivipollensis, Pseudomonas baetica, Aeromonas hydrophyla, Flavobacterium plurextorum, Acinetobacter sp., Flavobacterium plurextorum and Pseudomonas sp.

[0127] - Related to zebrafish (zebrafish in this specification): Chryseobacterium massiliense, Aeromonas veronii, Pseudomonas mossellii, Stenotrophomonas maltophilia, Aeromonas caviae, Pseudomonas peli, Pseudomonas sediminis, Phyllobacterium myrsinacearum, Pseudomonas nitroreducens.

[0128] According to another aspect, the bacterial strain or combination of bacterial strains of the present invention is further suitable as a probiotic or for use by themselves (one or more of the following list according to all their possible combinations):

[0129] a. Preventing the occurrence of Flavobacterium columnare pathogen infection in a host fish species or a treated host fish population;

[0130] b. Preventing further infection by Flavobacterium columnare pathogen in a host fish species or a treated host fish population, or minimizing the consequences of persistent infection; and / or

[0131] c. Enhancing the resistance of a treated host fish species or a treated host fish population to Flavobacterium columnare pathogen; and / or

[0132] d. Preventing or minimizing the extent (transmission and consequences) of subsequent Flavobacterium columnare pathogen infection in a host fish species or a treated host fish population, or enhancing the resistance of a treated host fish species or a treated host fish population to Flavobacterium columnare pathogen; and / or

[0133] d. Preventing or controlling diseases in a treated host fish species or a treated host fish population, especially diseases caused by Flavobacterium columnare pathogen infection, particularly preventing or minimizing disease transmission within a treated host fish population; and / or

[0134] e. Prolonging the lifespan of a treated host fish species or a treated host fish population, or reducing the mortality rate of a treated host fish species or a treated host fish population.

[0135] The present invention relates to "pathogens" that persist, occur regularly, or are liable to occur in aquaculture facilities, the pathogen Flavobacterium columnare (belonging to the genus Flavobacterium). For example, information on this pathogen can be found 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).

[0136] Infections of fish with Flavobacterium columnare can occur in the context of seasonal disease outbreaks in aquaculture environments: these infections that occur in the context of seasonal disease outbreaks are also the infections of interest in the context of the present invention.

[0137] According to a specific aspect, the present invention relates to preventing the occurrence of infections of fish with the pathogen Flavobacterium columnare. In this regard, the administration of the active ingredient of the present invention is envisaged in a preventive manner, where the host has not yet been infected by the pathogen.

[0138] As used herein, "infection" refers to the invasion and multiplication of a pathogen within a host. It should be understood that an infection may or may not cause symptoms, being subclinically or clinically apparent respectively. An infection can remain local or become systemic (generalized) by spreading through blood vessels or lymphatic vessels.

[0139] Whether the fish under consideration has been infected with the pathogen Flavobacterium columnare or is at risk of being infected, and whether the fish involved is asymptomatic or symptomatic for Flavobacterium columnare infection, the present invention also relates to enhancing the resistance of the fish under consideration to the pathogen Flavobacterium columnare.

[0140] According to another aspect, the present invention also relates to the prevention and / or control of diseases, particularly diseases caused by infections with the pathogen Flavobacterium columnare (i.e., the initial cause of the disease is the presence of an infection with this pathogen), or diseases known to be caused by or strongly associated with a pre-infection of the host with this pathogen (in terms of causality).

[0141] Thus, according to a specific embodiment, the bacterial strain or combination of bacterial strains of the present invention is used to combat diseases caused (or induced or led) by Flavobacterium columnare infection, which disease is particularly columnaris disease.

[0142] The most well-known disease directly caused by Flavobacterium columnare infection is columnaris disease (also written as Columnariosis Disease in the literature).

[0143] According to a specific embodiment, the present invention aims to mitigate diseases in rainbow trout, particularly Columnaris disease (also known as Columnariosis Disease in the literature). In this case, according to a specific embodiment, the disease is Columnaris disease, such that the present invention aims to mitigate Columnaris disease in fish affected by Flavobacterium columnare. When the fish is rainbow trout, the present invention aims to mitigate Columnaris disease in rainbow trout.

[0144] For "extending the lifespan of a treated host species or a population of treated host species", it can also be expressed as "reducing the mortality rate of a treated host species or a population of treated host species" because it should be understood that the active ingredient of the present invention has a beneficial effect on the host organism, thereby minimizing or mitigating co-disease events caused by pathogen infection at the individual level and / or at the level of the entire host population. A person skilled in the art can easily assess the reduction in mortality rate by observing the treated host species or the population of treated host species. A person skilled in the art can also easily assess the extension of lifespan based on the previous occurrence of events known to be harmful to the health status of the treated fish, or through statistical evaluation of different observations made on untreated fish, or similar information that can be inferred or derived from the common knowledge of a person skilled in the art.

[0145] "Extending lifespan" or "reducing mortality rate" can also be achieved at the level of the entire fish population through the mitigation, alleviation, cure, management, or control of diseases. Diseases have been discussed above.

[0146] According to a specific embodiment, the present invention aims to mitigate diseases in rainbow trout, particularly Columnaris disease, in the context of aquaculture research or animal husbandry.

[0147] According to a specific embodiment, a bacterial strain or a combination of bacterial strains is administered to a teleost fish or a population containing teleost fish, more particularly rainbow trout or a population containing rainbow trout, or is applied in an aquaculture environment or a fish rearing environment. In addition to trout, a list of fish is provided in the table above (which is incorporated into this paragraph). Specific fish have also been indicated above.

[0148] The aquaculture environment can be a natural environment or a tank, in fresh water or sea water, in flowing water or recirculating water rearing management.

[0149] According to a specific embodiment, a bacterial strain or a combination of bacterial strains is used to prevent or mitigate diseases in rainbow trout in an aquaculture environment or a fish rearing environment when a pathogen, particularly the pathogen defined in any embodiment herein, has been detected in the aquaculture environment or the fish rearing environment. In particular, the rainbow trout disease is Columnaris disease.

[0150] According to a specific embodiment, a bacterial strain or a combination of bacterial strains (including their variants) is introduced into the symbiotic microbial community of fish by directly adding the bacterial strain or the combination of bacterial strains in the form of live bacteria to the fish-raising water.

[0151] The possible administration routes and administration modes of the host to be treated can be deduced from different embodiments and / or formulations of the active ingredient to be delivered, which are detailed herein and above in this specification:

[0152] - The active ingredient of the present invention can be delivered in an individualized form or in a mixture of active ingredients, with or without additional components. The active ingredient can be encapsulated; and / or

[0153] - The active ingredient can be administered as a capsule, liquid, powder, powder particle, tablet, etc. (non-limiting citation); and / or

[0154] - The active ingredient can be administered together with the so-called fish food and / or fish oil in any form or type of preparation described herein. Accordingly, the present invention also relates to a kit, which comprises (or consists essentially of or consists of): the active ingredient of the present invention according to any embodiment described herein, at least one fish food product, and an instruction booklet for administration (if appropriate) provided for achieving the effects described herein.

[0155] Therefore, the active ingredient of the present invention can be directly administered to the environment of the host, for example, added to the water in which fish (including larvae) live and grow, or directly administered to the body of the host by any route, especially a route (liquid or food) involving (i.e., allowing) contact between the bacterial strain and the gastrointestinal tract (GIT) of the fish.

[0156] When considering the application, the term "for" is generally used herein to describe the present invention. Using synonyms, the present invention also relates to a method for administering a probiotic active ingredient (including variants) defined in any embodiment described herein to a host in need, especially a fish. Therefore, all instances of "for" can be rephrased as: a method for administering the active ingredient (including variants) described herein to a host according to any embodiment described for the purposes described herein.

[0157] The present invention also relates to a method for:

[0158] a. Preventing the occurrence of further infection by the pathogen Flavobacterium columnare, or minimizing the consequences of a persistent infection by the pathogen Flavobacterium columnare; and / or

[0159] b. Enhancing the resistance to the pathogen Flavobacterium columnare; and / or

[0160] c. Prevent or minimize the extent (transmission and consequences) of subsequent infection by Flavobacterium columnare pathogens, or enhance resistance to Flavobacterium columnare pathogens; and / or

[0161] d. Prevent or control diseases, especially diseases caused by Flavobacterium columnare pathogen infections, particularly prevent or minimize disease transmission within the treated host species population; and / or

[0162] e. Prolong the lifespan of the treated host species or the treated host species population, or reduce the mortality rate of the treated host species or the treated host species population, which species is preferably a fish.

[0163] Administer the active ingredient of the present invention to a host species or a treated host species population (preferably a fish) according to any of the embodiments of administration to a host described herein.

[0164] The present invention also relates to bacterial strains selected from: Chryseobacterium massiliense strain (SEQ ID NO: 2) identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020, and Flavobacterium sp. strain (SEQ ID NO: 1) identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020, or variants thereof, in which more than one virulence factor encoding gene and / or antibiotic resistance gene is deleted or inactivated.

[0165] When considering the complete or long genomic sequences, especially by reference to the SEQ ID NO of the present invention and by reference to the sequences obtained by sequencing the genomes of the deposited strains described herein, it should be understood that the claimed invention should not be affected by errors generated during the sequencing process. Examples of protocols and sequencing methods are provided herein and are well known to those skilled in the art, and they can easily determine sequencing errors based on common general knowledge.

[0166] According to a specific embodiment, the bacterial strain is an isolated bacterial strain.

[0167] According to a specific embodiment, the bacterial strain is an isolated live bacterium.

[0168] According to a specific embodiment, the bacterial strain is an isolated and lyophilized bacterial strain, but they retain the characteristics of being rehydrated and / or reactivated and / or resuscitated when appropriate.

[0169] According to another aspect, the present invention further includes:

[0170] (i) A Chryseobacterium massiliense strain that has an average nucleotide identity (ANI) value of at least 95% or more with the Chryseobacterium massiliense strain identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020 (also disclosed as SEQ ID NO: 2), but is different from the latter in that it has at least one (i.e., one, two, three or more) nucleotide deletion or substitution, especially nucleotide deletion or substitution achieved by non-natural sequence engineering, compared to the naturally deposited strain, such that the resulting strain has no natural counterpart, especially no genetically identical counterpart, but retains the characteristics related to the No. I-5479 strain detailed herein;

[0171] (ii) A Flavobacterium sp. strain that has an average nucleotide identity (ANI) value of at least 95% or more with the Flavobacterium sp. strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020 (also disclosed as SEQ ID NO: 1), but is different from the latter in that it has at least one (i.e., one, two, three or more) nucleotide deletion or substitution, especially nucleotide deletion or substitution achieved by non-natural sequence engineering, compared to the naturally deposited strain, such that the resulting strain has no natural counterpart, especially no genetically identical counterpart, but retains the characteristics related to the No. I-5481 strain detailed herein;

[0172] (iii) Alternatively, a variant of the Chryseobacterium massiliense strain that has an average nucleotide identity (ANI) value of at least 95% or more with the Chryseobacterium massiliense strain identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020 (also disclosed as SEQ ID NO: 2); or a variant of the Flavobacterium sp. strain that has an average nucleotide identity (ANI) value of at least 95% or more with the Flavobacterium sp. strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020 (also disclosed as SEQ ID NO: 1); or a variant of any one of the strains in (i) or (ii) above, according to all the described embodiments, in which more than one virulence factor encoding gene and / or antibiotic resistance gene is deleted or inactivated.

[0173] It should be understood that the present invention also includes modified and engineered strains that are different from any naturally occurring strains, although they are structurally similar to the naturally identified strains and retain the characteristics related to the deposited strains described in detail herein by reference to the CNCM accession numbers or by reference to the whole genome sequences. The variants described herein fall within this embodiment.

[0174] Furthermore, the present invention also relates to fish food products (solid or liquid) that comprise the bacterial strains of the present invention (as described in any embodiment herein, including variants) or mixtures thereof.

[0175] The present invention also relates to encapsulated bacterial strains of the present invention (as described in any embodiment herein, including variants) or mixtures thereof.

[0176] The present invention also relates to the use of the bacterial strains of the present invention (as described in any embodiment herein, including variants) or mixtures thereof, or encapsulated bacterial strains of the present invention (as described in any embodiment herein, including variants) or mixtures thereof, or the compositions of the present invention as described herein, as additives.

[0177] According to the conventional practice in the art, the carriers or delivery vehicles as described herein are adjusted accordingly. When administered, the bacterial strains can be encapsulated.

[0178] As is conventional in the art, the dosage can be readily determined by a person skilled in the art.

[0179] According to a specific embodiment, the bacterial strain or combination of bacterial strains (including variants) is administered to a subject in need thereof in the form of a dosage or formulation such that the final liquid dosage is 5×10 4 cfu / mL to 5×10 6 or up to 5×10 8 cfu / mL of the active ingredient, particularly a dosage of 5×10 5 cfu / mL. The present invention extends to dry / solid dosages that render the liquid dosage equivalent to the above dosages. The dosage for the dry form can be expressed in grams instead of milliliters. This range and specific values are readily capable of conferring comprehensive protection to the host (especially a fish host) against subsequent Flavobacterium columnare pathogen infections. In this case, the level of protection achieved can be demonstrated by in vivo infection challenges using gnotobiotic zebrafish or rainbow trout as animal models, examples of which are described herein or in the literature. The examples described herein show that Flavobacterium columnare killed axenic (GF) fish larvae within 48 hours, while fish exposed to the Chryseobacterium massiliense or Flavobacterium species strains of the present invention were more resistant to infection, with up to a 90% increase in survival, by reference to experiments in which the protective bacterial strains of the present invention were not administered to the models. Thus, it can be defined that "comprehensive" protection can be evaluated by in vivo infection challenge tests using gnotobiotic zebrafish or rainbow trout as animal models, in which up to a 90% increase in survival of the models can be observed after administering the strains contemplated by the present invention to the models. Guidance related to conducting this test can be found in the experimental section.

[0180] It should be noted that such a dose capable of achieving "comprehensive" protection requires a concentration of the active ingredient, which is a hallmark of the technical engineering of the product being administered.

[0181] The present invention also relates to a probiotic composition comprising at least one bacterial strain and other acceptable carriers or delivery vehicles and optional adjuvant components (or consisting essentially of or consisting of at least one bacterial strain and other acceptable carriers or delivery vehicles and optional adjuvant components), said at least one bacterial strain being selected from the group consisting of: Chryseobacterium massiliense strains and Flavobacterium species strains having an average nucleotide identity (ANI) value of at least 95% or more with the Flavobacterium species strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020, according to any combination thereof, in particular at least two or three different bacterial strains from said group.

[0182] The bacterial strains present in the compositions of the present invention are the same as those described in this specification (especially above), in the context of their intended administration, in particular including all the described variants and embodiments.

[0183] Specifically, the probiotic compositions of the present invention may include variants of naturally occurring strains, as defined herein especially with respect to modified strains, which are engineered to be different from any naturally occurring strain and retain the properties associated with the deposited strains described herein by reference to the CNCM accession numbers and described in detail herein.

[0184] The probiotic compositions of the present invention further include compositions comprising any of the strains described herein or mixtures thereof, or consisting essentially of or consisting of any of the strains described herein or mixtures thereof, in a dose capable of achieving "comprehensive" protection of the host as defined herein.

[0185] Acceptable carriers or delivery vehicles refer to any substance, reagent, ingredient that can be safely administered to a host for the purpose of delivering an active ingredient to said host (such that their functions, i.e., their probiotic functions, can be achieved). They can be water or other liquids, or other acceptable substances, reagents, ingredients. They can be pharmaceutical or veterinary carriers or delivery vehicles. Details regarding acceptable carriers or delivery vehicles can be found in the foregoing in this specification, which are also referred to in the context of the compositions described herein. When food is used as a carrier for the probiotics or compositions of the present invention, it can enable the probiotics to reach the intestine of the fish.

[0186] Also described is a probiotic composition comprising a strain of Flavobacterium species of the invention (characterized by reference to the deposited strain - see the present specification), which further comprises the following bacteria: a strain of Delftia acidovorans identified by accession number No. I-5480 deposited at the CNCM on January 24, 2020 ((the sequence is available in the ENA (European Nucleotide Archive) database under primary accession number ERS4574863 (version 1) and secondary accession number), Aeromonas rivipollensis, Pseudomonas salamanticensis, Aeromonas rivipollensis, Pseudomonas betica, Aeromonas hydrophila, Flavobacterium ploutonicum, Acinetobacter species, Flavobacterium ploutonicum, Pseudomonas species.

[0187] In this case, the bacteria can be isolated from the microbiota of trout, especially rainbow trout.

[0188] According to another aspect described herein, the composition comprises a strain of Chryseobacterium massiliense and further comprises the following bacteria: at least one strain of Aeromonas veronii (especially two different strains of Aeromonas veronii), Pseudomonas mosselii, Stenotrophomonas maltophilia, Aeromonas caviae, Pseudomonas perryensis, Pseudomonas cedrina, Phyllobacterium myrsinacearum, Pseudomonas nitroreducens.

[0189] In this case, the bacteria can be isolated from the microbiota of zebrafish.

[0190] According to a specific embodiment, at least one bacterial strain of the probiotic composition of the invention is selected from the following: a strain of Chryseobacterium massiliense deposited at the CNCM on January 24, 2020 and identified by accession number No. I-5479, a strain of Flavobacterium species deposited at the CNCM on January 24, 2020 and identified by accession number No. I-5481, and variants thereof, in which more than one virulence factor-encoding gene and / or antibiotic resistance gene is deleted or inactivated.

[0191] According to a specific embodiment, at least one bacterial strain of the probiotic composition of the invention is selected from the following: a strain of Chryseobacterium massiliense that comprises the genome of SEQ ID NO: 2, or consists essentially of the genome of SEQ ID NO: 2, or consists of the genome of SEQ ID NO: 2; and a strain of Flavobacterium species that comprises the genome of SEQ ID NO: 1, or consists essentially of the genome of SEQ ID NO: 1, or consists of the genome of SEQ ID NO: 1; and variants thereof, in which more than one virulence factor-encoding gene and / or antibiotic resistance gene is deleted or inactivated.

[0192] The probiotic compositions of the present invention described in any of the embodiments described herein (in particular in the preceding paragraphs relating to probiotic compositions) can be used for the purposes detailed in any of the embodiments described in this specification.

[0193] According to any of the embodiments disclosed herein, the present invention also relates to a method for manufacturing a composition, preparation or kit containing the active ingredients described herein.

[0194] According to any of the embodiments disclosed herein, the present invention also relates to a method of using any of the active ingredients described herein for producing or manufacturing a composition, preparation, food (liquid or dry) or kit suitable for administration to fish in need. According to any of the embodiments described herein, the composition, preparation or kit can be used for probiotic or prophylactic purposes, or to combat diseases of the host.

[0195] According to any of the embodiments disclosed herein, the present invention also relates to the use of any of the active ingredients described herein (including variants) for producing or manufacturing a composition, preparation, food (liquid or dry) or kit suitable for administration to fish in need.

[0196] According to any of the embodiments disclosed herein, the present invention also relates to the use of any of the active ingredients described herein (including variants) for administration to a host in need (in particular fish). According to any of the embodiments described herein, the administration can be for probiotic or prophylactic purposes, or to combat diseases of the host.

[0197] The present invention also relates to a method for identifying a bacterial strain that is a probiotic resistant to pathogen infection, the method comprising the following steps:

[0198] a) Providing a gnotobiotic trout model that is susceptible to infection by at least one defined pathogen and has an adverse effect on its health, the defined pathogen not infecting conventional trout and having an adverse effect on its health;

[0199] b) Determining the microbiota, in particular the culturable microbiota, of the conventional trout of step a), optionally identifying the most representative bacterial strains of the microbiota, further optionally after isolating the most representative bacterial strains of the microbiota (such that identification is possible);

[0200] c) After inoculating the gnotobiotic trout model with the microbiota determined in b) or the most representative bacterial strains of the microbiota identified in b) (when identification is carried out) and re-conventionalizing the gnotobiotic trout model, exposing the gnotobiotic trout model to at least one defined pathogen of step a);

[0201] d) Draw conclusions about the probiotic effect of the microbiota or its most representative bacterial strains inoculated into a re-conventionalized axenic trout model.

[0202] Regarding step a), the axenic trout model can include trout larvae. According to a specific embodiment, the axenic trout model is rainbow trout. This specification details how the inventors first obtained this new model.

[0203] According to a more specific embodiment, step a) alternatively includes identifying a pathogen that infects (especially kills) the axenic trout model but does not infect (especially kill) conventional trout.

[0204] The fact that the provided pathogen kills the axenic trout model used may increase the stringency of the method for identifying bacterial strains described above. In fact, by such a criterion, the method provides more relevant hits (i.e., can be called a "high-throughput" method). However, those skilled in the art should understand that observing symptoms less severe than fish death does not prevent the successful implementation of the method (i.e., by observing the infection symptoms of the fish in step c) and their possible alleviation), although with lower efficiency (low throughput, but still able to identify hits).

[0205] Specific examples detailing how steps a) to d) can be achieved are fully exemplified in the experimental section of this specification. It should be understood that depending on the experimental situation, the method for identifying bacterial strains described herein can be applied to a large number of different pathogens (in step a)) and a large number of different microbiota (possibly partly depending on the environment). Those skilled in the art can easily determine the experimental situation, and guidance is provided in the experimental section below.

[0206] "Re-conventionalization" refers to inoculating a relevant microbiota into a host lacking said microbiota. Extensive examples and the guidance that can be derived therefrom are provided herein.

[0207] The conclusions in step d) can be drawn by observing the biological effects obtained in the re-conventionalized trout model after the challenge in step c). Extensive examples and the guidance that can be derived therefrom are provided herein.

[0208] As used herein, the term "comprising", which is synonymous with "including" or "containing", is open-ended and does not exclude additional, unenumerated elements, steps or components, while the term "consisting of" is a closed term and does not include any additional unenumerated elements, steps or components.

[0209] The term "consisting essentially of" is a partially open term that does not exclude additional, unenumerated elements, steps or components, provided that these additional elements, steps or components do not materially affect the basic and novel characteristics of the present application.

[0210] Thus, the term "comprising" (or "including") includes the terms "consisting of" and "consisting essentially of". Accordingly, in this application, the term "comprising" (or "including") is intended to more specifically encompass the terms "consisting of" and "consisting essentially of".

[0211] To assist the reader of this application, the specification is divided into different paragraphs or sections. These divisions should not be regarded as separating the content of one paragraph or section from that of another. On the contrary, this specification encompasses all combinations of the various parts, paragraphs, and sentences that can be contemplated.

[0212] The relevant disclosures of all references cited herein are specifically incorporated by reference.

[0213] When reading the examples and the drawings (which illustrate the experiments conducted by the inventors), the above and other features of the invention will be apparent, supplementing the features and definitions given in this specification. The following examples are provided by way of illustration. However, these examples are not restrictive of the invention described. BRIEF DESCRIPTION OF THE DRAWINGS

[0214] Figure 1 : Flavobacterium columnare kills germ-free zebrafish but not conventional zebrafish. Germ-free (GF) and conventional (Conv) zebrafish larvae at 6 dpf were exposed to different doses of F. columnare by immersion ALG , and transferred to sterile water after 3 h. Mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. Statistics correspond to unpaired, non-parametric Mann-Whitney tests comparing all conditions to non-infected GF (left) or Conv (right). ****: p < 0.0001; no *: not significant. Blue (gray in black and white version) mean bars correspond to non-exposed larvae, and red (light gray in black and white version) mean bars correspond to larvae exposed to F. columnare.

[0215] Figure 2: Screening of F. columnare strains that kill germ-free zebrafish. A: Survival of germ-free zebrafish larvae exposed to a collection of 28 F. columnare strains. B: Survival of germ-free and conventional zebrafish larvae exposed to the 7 most virulent F. columnare strains. Zebrafish larvae at 6 dpf were exposed at 5×10 2were infected by soaking in cfu / mL for 3 hours. The mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. Statistical data correspond to unpaired, non-parametric Mann-Whitney tests comparing all conditions to uninfected GF. ****: p < 0.0001; ***: p < 0.001; *: p < 0.005; *: p < 0.05; no *: not significant. Blue / grey mean bars correspond to uninfected larvae, and red (light grey in black / white version) mean bars correspond to infected larvae.

[0216] Figure 3 : Conventional larvae from 4 different zebrafish facilities were also protected against Flavobacterium columnare infection. AB-strain zebrafish eggs were collected from 4 different zebrafish facilities: Facility 1 - the academic facility of the Hospital Robert Debré (Paris, France); Facilities 2 and 3 - two academic research facilities of the University of Paris VI (Paris, France); Facility 4 - the commercial Amagen facility in Gif-sur-Yvette (France) located on the Yvette River. Axenic zebrafish larvae or conventional zebrafish larvae at 6 dpf from each facility were exposed to Flavobacterium columnare by immersion ALG and transferred to sterile water 3 hours later. The mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Blue (grey in black / white version) mean bars correspond to uninfected larvae, and red (light grey in black / white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. The statistical data shown correspond to unpaired, non-parametric Mann-Whitney tests. ****: p < 0.0001; no *: not significant.

[0217] Figure 4 : Re-conventionalization of axenic zebrafish larvae conferred protection against Flavobacterium columnare infection. At 0 dpf (sterilization day) or 4 dpf (hatching day), axenic zebrafish larvae and conventional zebrafish larvae in contact with the water in the fish tank facility tank or mashed non-sterile eggs were resistant to Flavobacterium columnare ALG The inoculation dose of Flavobacterium columnare ALG = 5 × 10 5cfu / mL. The mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. Statistical data correspond to unpaired, non-parametric Mann-Whitney tests comparing all conditions to uninfected GF. ****: p < 0.0001; no *: not significant. Blue (gray in black and white version) mean bars correspond to uninfected larvae, and red (light gray in black and white version) mean bars correspond to infected larvae.

[0218] Figure 5 : A mixture of 10 culturable bacteria that constitute the core microbiota of zebrafish larvae conferred protection against infection by Flavobacterium columnare. The 10 culturable strains identified as the core conventional microbiota were added to larvae at 4 dpf as a mixture at an equivalent concentration of 5×10 5 cfu / mL, and then infection challenge was performed at 6 dpf. The mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. The statistical data shown correspond to unpaired, non-parametric Mann-Whitney tests. ****: p < 0.0001; no *: not significant. Blue (gray in black and white version) mean bars correspond to uninfected larvae, and red (light gray in black and white version) mean bars correspond to infected larvae.

[0219] Figure 6 : Kinetics of bacterial community establishment in re-normalized zebrafish larvae. Relative abundances of the 10 species that constitute the identified core protective zebrafish microbiota Mix10 at different time points, using 16S rDNA gene amplicon sequencing of 10 larval pools. The bars on the left represent the relative abundances of microbiota species in conventional zebrafish larvae at hatching day (4 dpf). For the re-normalized Mix10 population, axenic larvae fed Tetrahymena were incubated at 4 dpf with an equimolar combination of 10 species that make up the core of the microbiota (5×10 5 cfu / mL each).

[0220] Figure 7 : Effect of antibiotic treatment on zebrafish survival. Axenic zebrafish larvae and conventional zebrafish larvae were exposed to antibiotic treatment for 16 hours at 4 dpf. Then the antibiotics were washed off, and Flavobacterium columnare ALGInfected zebrafish larvae. Non-toxic antibiotic treatments that resulted in microbiome dysbiosis were determined using different concentrations of penicillin / streptomycin or kanamycin. Penicillin / streptomycin dose 1 = 250 μg / mL, dose 2 = 15.6 μg / mL; Kanamycin dose 1 = 200 μg / mL, dose 2 = 50 μg / mL, dose 3 = 25 μg / mL. Mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Blue (gray in black and white version) mean bars correspond to uninfected larvae, and red (light gray in black and white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. The statistics shown correspond to unpaired, non-parametric Mann-Whitney tests. ****: p < 0.0001; ***: p < 0.001; **: p < 0.005; *: p < 0.05; no *: not significant.

[0221] Figure 8: Analysis of protection against Flavobacterium columnare infection after antibiotic dysbiosis. A: Response of zebrafish larvae to F. columnare infection after antibiotic-induced dysbiosis, the figure shows the time and treatment of the experiment. ALG Response to infection, the figure shows the time and treatment. Mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Blue (gray in black and white version) mean bars correspond to uninfected larvae, and red (light gray in black and white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. The statistics shown correspond to unpaired, non-parametric Mann-Whitney tests. ****: p < 0.0001; no *: not significant. C: Profile of community recovery in streptomycin / penicillin-treated. D: Profile of community recovery in kanamycin-treated. Pooled samples of 10 larvae were collected for 16S rDNA sequencing.

[0222] Figure 9 : Protection of zebrafish against F. columnare (recolonized with individual or mixed bacterial strains isolated from zebrafish larvae). A: Determination of the level of protection provided by each of the 10 bacterial species that make up the core protective zebrafish microbiome. Bacteria were added individually to the water on the day of hatching (dose 5×10 5cfu / mL). B: Protection levels provided by different amounts of Chryseobacterium massiliense and Mix9. Mix9 provided protection only at the highest inoculation dose. Mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Blue (gray in black-and-white version) mean bars correspond to uninfected larvae, and red (light gray in black-and-white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. The statistics shown correspond to unpaired, non-parametric Mann-Whitney tests. ****: p < 0.0001; no *: not significant.

[0223] Figure 10: Combinations of 8 species from protective Mix9 do not defend against Flavobacterium columnare infection. A: Survival of zebrafish larvae at 6 dpf after reconstitution with different possible mixtures, including the 8 species in the Mix9 consortium, under Flavobacterium columnare ALG infection. Mix10 = reconstitution Mix10 , Mix9 = reconstitution Mix9 , Mix8 = reconstitution Mix8 . B: Table shows the different combinations used for reconstitution at 4 dpf. C: Survival of zebrafish larvae at 6 dpf was tested after reconstitution with 8 different combinations of non-protective Mix8a under Flavobacterium columnare ALG infection, where the amount of 1 species out of 8 species was doubled in each combination (shown in yellow in D). D: Table shows the different combinations used for reconstitution at 4 dpf. Mix10 = reconstitution Mix10 , Mix9 = reconstitution Mix9 , Mix8 = reconstitution Mix8 . A and C: Mean survival is represented by thick horizontal bars with standard deviation. For each condition, n = 12 zebrafish larvae. Blue (gray in black-and-white version) mean bars correspond to uninfected larvae, and red (light gray in black-and-white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. The statistics shown correspond to unpaired, non-parametric Mann-Whitney tests. ****: p < 0.0001; no *: not significant.

[0224] Figure 11: Zebrafish immune response to infection with F. columnaris. AC: qRT-PCR analysis of host gene expression in larvae reconventionalized with the indicated bacteria or bacterial mixtures, 18 h after exposure to F. columnaris; each dot corresponds to an individual larva. Expression of il10 (A), il1b (B), and il22 (C) in wild-type AB zebrafish; D: Expression of myd88- / - zebrafish and background-matched myd88+ / + zebrafish after reconventionalization and exposure to F. columnaris. ALG Comparison of survival after infection. Survival means are represented by thick horizontal bars with standard deviations. For each condition, n=12 zebrafish larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 after infection. AD: blue (grey in the black and white version) mean bars correspond to uninfected larvae, and red (light grey in the black and white version) mean bars correspond to infected zebrafish. The statistics shown correspond to the unpaired, non-parametric Mann-Whitney test. ****: p<0.0001; ***: p<0.001; **: p<0.005; *: p<0.05; No *: not significant.

[0225] Figure 12 : In WT zebrafish and myd88 reconstituted with various bacteria or bacterial mixtures as shown - / - il1b expression in zebrafish mutants. ALG qRT-PCR analysis of il1b gene expression was performed 18 hours later. Each point corresponds to a single larva. The blue (grey in the black and white version) mean bars correspond to uninfected larvae, and the red (light grey in the black and white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 after infection. The statistics shown correspond to the unpaired, non-parametric Mann-Whitney test. ****: p<0.0001; ***: p<0.001; *: p<0.005; *: p<0.05; No *: not significant. Survival means are represented by thick horizontal bars. The blue (grey in the black and white version) mean bars correspond to uninfected larvae, and the red (light grey in the black and white version) mean bars correspond to infected zebrafish.

[0226] Figure 13 : The intestine of infected germ-free zebrafish shows severe disarray. Germ-free, conventionalized and reconventionalized zebrafish larvae. Reconventionalized zebrafish were inoculated with Mix9 or Chryseobacterium martensii at 4 dpf. A: Representative images of the intestine of uninfected larvae. Fish were fixed at 7 dpf for histological analysis or electron microscopy. Left: Giemsa staining, L = intestinal lumen, right: transmission electron microscopy. B: Exposure to F. columnaris at 7 dpf ALGRepresentative images of infected larval intestines. Epoxy-embedded zebrafish larvae at 7 dpf stained with toluidine blue and imaged by light microscopy (left) or transmission electron microscopy (right).

[0227] Figure 14 :Infection of larvae by Flavobacterium columnaris requires feeding. Feeding sterile zebrafish larvae with sterile thermophilic Tetrahymena thermophila (sterile + Tetrahymena) or sterile fish meal (sterile + meal) or with Flavobacterium columnaris ALG No feeding was done before infection. However, the fed larvae were sensitive to Flavobacterium columnaris ALG Infection-susceptible, unfed, axenic larvae did not die after infection with fish pathogens. Survival means are represented by thick horizontal bars with standard deviations. For each condition, n=12 zebrafish larvae. Blue (grey in the black and white version) mean bars correspond to uninfected larvae, and red (light grey in the black and white version) mean bars correspond to infected larvae. Larval mortality was monitored daily, and surviving fish were euthanized on day 10 after infection. The statistics shown correspond to the unpaired, non-parametric Mann-Whitney test. ****: p<0.0001; ***: p<0.001; *: p<0.005; *: p<0.05; No *: not significant.

[0228] Figure 15 :Preexposure to Chryseobacterium martensii protects larval and adult zebrafish from infection with Flavobacterium columnaris. A:Zebrafish larvae were inoculated with 5×10 5 cfu / mL of Chryseobacterium martensii for 48 hours, and then infected with virulent Flavobacterium columnaris strains at 6 dpf. B: Pre-exposure to or without pre-exposure to Chryseobacterium martensii (2×10 6 cfu / mL, 48 h), followed by exposure to Flavobacterium columnaris ALG (5×10 6 cfu / mL, 1 hour). Survival means are represented by thick horizontal bars with standard deviations. For each condition, n=12 zebrafish larvae or adults. Zebrafish mortality was monitored daily, and surviving fish were euthanized on day 10 post-infection. Blue (grey in the black and white version) mean bars correspond to uninfected larvae, and red (light grey in the black and white version) mean bars correspond to infected zebrafish. The statistics shown correspond to the unpaired, non-parametric Mann-Whitney test. ****: p<0.0001; **: p<0.005*; No *: not significant.

[0229] Figure 16 : Protocol used to rear and infect or reconventionalize germ-free trout larvae in this study

[0230] Figure 17: Protocol used in this study for rearing and infecting or re - conventionalizing axenic rainbow trout larvae. After fertilization, the eggs were sterilized (-5 dph) and kept in sterile, autoclaved mineral water in Petri dishes at 16 °C until hatching. After hatching, the rainbow trout larvae were transferred to vented - cap cell culture flasks where they were maintained throughout all experiments. The larvae were fed sterile food powder every 2 days after 21 dph until the end of the experiment; the water was changed 30 minutes after feeding the animals. To test the protective effect of potential probiotic strains, the larvae were re - conventionalized with one or several symbiotic bacteria diluted in water at 22 dph. The pathogenic bacteria were added to the water for 24 hours at 24 dph, and then the larvae were washed with fresh sterile water.

[0231] Figure 18 : Growth performance of axenic and conventional rainbow trout larvae. Body size (A) and body weight (B) of conventional and axenic fish measured at 35 dph (n = 6).

[0232] Figure 19 : Anatomical comparison of conventional and axenic rainbow trout larvae. Whole - mount specimens were analyzed by optical projection tomography after clearing the fish, based on the solvent - cleared organ immunolabeling 3D imaging procedure (iDISCO+). Lateral views of conventional (A, B, C, and D) and axenic (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 head kidney (E and J). Images represent two different fish for each condition.

[0233] Figure 20 : Anatomical comparison of the intestine of conventional and axenic rainbow trout larvae. Whole - mount specimens were analyzed by optical projection tomography after clearing the fish, based on the solvent - cleared organ immunolabeling 3D imaging procedure (iDISCO+). Lateral views of conventional (A and B) and axenic (C and D) rainbow trout larvae imaged at 21 dph. Mid - intestine (A and C) and hind - intestine (B and D). Images represent two different fish for each condition.

[0234] Figure 21: Survival of axenic rainbow trout larvae and conventional rainbow trout larvae infected with different pathogens. A: Survival of axenic larvae exposed to Flavobacterium psychrophilum strain THCO2-90, Flavobacterium columnare strain Fc7, Lactococcus garvieae, Vibrio anguillarum, and Yersinia ruckeri strain JIP 27 / 88 by bath exposure. B: Flavobacterium columnare strain Fc7 kills axenic rainbow trout but not conventional rainbow trout. Mean and SD plots represent the mean post-infection days to death of infected fish. For each condition, n = 12 larvae. Surviving fish were euthanized on day 10 post-infection. Asterisks indicate significant differences from the uninfected population (****p < 0.0001).

[0235] Figure 22 : Survival of recolonized trout larvae upon infection with Flavobacterium columnare Fc7. A: Axenic trout larvae exposed to water used for rearing conventional fish showed similar survival upon Flavobacterium columnare infection compared to conventional trout larvae at 21 dph. B: Eleven species identified from the microbiota of conventional fish were added to rainbow trout larvae at 22 dph, followed by Flavobacterium columnare infection at 24 dph. This bacterial mixture was able to protect the recolonized larvae from infection. For each condition, n = 12 larvae. All surviving fish were euthanized on day 10 post-infection. (****p < 0.0001).

[0236] Figure 23: Protection against Flavobacterium columnare infection provided by individual species isolated from the microbiota of conventional rainbow trout to axenic trout larvae. A: Eleven species (Table 2) isolated from the microbiota of conventional fish were added individually to rainbow trout larvae at 22 dph, followed by Flavobacterium columnare Fc7 infection at 24 dph. Among the 11 different strains, only Flavobacterium sp. strain 4466 protected the recolonized larvae from infection. B: Mix11, a mixture of all identified strains except Flavobacterium strain 4466, and Mix10 were added to rainbow trout larvae at 22 dph, followed by Flavobacterium columnare infection at 24 dph. Mix11 protected the recolonized larvae from infection, while Mix10 did not. For each condition, n = 10 larvae. All surviving fish were euthanized on day 10 post-infection. C: CFU / mL recovered from the dissected intestines of axenic fish exposed to Flavobacterium columnare Fc7, Flavobacterium sp. strain 4466, or both (Flavobacterium columnare Fc7 and Flavobacterium sp. strain 4466) 24 hours post-infection. (****p < 0.0001).

[0237] Figure 24: Chryseobacterium massiliae provides complete protection against Flavobacterium columnare infection in trout. Survival of axenic trout larvae exposed to Chryseobacterium massiliae 48 h before infection with Flavobacterium columnare strains Fc7, IA-S-4, Ms-Fc-4, and ALG-00-530. Flavobacterium columnare kills axenic trout but not trout previously recolonized with Chryseobacterium massiliae. Mean and SD plots represent mean post-infection days to death of infected fish. For each condition, n = 12 larvae. All surviving fish were euthanized on day 10. Asterisks indicate significant differences from the uninfected population (****p < 0.0001; **p < 0.01).

[0238] Figure 25 : Survival of axenic and conventional rainbow trout larvae infected with different fish pathogens. Kaplan-Meier plots of axenic larvae survival after bath exposure to Flavobacterium psychrophilum strain THCO2-90, Flavobacterium columnare strain Fc7, Lactococcus garvieae strain JIP 28 / 99, Vibrio anguillarum strain 1669, and Yersinia ruckeri strain JIP 27 / 88. Mean and SD plots represent mean percentage survival of fish 10 days after exposure to different pathogenic microorganisms. For each condition, n = 10 larvae. All surviving fish were euthanized 10 days post-infection. Asterisks indicate significant differences from the uninfected population (**p < 0.01; ***p < 0.001; ****p < 0.0001).

[0239] Figure 26 : Survival of recolonized trout larvae against Flavobacterium columnare Fc7 infection. Kaplan-Meier plots of axenic larvae survival after bath exposure to Flavobacterium columnare strain Fc7. A: Flavobacterium columnare strain Fc7 kills axenic rainbow trout but not conventional rainbow trout. B: Axenic trout larvae exposed to water used to rear conventional fish showed similar survival rates under Flavobacterium columnare infection compared to conventional trout larvae at 22 dph. C: Eleven strains identified from the conventional fish microbiota were added to rainbow trout larvae at 22 dph, followed by Flavobacterium columnare infection at 24 dph. This bacterial mixture was able to protect recolonized larvae from infection. Mean and SD plots represent mean percentage survival of fish 10 days after exposure to different pathogenic microorganisms. For each condition, n = 10 larvae. All surviving fish were euthanized 10 days post-infection. Asterisks indicate significant differences from the uninfected population (****p < 0.0001).

[0240] Figure 27: Histological comparison of the posterior intestine of infected and uninfected conventional and axenic rainbow trout larvae. A: Representative images of the intestine of uninfected axenic trout larvae and conventional trout larvae. B: Representative images of the intestine of infected axenic and conventional larvae exposed to the Flavobacterium columnare strain Fc7. Fish were fixed at 1 day post-infection (dpi) for histological analysis. C: Mean number of goblet cells / microvilli in the posterior intestine. Bars represent the mean ± SD / villus of the same region of the posterior intestine of three fish per condition. Combined Alcian blue and PAS staining of paraffin-embedded rainbow trout larvae was used for light microscopy. Images and quantification data represent three different fish per condition.

[0241] Figure 28 : Representative images of the in vitro growth inhibitory activity of Flavobacterium sp. strain 4466 against different virulent Flavobacterium columnare strains. A: Lack of growth inhibition of Flavobacterium columnare Fc7 after addition of 5 μL of Flavobacterium sp. culture supernatant. B: Growth inhibition halo of Flavobacterium columnare FC7 around the Flavobacterium sp. colony on a Flavobacterium columnare strain Fc7 overlay. C: Growth inhibition halos of Flavobacterium columnare ALG-00-530, IA-S-4, and Ms-Fc4. The agar overlay technique was performed by spreading a Flavobacterium columnare bacterial suspension on a soft agar solution on TYES agar and then dropping 5 μL of an overnight culture of Flavobacterium sp. strain 4466. Incubation was carried out at 28 °C for 24 h. The experiment was performed in triplicate.

[0242] Figure 29 : Phylogenetic tree (based on ANI analysis) showing the relationship between Flavobacterium sp. strain 4466 and the 15 closest Flavobacterium species. The tree was constructed by RAxML (version 8.2.8) using the 400 most conserved proteins in the proteome of each strain. Support values of bootstrap are indicated in the nodes.

[0243] Figure 30 : Flavobacterium sp. strain 4466 confers protection to rainbow trout larvae against all Flavobacterium columnare strains. Detailed Description

[0244] Results

[0245] Flavobacterium columnare kills axenic zebrafish but not conventional zebrafish

[0246] To study microbiota-based anti-infectivity in zebrafish, we compared the susceptibility of germ-free (GF) and conventional (Conv) zebrafish larvae to Flavobacterium columnare, an important fish pathogen that has previously been shown to infect and kill adult zebrafish [33, 34]. We used immersion to expose 6-day postfertilization (dpf) germ-free and conventional zebrafish larvae to a collection of 28 Flavobacterium columnare strains (belonging to four different genomospecies) for 3 hours at 5×10 5 colony-forming units (cfu) / mL. Daily monitoring showed that 16 out of 28 Flavobacterium columnare strains killed germ-free larvae within 48 hours ( Figure 2A ), while conventional larvae exposed to all tested virulent Flavobacterium columnare strains survived ( Figure 2B ). Exposure to the highly virulent strain ALG-00-530 (hereafter referred to as Flavobacterium columnare ALG ) also showed that germ-free zebrafish died rapidly (within 1 day) and in a dose-dependent manner, while conventional zebrafish survived at all doses except the highest (10 7 cfu / mL) ( Figure 1 ). Similar survival of infected conventional larvae obtained from AB-strain zebrafish eggs from 4 different zebrafish facilities was observed ( Figure 3 ), indicating that the conventional zebrafish microbiota can provide protection against Flavobacterium columnare infection.

[0247] Ten culturable bacterial strains are sufficient to prevent Flavobacterium columnare infection

[0248] Under our rearing conditions, the conventional larval microbiota is acquired from microorganisms present on the egg membrane and in the fish facility water after hatching. To test the hypothesis that microorganisms associated with conventional eggs provide protection against Flavobacterium columnare ALG , we exposed sterilized eggs at 0 or 4 dpf (before or after hatching, respectively) to water from the fish tank facility or mashed unsterilized conventional eggs. In both cases, these re-conventionalized (Re-Conv) zebrafish and conventional zebrafish survived Flavobacterium columnare ALG infection (Figure 2). To determine the composition of the conventional zebrafish microbiota, we generated clone libraries from 10 conventional zebrafish larvae (aged 4, 6, and 11 dpf, exposed or not exposed to Flavobacterium columnare ALG, the 16S rDNA gene content of the homogenate composites sampled from larvae of different batches within 3 months) was sequenced. We identified 13 major OTUs, 10 of which were identified in all experiments (Table 2). In addition, deep sequencing of the V3-V4 region of 16S rDNA of gDNA obtained from larvae sourced from the other 4 zebrafish facilities mentioned above indicated that most of these 10 OTUs were also detected in conventional larvae (Table 3).

[0249] Table 2: Ten strains that constitute the core of the zebrafish larval microbiota

[0250] Bacterial strains that were consistently detected at all time points (4, 6, and 11 dpf) in all experimental runs and constitute the core of the conventional zebrafish larval microbiota, and their taxonomic affiliations.

[0251]

[0252] a Average nucleotide identity value

[0253] b 16S rRNA gene sequence similarity

[0254] c recA gene sequence similarity

[0255] d rplC gene sequence similarity

[0256] * Species identified ambiguously

[0257]

[0258]

[0259] To isolate culturable zebrafish microbiota bacteria, we plated dilutions of homogenized composites of 6-dpf and 11-dpf larvae on various growth media, and we identified 10 different bacterial morphological types. Using 16S-based analysis followed by whole-genome sequencing, 10 bacteria were identified, corresponding to 10 strains of 9 different species, and these strains were also consistently detected by the culture-free method (Table 2). Then, we re-conventionalized germ-free zebrafish at 4 dpf with a mixture of all 10 identified culturable bacterial species (each at a concentration of 5×10 5 cfu / mL) (hereinafter referred to as Mix10), and we monitored exposure to Flavobacterium columnare at 6 dpf ALGSurvival of zebrafish larvae. We demonstrated that zebrafish reconstituted with Mix10 (reconstitution Mix10 ) exhibited a strong level of protection against all identified highly virulent Flavobacterium columnare strains ( Figure 5 ). These results suggest that Mix10 constitutes a core protective bacterial community that provides comprehensive protection for zebrafish larvae against F. columnare infection.

[0260] Community dynamics under antibiotic dysbiosis revealed key factors for resistance to F. columnare infection

[0261] To further analyze the determinants of the protective effect of Mix10 against F. columnare ALG infection, we inoculated 4 dpf larvae with an equimolar mixture of 10 bacteria (5×10 5 cfu / mL each) and monitored their establishment over 8 h. We first verified that there was no significant difference in the bacterial content of whole larvae and dissected intestines (p = 0.99). Then, we collected pools of 10 larvae immediately after reconstitution (t0), 20 min, 2 h, 4 h, and 8 h later, and used 16S rDNA sequencing to track the relative abundances of bacteria. At t0, all species in zebrafish were >4% ( Figure 6 ) except Aeromonas veronii strains 1 (0.2%) and 2 (not detected). Aeromonas caviae was detected as the most abundant species, followed by Stenotrophomonas maltophilia and Chryseobacterium massiliae, together accounting for 67.5% of the community ( Figure 6 ). The relative species abundances of most species (which may reflect initial colonization ability) were relatively stable during community establishment, with similar species evenness at t0 (E = 0.84) and t8h (E = 0.85). However, although both conventional and reconstituted Mix10 larvae were protected against F. columnare ALG infection, the overall structure of the reconstituted Mix10 population at 4 dpf was different from the conventional structure ( Figure 6 ).

[0262] To test the sensitivity to perturbation and protective resilience provided by the Mix10 bacterial community, we treated reconstituted Mix10 zebrafish with non-lethal antibiotics at 4 dpf using a combination of 250 μg / mL penicillin / streptomycin (all members of the Mix10 bacteria are sensitive to penicillin / streptomycin) or 50 μg / mL kanamycin (which affects all members of the Mix10 bacteria except Chryseobacterium massiliae, Phyllobacterium myrsinacearum, and Stenotrophomonas maltophilia) ( Figure 7 ). At 5 dpf, 16 h after exposure, the antibiotics were washed off and the zebrafish were immediately exposed to F. columnare ALG。In re-normalization Mix10 it was observed that both antibiotic treatments led to a complete loss of protection against ALG Flavobacterium columnare Figure 8A infection ( Figure 8B ). Then we used the same antibiotic treatments but with a 24-hour recovery period after washing off the antibiotics at 5 dpf, so that the infection was performed only at 6 dpf ( Mix10 ). Although the re-normalized Mix10 larvae treated with penicillin / streptomycin still showed a similar protection to the infected germ-free larvae, the re-normalized Figure 8B zebrafish treated with kanamycin showed a restored protection after the 24-hour recovery period and survived similarly to the untreated re-normalized fish ( Figure 8C ). Sampling at different time points and 16S analysis in the recovery experiment showed that the bacterial community evenness decreased similarly after the antibiotic application in both treatments (for the 4 dpf control, E = 0.85; for t0 kanamycin, E = 0.72; for t0 penicillin / streptomycin, E = 0.7), and continued to decrease during the recovery period (for kanamycin and penicillin / streptomycin treatments after the 24 h recovery period, E = 0.6 and E = 0.64, respectively). Notably, although Chryseobacterium massiliense could still be detected immediately similarly after both antibiotic treatments, the penicillin / streptomycin treatment led to a significant decrease in its relative abundance ( Figure 8D ). In contrast, the relative abundance of Chryseobacterium massiliense rebounded 6 hours after the cessation of kanamycin treatment and was the main member of the reconstituted microbiota after the 24-hour recovery period (

[0263] ), suggesting that the protective effect observed in the kanamycin-treated larvae might be due to the recovery of Chryseobacterium massiliense.

[0264] Both individual-level and community-level protection provide resistance against Flavobacterium columnare ALG infection. To test the potential key role of Chryseobacterium massiliense in the protection against 2 Flavobacterium columnare Figure 9 infection, we exposed 4 dpf germ-free zebrafish only to Chryseobacterium massiliense, and the results showed that it conferred individual protection at a dose as low as 5×10 Figure 9 cfu / mL ( 4 ). Interestingly, although none of the other 9 species that composed Mix10 provided individual protection ( Figure 9 A), their equimolar combination (referred to as Mix9) conferred protection to zebrafish, although the dose was not lower than 5×10 Mix9For species or species combinations related to protecting zebrafish from infection by Flavobacterium columnare, we tested all nine combinations of eight species (Mix8), as well as several combinations of seven, six, four, or three species, and the results showed no protection (Figure 10 and Table 4). Then, by doubling the concentration of any one species in the non-protective Mix8a, we tested whether the lack of protection of Mix8 compared to Mix9 might depend on density effects ( Figure 10B ), and the results showed no protection. Therefore, these results suggest that microbiota-based protection against Flavobacterium columnare infection depends on Chryseobacterium massiliense-dependent member effects or on community-dependent effects mediated by the Mix9 consortium.

[0265] Table 4: Ability of combinations of three, four, six, and seven core zebrafish microbiota species tested to prevent Flavobacterium columnare ALG infection. These tested combinations did not include Chryseobacterium massiliense. None of these tested combinations showed significant protective activity against Flavobacterium columnare ALG .

[0266]

[0267]

[0268]

[0269]

[0270]

[0271] The production of pro-inflammatory and anti-inflammatory cytokines does not contribute to microbiota-mediated protection against Flavobacterium columnare ALG infection

[0272] To test the contribution of the innate immune response of larval zebrafish to resistance against Flavobacterium columnare infection, we used qRT-PCR to measure the expression of cytokine mRNAs in germ-free and conventional zebrafish, and in larvae reconstituted with Chryseobacterium massiliense (re-conventionalized Cm ), Mix10 (re-conventionalized Mix10 ), or larvae reconstituted with Mix4 (Aeromonas caviae, Aeromonas veronii spp., Pseudomonas mosselii) (as a non-protective control) (Table 4), with or without exposure to Flavobacterium columnare ALG。We tested genes encoding IL1β (pro-inflammatory), IL22 (promotes intestinal repair), and IL10 (anti-inflammatory). Although we observed some changes in il10 expression in uninfected, re-conventionalized larvae, this was not associated with protection. Additionally, il10 expression was not regulated by infection under any of the tested conditions( Figure 11A ). In contrast, we observed strong induction of il1b and il22 in germ-free zebrafish exposed to ALG Flavobacterium columnare( Figure 11B C). However, this induction was not seen in protected conventional, re-conventionalized Cm , re-conventionalized Mix10 or non-protective re-conventionalized Mix4 larvae. These observations suggest that the presence of the gut microbiota significantly (down-) regulates the inflammatory response induced by Flavobacterium columnare infection. However, this effect is not associated with protection, suggesting that inflammatory regulation is not the main mechanism of microbiota-induced resistance.

[0273] Given that Myd88 is a key adaptor downstream of IL-1 and toll-like receptor signaling, we also generated germ-free and conventional larvae from the null myd88 zebrafish mutant

[35] and exposed them to Flavobacterium columnare ALG infection. Notably, despite their defects in innate immunity, conventional or re-conventionalized Mix10 (but not germ-free myd88 mutants) and wild-type zebrafish survived under Flavobacterium columnare conditions( Figure 11D ). Additionally, il1b induction by Flavobacterium columnare infection was only observed in germ-free larvae and was independent of myd88( Figure 12 ). Thus, these results are inconsistent with an important role for cytokine contributions in microbiota-mediated protection against Flavobacterium columnare infection by regulating pathogen-induced inflammatory responses.

[0274] Chryseobacterium massiliense and Mix9 protect zebrafish from intestinal damage caused by Flavobacterium columnare infection

[0275] Histological analysis of germ-free larvae fixed 24 h after exposure to Flavobacterium columnare ALG revealed extensive intestinal damage before any obvious signs appeared in other potential target organs such as gills or skin( Figure 13 A). To test the requirement for entry into the intestine during Flavobacterium columnare ALG infection, we modified our standard rearing protocol for germ-free fish to include feeding with live, germ-free Tetrahymena thermophila. We found that if not fed, germ-free zebrafish died within 24 h of exposure to Flavobacterium columnareALG did not die afterwards, and feeding with *Tetrahymena thermophila* or other food sources (e.g., sterile fish food powder) restored susceptibility to *Flavobacterium columnare* ALG infection ( Figure 14 ), indicating that successful infection requires feeding and ingestion.

[0276] Histological sections consistently showed severe intestinal disorders, vacuolization within microvilli, and formation of vacuoles in the sterile larvae infected with *Flavobacterium columnare* ( Figure 13 ). In contrast, zebrafish pre-incubated with *Chryseobacterium massiliense* or the Mix9 consortium at 4 dpf and then exposed to *Flavobacterium columnare* at 6 dpf showed no differences compared to uninfected or conventionally infected larvae ( Figure 13 ), confirming overall protection against *Flavobacterium columnare* at the intestinal level.

[0277] *Chryseobacterium massiliense* protects larval and adult zebrafish against virulent *Flavobacterium columnare* strains

[0278] The clear protection provided by *Chryseobacterium massiliense* against *Flavobacterium columnare* infection prompted us to test whether exogenous addition of this bacterium could improve microbiota-based protection against this widespread fish pathogen. We first demonstrated that zebrafish larvae colonized with *Chryseobacterium massiliense* were fully protected against all virulent *Flavobacterium columnare* strains identified in this study ( Figure 15 A). To test whether *Chryseobacterium massiliense* could also protect adult zebrafish against *Flavobacterium columnare* infection, we pre-treated conventional 3-4-month-old adult zebrafish with *Chryseobacterium massiliense* for 48 h and then challenged them with a high dose (5×10 6 cfu / mL) of *Flavobacterium columnare* ALG . Monitoring of mortality showed that pre-treatment with *Chryseobacterium massiliense* significantly increased (p = 0.0084) the survival rate of adult zebrafish challenged with *Flavobacterium columnare* compared to untreated conventional fish ( ALG B). Collectively, these results indicate that *Chryseobacterium massiliense* is a putative broad-spectrum probiotic that protects zebrafish against columnaris disease caused by *Flavobacterium columnare*. Figure 15 B). Collectively, these results indicate that *Chryseobacterium massiliense* is a putative broad-spectrum probiotic that protects zebrafish against columnaris disease caused by *Flavobacterium columnare*.

[0279] Generation of germ-free rainbow trout larvae

[0280] To study the potential protective effect of endogenous or exogenous bacteria against incoming pathogens in a microbiologically controlled rainbow trout host (Oncorhynchus mykiss), we first planned to produce germ-free (GF) trout larvae. To this end, we exposed freshly fertilized eggs to a previously described mixture of antibiotics and antifungals for 5 h

[136] , then to 0.005% bleach for 15 min, and then treated them with the iodophor disinfection solution Romeiod for 10 min. The sterile eggs were then stored under sterile conditions in a class II laminar flow cabinet in an aqueous solution containing antibiotics at 16 °C. Then, we took 50 μL samples of the rearing water and performed culture-based and 16S PCR-based tests to assess the sterility of the treated eggs 24 h after treatment ( Figure 16 ).

[0281] The sterile eggs hatched spontaneously after fertilization (dpf), similar to untreated conventional (Conv) eggs, indicating that our sterilization protocol did not affect egg viability. Instead, we determined that egg sterilization had a positive effect on hatching efficiency, with 72 ± 5.54% of the treated eggs and 48.6 ± 6.2% of the conventional eggs hatching. After hatching, up to 12 larvae were transferred to 75 cm 3 vented-cap cell culture flasks containing fresh sterile water without antibiotics ( Figure 17 ). The flask water was changed every 48 h, and the germ-free and conventional fish relied on their yolk reserves until 21 days post-hatching (dph), after which they were fed gamma-irradiated fish meal powder 30 min before each 48-h water change ( Figure 17 ). For the germ-free fish, we took 50 μL samples of the fish rearing water before each water change and 1 larval sample per week for culture-based and 16S PCR-based sterility tests until the end of the experiment (35 dph).

[0282] Compared with conventional larvae, the germ-free trout at 35 dph showed normal development and growth

[0283] To test the consequences of rearing germ-free larvae under sterile conditions, we compared the growth performance of conventional and germ-free larvae cultured from the same batch of eggs and observed no significant differences in standard body length and weight at 35 dph, with 2.33 ± 0.20 cm vs 2.16 ± 0.11 cm and 0.72 ± 0.21 g vs 0.64 ± 0.19 g for conventional and germ-free larvae, respectively ( Figure 18 ).

[0284] Consistently, anatomical comparisons of conventional and germ-free trout by optical projection tomography

[138] showed that at 21 dph, there were no anatomical differences in the organ development of conventional or germ-free fish, even in organs in direct contact with the fish microbiota, such as the gills ( Figure 19 D and 19I) and the intestine ( Figure 19 C and 19H; Figure 20 ), or in organs that may be affected by the gut microbiota, such as the brain ( Figure 19 A and 19F), spleen ( Figure 19 B and 19G) or head kidney ( Figure 19 E and 19J)

[139] . These results suggest that at this stage of the rainbow trout life cycle, the natural microbiota has no major impact on development and growth.

[0285] Identification of fish pathogens that kill germ-free trout larvae but not conventional trout larvae

[0286] To identify pathogens capable of infecting germ-free rainbow trout larvae via the natural infection route, we tested several trout bacterial pathogens, including Flavobacterium psychrophilum strain THC-O2 / 90, Flavobacterium columnare strain Fc7, Lactococcus garvieae, Vibrio anguillarum strain 1669, and Yersinia ruckeri strain JIP27 / 88. At 24 dph, germ-free rainbow trout larvae were exposed to water containing 10 7 CFU / mL of the test pathogen for 24 h. The fish were then washed three times by replacing 90% of the infection bath with fresh sterile water and then kept at 16 °C under sterile conditions. Among all the test pathogens, Flavobacterium columnare strain Fc7 was the most virulent pathogen, causing high and reproducible mortality in germ-free trout within 48 h after exposure ( Figure 21 A and Figure 25 ). Compared with the susceptibility of germ-free trout to Flavobacterium columnare, conventional larvae cultured from non-sterilized eggs resisted infection under all test conditions ( Figure 21 B and Figure 26 ). Consistently, histological analysis performed at 25 dph (24 h post-infection) showed that infected germ-free fish exhibited dissociation of the gill epithelium and severe disruption of the intestinal region. In contrast, infected conventional rainbow trout larvae exposed to Flavobacterium columnare were not different from uninfected germ-free or conventional larvae. More precisely, histological analysis of germ-free and conventional larvae at 25 dph (24 h post-infection) did not show any signs of intestinal damage ( Figure 27 ). However, we observed an increase in the number of goblet cells in infected germ-free larvae compared with uninfected germ-free larvae, while infected conventional larvae showed the opposite phenotype compared with uninfected conventional larvae ( Figure 27 ).

[0287] Microbiota Defense of Conventional Rainbow Trout against Flavobacterium columnare Infection

[0288] Based on the high susceptibility of germ-free rather than conventional rainbow trout to Flavobacterium columnare Fc7, we hypothesized that the observed anti-infectivity could be provided by the conventional microbiota. To test this, one week before the Flavobacterium columnare Fc7 infection challenge, we exposed 21-dph germ-free rainbow trout larvae to the water used for rearing conventional fish. The recolonized rainbow trout larvae survived as well as conventional rainbow trout larvae under Flavobacterium columnare conditions, while those kept under germ-free conditions died rapidly within the first 24 hours after infection ( Figure 22 A), indicating that the microbiota of conventional rainbow trout provides anti-infectivity against Flavobacterium columnare Fc7. To identify the culturable species in the microbiota of conventional trout, we plated dilutions of material extracted from three 35-dph conventional rainbow trout larvae on various agar growth media. Individual 16S-based determination of the bacterial diversity forming colonies on the test media identified 11 different bacterial strains, which were isolated and stored individually (Table 5).

[0289] Table 5: Eleven species isolated from conventional rainbow trout larvae

[0290]

[0291] To test whether these 11 culturable strains contribute to the defense against Flavobacterium columnare infection observed in conventional trout, we recolonized 22-dph germ-free rainbow trout larvae with a mixture of all 11 bacterial strains (hereinafter referred to as Mix11), each at a concentration of 5×10 5 CFU / mL. The survival of these recolonized trout was monitored after exposure to the Flavobacterium columnare strain Fc7, and the results showed that the recolonized Mix11 larvae survived as well as conventional fish ( Figure 22 B). These results indicate that the presence of 11 bacterial strains isolated from the rainbow trout microbiota recapitulates the overall protection against Flavobacterium columnare infection observed in conventional fish.

[0292] Resistance to Flavobacterium columnare infection is conferred by a member of the trout microbiota

[0293] To determine whether some individual members of the protective Mix11 could play a key role in anti-infection, we individually recolonized 22-dph germ-free trout with each of the 11 isolated bacterial strains at 5×10 5 CFU / mL and then challenged them with Flavobacterium columnare Fc7. We found that whether added individually ( Figure 23A ) or as a mixture ( Figure 23BAmong the Mix10), only Flavobacterium sp. strain 4466 restored the conventional level of protection, while the other 10 strains did not show protection. To evaluate the colonization of Flavobacterium sp. strain 4466 and / or Flavobacterium columnare Fc7 in the gastrointestinal tract, we plated the intestines taken from singly re-normalized fish 24 hours post-infection on TYES agar after dissection under aseptic conditions. Interestingly, while both Flavobacterium sp. strain 4466 and Flavobacterium columnare Fc7 were able to successfully colonize the intestines of singly exposed rainbow trout ( Figure 23C ), after rainbow trout were infected with Flavobacterium columnare Fc7, we only detected Flavobacterium sp.( Figure 23C ), indicating potential competition between the two bacterial species.

[0294] Consistently, although the cell-free spent supernatant of Flavobacterium sp. strain 4466 had no inhibitory activity against Flavobacterium columnare Fc7 in the overlay assay( Figure 28 A), the growth of Flavobacterium sp. strain 4466 colonies inhibited the growth of Flavobacterium columnare Fc7( Figure 28 B) and all tested Flavobacterium columnare strains( Figure 28 C), indicating potential contact-dependent inhibition. We identified a cluster of 12 genes (tssB, tssC, tssD, tssE, tssF, tssG, tssH, tssI, tssK, tssN, tssP, and tssQ) in the genome of Flavobacterium sp. strain 4466 that are likely related to this phenotype and have the characteristics of a type VI secretion system (T6SS), T6SS iii , a contact-dependent antagonistic system that is only present in the phylum Bacteroidetes

[32] . To improve the taxonomic identification of the protective Flavobacterium isolated from the microbiota of juvenile trout, we performed whole-genome sequencing and then average nucleotide identity (ANI) analysis. We determined that, although there were similarities to Flavobacterium spartansii (94.65%) and Flavobacterium structuratum (94.62%), these values were below the 95% ANI required to identify two organisms as the same species

[204] . In addition, full-length 16S rRNA and recA gene comparisons also showed high similarities to Flavobacterium spartansii and Flavobacterium structuratum. However, the values obtained were also below the 99% similarity threshold required to consider two organisms to belong to the same species (see the table on page 5 of this article). Similarly, a maximum likelihood-based phylogenetic tree generated from the sequences of 15 bacterial strains from the genus Flavobacterium( Figure 29 ) revealed that the sequence of Flavobacterium sp. strain 4466 clustered with the sequences of Flavobacterium spartansii and Flavobacterium structuratum, but did not allow for the identification of Flavobacterium sp. strain 4466 at the species level.

[0295] Endogenous Flavobacterium species strain 4466 protects axenic rainbow trout from infection by different Flavobacterium columnare strains

[0296] To test whether a protective Flavobacterium species isolated from the conventional rainbow trout microbiome could protect rainbow trout, we recolonized axenic fish larvae with Flavobacterium species 48 h prior to exposure to four virulent Flavobacterium columnare strains (Fc7, ALG-00-530, IA-S-4, and Ms-Fc-4; belonging to genotypes I and II and isolated from different geographical sources and host fish species). Flavobacterium species strain 4466 conferred protection to rainbow trout larvae against all Flavobacterium columnare strains ( Figure 30 ). Thus, the Flavobacterium species strain identified from trout Mix11 is a putative probiotic that may protect trout and other fish from columnaris disease.

[0297] Identification of exogenous probiotics that resist Flavobacterium columnare infection using axenic trout

[0298] Our results show that axenic trout can be used as a gnotobiotic model to identify bacteria that defend against Flavobacterium columnare infection. To determine whether this controlled gnotobiotic approach can be used to identify probiotics other than bacteria present in the trout microbiota, we pre-exposed 22-dph axenic rainbow trout larvae to Chryseobacterium massiliense, a bacterium previously shown to protect larval and adult zebrafish from Flavobacterium columnare infection [Stressmann et al.]. After 48 h bath treatment with 10 6 cfu / mL of Chryseobacterium massiliense, we infected the trout larvae with four Flavobacterium columnare strains: Fc7, ALG-00-530, IA-S-4, and Ms-Fc-4, which belong to genotypes I and II and were isolated from different geographical sources and hosts (Table 3). As previously observed in the axenic zebrafish model, Chryseobacterium massiliense also protected rainbow trout larvae from Flavobacterium columnare infection ( Figure 24 ). Collectively, these results show that axenic rainbow trout allow for the rational identification of bacterial species (whether or not non-trout endogenous) with probiotic potential against highly virulent Flavobacterium pathogens.

[0299] Bacterial taxonomic identification of protective microorganisms

[0300] To taxonomically identify the protective Chryseobacterium and Flavobacterium strain 4466 isolated from the larval microbiota of zebrafish and trout, we performed whole-genome sequencing followed by average nucleotide identity (ANI) analysis. The morphological type corresponding to Chryseobacterium was identified at the species level as Chryseobacterium massiliense, with 95.85% whole-genome similarity (see the table on pages 5 to 6 of this specification). Regarding Flavobacterium strain 4466, we determined that although there were similarities with Chryseobacterium spartanium (94.65%) and Chryseobacterium proteolyticum (94.62%), these values were below the 95% ANI required to identify two organisms as the same species

[204] . Additionally, full-length 16S rRNA and recA gene comparisons also showed high similarities with Chryseobacterium spartanium and Chryseobacterium proteolyticum; however, the values obtained were also below the 99% similarity threshold required to consider two organisms to belong to the same species (see the table on pages 5 - 6 herein).

[0301] Antibacterial gene prediction

[0302] Antibiotic resistance (AMR) genes were discovered using AMRFinderPlus from the fully sequenced genomes of each strain. This tool is documented in Feldgarden, M., Brover, V., Haft, D.H., Prasad, A.B., Slotta, D.J., Tolstoy, I., Tyson, G.H., Zhao, S., Hsu, C.-H., McDermott, P.F., Tadesse, D.A., Morales, C., Simmons, M., Tillman, G., Wasilenko, J., Folster, J.P., and Klimke, W., “Validating the NCBI AMRFinder Tool and Resistance Gene Database Using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of NARMS Isolates”, Antimicrob. Agents Chemother. 2019, Vol. 63, No. 11 (November 1, 2019): e00483-19, https: / / doi.org / 10.1128 / AAC.00483-19 , so that technicians can obtain its implementation.

[0303] No evidence of AMR genes was detected in the genome of Chryseobacterium.

[0304] The chromosome of Flavobacterium species strain 4466 revealed genes encoding resistance to carbapenems, lincosamides, streptogramins, pleuromutilins, and fluoroquinolones (Table 6).

[0305] Table 6: Identification of antibiotic resistance genes. Using AMRFinderPlus, antibiotic resistance (AMR) genes were discovered using protein annotations or nucleotide sequences from the whole genome of Flavobacterium species strain 4466.

[0306]

[0307]

[0308] Virulence gene prediction

[0309] The isolated Chryseobacterium species strain contains five predicted virulence factors, including some proteins involved in capsule biosynthesis, the HtpB subunit of heat shock protein, KatA catalase, and the ClpP protease proteolytic subunit.

[0310] Identification of virulence genes of Chryseobacterium massiliense. Using the Virulence Factor Database, virulence factor-encoding genes were predicted using protein annotations or nucleotide sequences from the whole genome of Chryseobacterium massiliense.

[0311] For Flavobacterium species 4466, genes encoding capsule, sialic acid synthase, type IV and type VI secretion system effectors, and catalase were found to be potential virulence factors (Table 7).

[0312] Table 7: Identification of virulence genes of Flavobacterium species strain 4466. Using the Virulence Factor Database, virulence factor-encoding genes were predicted using protein annotations or nucleotide sequences from the whole genome of Flavobacterium species strain 4466.

[0313]

[0314] Discussion

[0315] Many studies have focused on the influence of microbial diversity on higher-order bacterial community properties. In this study, we demonstrate a new community-level protective effect of the resident microbiota against lethal infection. More specifically, we show that recolonization of germ-free zebrafish larvae with a consortium of 10 cultivable bacterial strains (belonging to 9 different species from the standard laboratory zebrafish microbiota) provides protection at a conventional level against infection with a wide range of highly virulent strains of Flavobacterium columnare. The protective consortium is dominated by Proteobacteria (such as Pseudomonas and Aeromonas spp.) and bacteria common in aquatic environments, in addition to Chryseobacterium massiliense of the phylum Bacteroidetes. Although the zebrafish larval microbiota is relatively tolerant to environmental changes and intrinsic differences between samples

[36] , we found that these 10 bacteria were also identified as dominant in 4 different zebrafish facilities, indicating the presence of a core microbiota with important functions.

[0316] Using germ-free and gnotobiotic zebrafish larvae exposed to defined combinations of bacterial species, we were able to demonstrate strong species-specific protection in larvae mono-associated with C. massiliense. We also identified community-level protection provided by a combination of the other 9 species that make up the protective zebrafish larval microbiota, which were unable to defend against F. columnare when provided individually. However, the reproducibility of this protection was low (complete protection was observed in only 50% of the tests performed), and a minimum inoculum of 5 × 10 4 cfu / mL was required, compared with a minimum inoculum of 5 × 10 2 cfu / mL for C. massiliense. Thus, these results suggest the existence of two different microbiota-based protection scenarios against F. columnare infection: a member effect provided by C. massiliense and a threshold effect mediated by the Mix9 consortium.

[0317] Neither of these two protection mechanisms against *Flavobacterium columnare* infection seems to rely on microbiota-based immune modulation. However, we cannot rule out that some members of the protective Mix10 can individually induce pro-inflammatory or anti-inflammatory responses that are masked in the presence of a mixed microbiota [1]. Although the identification of the mechanisms involved in community-level Mix9 protection requires further investigation, the recolonization and dysbiosis and recovery experiments demonstrated the key role of *Chryseobacterium massiliense* in protection against *Flavobacterium columnare*. This protection can be provided by multiple mechanisms, including nutrient depletion or competition, adhesion inhibition, release of inhibitory metabolites, and stimulation of host immune defenses [6, 12, 37]

[37] . Mix9 and *Chryseobacterium massiliense* showed differences in the minimum cell density required for protection, and it is also possible that for the latter, *Flavobacterium columnare* infection triggers a relatively density-independent protection mechanism in *Chryseobacterium massiliense* through direct antagonistic niche exclusion. *Flavobacterium columnare* and *Chryseobacterium massiliense* are both Bacteroidetes, and in addition to direct resource competition, several niche exclusion mechanisms have been shown 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 massiliense* mutants to further analyze its protection mechanism. Interestingly, infected juveniles recolonized with *Chryseobacterium massiliense* or Mix9 did not show signs of intestinal damage exhibited by germ-free juveniles, indicating that both *Chryseobacterium massiliense* and Mix9 provide similar intestinal resistance against *Flavobacterium columnare* infection. Although microbial colonization contributes to intestinal maturation and stimulates the production of epithelial passive defenses (e.g., mucus) [41, 42], the lack of intestinal maturation is unlikely to cause mortality due to *Flavobacterium columnare*, as mono-colonized juveniles or juveniles recolonized with a non-protective mixture died as fast as germ-free juveniles.

[0318] Several studies have monitored the long-term assembly and development of the zebrafish microbiota from larvae to sexually mature adults, but little is known about the initial colonization of larvae after hatching [43, 44]. Neutral (random) and deterministic (host niche-based) processes [45 - 47] lead to microbial communities being mainly represented by a limited number of high-abundance species and a highly diverse low-abundance population. In our experiment, the Mix10 species inoculum corresponded to an equal-proportion bacterial mixture and thus started from a presumed total evenness (E = 1) [48, 49]. Since the evenness remained relatively high (0.84) and was very similar throughout our study until 8 h, this indicates that most of the 10 species were able to colonize the larvae. From the perspective of community composition, loss of diversity is generally associated with reduced colonization resistance, but it is unclear whether this increased susceptibility is due to the loss of certain key member species of the microbial community and / or changes in their prevalence [7, 8]. We investigated the resistance to infection by exposing established bacterial communities to different antibiotic perturbations and then directly performing a challenge with Flavobacterium columnare (to study the sensitivity of the core microbiota to perturbations) or a challenge with Flavobacterium columnare after recovery (to study its resilience) [11, 50]. It is known that antibiotics alter the composition and relative abundance of the microbiota according to their antibacterial spectra [12, 51]. We observed that penicillin / streptomycin treatment, which affected most of the core species, reduced the abundances of all species except two (Aeromonas veronii 1 and Phyllobacterium myrsinacearum), which became relatively dominant during the recovery process but failed to provide protection against Flavobacterium columnare. After treatment with kanamycin, the colonization resistance fully recovered by the end of the recovery period, indicating the resilience that species may have due to rapid recovery to pre-perturbation levels (due to rapid growth rates, physiological flexibility, or mutations)

[52] . Interestingly, even considering the potential biases associated with using 16S rDNA as a representative index for determining relative abundances [53, 54], the evenness decreased similarly during the recovery process for both treatments, but for Proteobacteria, the phylum-level abundance became 48% and for Bacteroidetes 52%, compared with >98% Proteobacteria for the penicillin / streptomycin treatment. In addition, Chryseobacterium massiliense was detected as rare (<1%) in conventional larvae, suggesting that it may have a disproportionate impact on the community or that community-level protection provided by the other nine bacteria is also responsible for protecting conventional larvae from Flavobacterium columnare infection.

[0319] Although the symptoms associated with columnaris disease in cold- and warm-water fish caused by *Flavobacterium columnare* are phenotypically homogeneous, *F. columnare* strains exhibit a high degree of genetic diversity, making it difficult to standardize animal models of infection [34, 55, 56]. Our study shows that germ-free zebrafish larvae are highly susceptible to a variety of different genotypes isolated from different hosts, demonstrating that they are a reliable animal model for studying *F. columnare* pathogenesis. Although *F. columnare* infections result in significant losses in aquaculture, there is no consensus on the molecular basis of *F. columnare* virulence. Secreted enzymes, such as chondroitin AC lyase, which acts on connective tissue [33, 57–59], and collagenase

[60] , have been proposed as possible virulence factors

[31] . Recently, mutants of the type IX secretion system (T9SS) of *F. columnare* were shown to be avirulent in adult zebrafish, suggesting that proteins secreted by the T9SS may be key to virulence

[33] . The colonization process of *F. columnare* remains largely undefined

[31] . In salmon, the gills are the main site of infection, but the body skin, fins, and tail are also frequently damaged, and septicemia can occur in severe cases

[57] . In salmon, general tissue damage in several organs was associated with low-virulence strains, while highly virulent strains were killed before such damage occurred

[31] . We were unable to identify a clear site of *F. columnare* infection in zebrafish larvae by histology, probably because the infection dose was very low, less than 100 cfu obtained from infected moribund larvae. However, several lines of evidence suggest that the intestine is the main target of *F. columnare* infection in our model: (i) unfed germ-free larvae survived after exposure; (ii) histological analysis showed severe disruption of the intestinal region a few hours after infection of germ-free larvae; (iii) induction of il22 in germ-free larvae exposed to *F. columnare*, as the main function of IL-22 is to promote intestinal repair

[61] . This induction seems to be a consequence of pathogen-mediated damage, as no induction was observed in conventional or re-conventionalized larvae. This severe intestinal damage may have led to very rapid death of the larvae, which may explain why little damage was observed in other common target organs of columnaris bacteria, such as the gills, skin, and fins.

[0320] Many bacterial diseases affect aquaculture and cannot be controlled by vaccination because they affect young fry or because there are no effective vaccines available (e.g., Flavobacterium columnare disease). In these cases, the use of antibiotics remains the only option, which may lead to the spread of antibiotic resistance. There is an urgent need to develop alternative therapies

[11] . In the case of F. columnare infection, high genetic variability and a broad host range constitute important limitations in identifying effective probiotics against this widespread pathogen. In this study, we demonstrated that Chryseobacterium massiliense might be a promising probiotic candidate for preventing columnaris disease because it provided comprehensive and robust protection against all tested virulent F. columnare genotypes and was also able to significantly improve the survival of adult conventional zebrafish exposed to this pathogen. Although further studies are needed to elucidate the protective potential of C. massiliense in other teleosts, the endogenous nature of C. massiliense suggests that it can establish itself as a long-term resident of the microbiota of zebrafish larvae and adults, which is an advantageous trait in the search for probiotic adaptation to target fish species

[62] . While short-lived probiotics may limit unforeseen consequences for the microbial community and host system, the use of endogenous commensals can stably modulate the community and provide long-term protection against recurrent disease outbreaks in fish

[63] .

[0321] In summary, the use of a simple and tractable fish model to explore the inherent fish microbiota as a source of protection against fish pathogens further highlights the ability of the zebrafish model to analyze microbiota function. Our study contributes to expanding knowledge on microbiota-mediated colonization and anti-infectivity against important fish pathogens. Further studies will determine the potential of endogenous bacteria as aquaculture probiotics to improve the health and production of other teleosts.

[0322] The use of probiotics to improve fish growth and health and to limit the use of chemical and antibiotic treatments is a common approach to controlling disease outbreaks in the fish farming industry [123, 140, 141]. However, the identification and characterization of protective bacteria are hampered by experimental variability associated with administration and infection challenges using poorly controlled conventional fish under open environmental conditions. Therefore, the development of robust and reproducible gnotobiotic models has facilitated the development of fish probiotics [125, 131]. In this article, we established a new axenic gnotobiotic rainbow trout model that enabled controlled studies of probiotic-based protection against infection by several fish bacterial pathogens.

[0323] The resistance of rainbow trout eggs to effective sterilization protocols eliminates the egg surface-associated microbial community, allowing for the routine rearing of axenic larvae at 16 °C for up to 35 dph without continuous exposure to antibiotics. Thus, our protocol is comparable to the gnotobiotic protocols used for zebrafish [136, 142], cod larvae

[128] , and stickleback (Gasterosteus aculeatus)

[143] , which do not rely on the continuous addition of antibiotics and thus avoid possible long-term effects on fish development

[131] . Additionally, immediately after hatching, the fish larvae were transferred to cell culture flasks with breathable caps. Rearing fish in flasks has several drawbacks that impede long-term experiments, such as the inability to aerate the water and perform automatic water changes

[131] . These constraints limit the model as an effective method for short-term experiments. The relatively short-term experiments conducted to study the anti-infectivity of trout larvae, while effectively controlling axenicity, have the drawback of studying larvae with a low-complexity microbiota. Axenic and gnotobiotic conditions are artificial compared to the conventional larval rearing conditions of fish farming or wild animals

[128] . However, even though adding bacterial strains as pure cultures may not represent the effects of the natural host-associated microbiota, the model remains an excellent available tool for studying the effects of specific bacterial additives without any microbial interference.

[0324] Rearing under axenic conditions had no major impact on the development and growth of rainbow trout larvae at 21 dph. Similar results were reported for axenic stickleback larvae at 14 dph

[143] . In sea bass (D. labrax L.), axenically reared larvae grew faster and had a more developed gut compared to conventionally reared larvae

[144] . This discrepancy may stem from the fact that in our study and in axenic sticklebacks, the anatomical analysis was performed before the first feeding, while axenic sea bass were fed externally. Fish initially obtain nutrients by absorbing their endogenous yolk until the gut is open from mouth to anus. We cannot rule out the possibility that differences may arise between axenic and conventional fish in terms of overall body weight and the structure and size of organs such as the gut at a later stage of development or after the first fish feeding.

[0325] Salmonids (including rainbow trout) are important economic species, and their production in intensive aquaculture environments is associated with an increased susceptibility to diseases caused by viruses, bacteria, fungi, and parasites

[145] . In this study, we tested the susceptibility of germ-free and conventional juvenile trout to the main salmonid freshwater pathogens. This led to the identification of Flavobacterium columnare as a highly virulent species that was lethal to germ-free juveniles. Flavobacterium columnare is the pathogen of columnaris disease, which affects several aquaculture fish species [137, 146], and is a new problem for rainbow trout juveniles and fry [147, 148]. Compared with the high susceptibility shown by germ-free trout, conventional juveniles reared from non-sterilized eggs were completely resistant to Flavobacterium columnare infection, and we demonstrated that the symbiotic microbiota carried by conventional juvenile trout played a crucial role in defense against Flavobacterium columnare infection.

[0326] Although germ-free conditions cannot be compared with those prevalent in the wild or used in fish farming

[205] , our results showed that germ-free rainbow trout juveniles were highly susceptible to Flavobacterium columnare, the pathogen of columnaris disease that affects many aquaculture fish species [206, 207]. Although our histological analysis comparing germ-free and conventional juveniles infected or not infected with Flavobacterium columnare Fc7 did not show any major signs of inflammatory damage, we observed an increase in the number of goblet cells per crypt in infected germ-free juveniles and a decrease in conventional juveniles. Goblet cells, which secrete mucus with bactericidal properties

[208] , are used as biomarkers to determine a healthy gut. Interestingly, as previously reported in zebrafish, a significant decrease in the number of goblet cells in uninfected germ-free juveniles compared with those in conventional juveniles was also observed

[209] . The lack of stimulating microorganisms in germ-free juveniles can lead to a dysregulated acute immune response after Flavobacterium columnare infection. These results suggest that the microbiota affects cell differentiation (or maturation) in the trout intestinal epithelium

[210] , potentially influencing protection against Flavobacterium columnare infection in some aspects.

[0327] Different studies have shown that highly diverse gut communities exert a higher protective effect on the host [149 - 151]. This forms the basis for the paradoxical negative impact on fish health associated with the extensive use of antibiotics in aquaculture, which in turn promotes the colonization of opportunistic pathogens by reducing microbiota diversity

[152] .

[0328] While this supports the practice of enriching fish microbial communities to minimize pathogen invasion in aquaculture

[122] , our findings suggest that resistance to infection can be achieved with a relatively less complex culturable microbiota identified in conventional juvenile trout. We did identify only 11 different bacterial species from the conventional rainbow trout juvenile microbiota, but conferred full protection to re-conventionalized germ-free trout. Although examples of anti-infective properties provided by defined bacterial consortia in gnotobiotic hosts generally rely more on community structure than on individual members of the microbiota [153 - 156], we demonstrated that the protective effect of the observed bacterial consortium composed of 11 identified microorganisms was mainly due to the presence of Flavobacterium sp. strain 4466. However, we cannot rule out that the presence of other bacterial species may be required at later developmental stages for the implantation or stabilization of protective members in the trout microbiota for higher efficiency.

[0329] Over the past 30 years, the fish farming industry has invested a great deal of effort in identifying probiotic microorganisms in rainbow trout, including Gram-positive and Gram-negative bacteria as well as yeasts

[157] . However, the irreproducibility of many in vivo experiments, the high inter-individual and seasonal variability in the composition of the trout microbiota, and the stochasticity of the limited colonization ability of exogenous microorganisms have rarely allowed the firm establishment of probiotic properties [158 - 160]. The identification of Flavobacterium sp. (an inherent member of the juvenile trout microbiota that defends against Flavobacterium columnare infection) suggests that this bacterium can be used as a probiotic to prevent infection. Although there is no clear evidence that probiotics of inherent origin perform better than exogenous probiotics of the target host

[161] , the use of beneficial inherent bacteria isolated from aquatic organisms to control pathogens in aquaculture is gaining acceptance

[162] .

[0330] The high genetic variability of Flavobacterium columnare and its broad host range constitute important limitations in the identification of effective probiotics against this widespread pathogen. Several probiotic candidates isolated from the host provided partial protection against F. columnare infection to other common fish species such as Sander vitreous and Salvelinus fontinalis [163, 164]. However, depending on the fish family used, high variability in the protective efficacy of probiotic strains used on S. fontinalis challenged with F. columnare was observed. As suggested by the authors, based on the fact that the microbiota composition is directly influenced by the host genotype, the genetic background of each family controls the efficiency of the probiotic action against the pathogen

[163] . As proposed in the present study, the use of gnotobiotic or axenic animal models should reduce this variability in order to more precisely evaluate probiotic candidate strains. According to different criteria defined for the selection of bacterial probiotics [157, 161], our study shows that Flavobacterium spp. can be considered as promising endogenous probiotics, and their potential in aquaculture needs to be further established at different stages of the trout life cycle.

[0331] More precisely, the inventors specifically demonstrated the ability of Flavobacterium spp. strain 4466 isolated from the microbiota of conventional trout larvae to defend against F. columnare infection. Moreover, the bacterium (but not its supernatant) inhibited the growth of F. columnare in vitro, indicating a direct interaction between Flavobacterium spp. strain 4466 and F. columnare. Interestingly, Flavobacterium spp. strain 4466 encodes a complete T6SS iii subtype, a molecular mechanism unique to the phylum Bacteroidetes that delivers antibacterial effector proteins upon contact with target cells

[211] . Members of the genus Flavobacterium are common inhabitants of the microbiota of freshwater and marine fish, and commensal and pathogenic Flavobacterium often share the same ecological niche [212 - 214]. Whether the T6SS iii contact-dependent killing system of Flavobacterium spp. strain 4466 contributes to colonization resistance by inhibiting the growth of F. columnare Fc7 is currently under investigation. However, we cannot rule out other mechanisms such as nutrient competition or the exclusion of the pathogen in direct competition for adhesion to host tissues. This has been suggested for zebrafish infected, which showed effective colonization by highly adhesive probiotic strains and an extended lifespan [215, 216, 217].

[0332] Interestingly, our axenic rainbow trout larva model also allowed us to demonstrate the protective activity of Chryseobacterium massiliense, a potential probiotic against different Flavobacterium columnare strains of diverse host and geographical origins isolated from conventional zebrafish [Stressman]. These results support C. massiliense as a potential probiotic for preventing columnaris disease in other teleosts besides its original host, the zebrafish. Moreover, this axenic fish model demonstrated the wide possibilities for studying potential anti-infective probiotics, both endogenous and exogenous.

[0333] In summary, by using experimental conditions that reduce microbiota variability, axenic rainbow trout larvae allow challenges under gnotobiotic conditions and clear analysis of protective phenotypes against fish pathogens. This approach will also contribute to the study of host-pathogen interactions under controlled conditions, thus leading to a better understanding of the virulence mechanisms used by fish pathogens. In summary, this model contributes to the mitigation of rainbow trout diseases in the context of aquaculture research and livestock farming.

[0334] Materials and methods

[0335] Bacterial strains and growth conditions. The bacterial strains used in this study are listed in Table 2. Flavobacterium columnare strains (Table 8) 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 genotypic groups, including genotypes I, I / II, II, and III

[64] using 16S rDNA restriction fragment length polymorphism analysis. All 10 Mix10 microbiota species were grown at 28 °C in Luria Bertani (LB).

[0336] Table 8: Flavobacterium columnare strains used in this study

[0337]

[0338]

[0339] Ethical statement

[0340] All animal experiments described in this study were conducted in accordance with EU guidelines for the handling of laboratory animals ( http: / / ec.europa.eu / env ironment / chemicals / lab_animals / home_en.htm ) at the Pasteur Institute (larvae) or INRA Jouy-en-Josas (adults) and were approved by the relevant institutional animal health and protection committees.

[0341] General treatment of zebrafish. Wild-type AB fish, originally purchased from the Zebrafish International Resource Center (Eugene, OR, USA), or myd88-null mutants (myd88 hu3568 / hu3568 )

[35] , kindly provided by AH Meijer (Leiden University, Netherlands), were raised in our facility. A few hours after spawning, eggs were collected, rinsed, and sorted under a dissecting microscope to remove feces and unfertilized eggs. All procedures below were performed in a laminar flow microbiological cabinet with disposable plasticware. Fish were kept at 28 °C in sterile 25 cm 3 vented-cap culture flasks containing 20 mL of water (0 - 6 fish at dpf - 15 per flask), or in autoclaved mineral water (Volvic) in 24-well microtiter plates (6 - 15 fish at dpf - 1 per 2 mL well). Fish were fed three times a week with sterile protozoan Tetrahymena thermophila starting from 4 dpf

[23] . Germ-free zebrafish were generated by sterilizing the egg envelopes with antibiotics and chemical treatments under otherwise germ-free conditions protecting the sterile eggs (see below), while conventional larvae (with the microbiota inherent to the facility) were directly cultured from non-sterilized eggs and then treated exactly like germ-free larvae.

[0342] Sterilization of zebrafish eggs. Eggs were sterilized as previously described with some modifications

[23] . First, freshly fertilized zebrafish eggs were bleached (0.003%) for 5 minutes, washed three times in sterile water with gentle agitation, and 100 eggs per group were maintained overnight in a 75 cm 3 vented-cap culture flask containing 100 mL of autoclaved Volvic mineral water supplemented with methylene blue solution (0.3 μg / mL). Thereafter, the eggs were transferred to 50 mL Falcon tubes (100 eggs per tube) and treated with a mixture of antibiotics (500 μL 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 antifungal drug (50 μL amphotericin B solution, Sigma-Aldrich (250 μg / mL)#A2942) and stirred for 2 hours at 28 °C. Then the eggs were washed three times in water with gentle agitation, bleached (0.003%) for 15 minutes, and the eggs were resuspended by inverting every 3 minutes. The eggs were washed three more times in water and incubated with 0.01% Romeiod (COFA, French Aquaculture Cooperative) for 10 minutes. Finally, the eggs were washed three times in water and then transferred to 25 cm containing 20 mL of water 3In vented-cap culture flasks. After sterilization, eggs were transferred at approximately 30 to 35 eggs / flask and then again at 4 dpf to new flasks at 10 to 15 eggs / flask prior to conventionalization. We monitored sterility at several points during the experiment as follows: 50 μL of water from each flask was dropped onto LB, TYES, and YPD agar plates, all of which were incubated aerobically at 28 °C. Plates were left for at least 3 days to allow slow-growing organisms to multiply. Bacterial contamination was also sampled by PCR amplification of water samples using 16S rDNA gene primers and the procedure detailed further below. If a particular flask was contaminated, those fish were removed from the experiment.

[0343] Procedures for rearing axenic zebrafish. After hatching, starting at 4 dpf, axenic zebrafish were fed three times a week with axenic Tetrahymena thermophila. (i) Tetrahymena storage. Axenic strains of Tetrahymena thermophila were stored at 28 °C in 20 mL of PPYE (0.25% peptone, BD Bact #211684; 0.25% yeast extract, BD Bacto #212750) supplemented with penicillin G (10 units / mL) and streptomycin (10 μg / mL). Media were inoculated with 100 μL of the previous Tetrahymena storage. After one week of growth, samples were taken and tested for sterility on LB, TYES, and YPD plates and then stored again. (ii) Growth. Tetrahymena thermophila was inoculated from the storage suspension at a ratio of 1:50 into MYE broth (1% milk powder, 1% yeast extract) and cultured at 28 °C. After 24 h of growth, Tetrahymena thermophila was transferred to Falcon tubes and washed three times (4400 rpm, 3 min, 25 °C) in 50 mL of autoclaved Volvic water. Finally, Tetrahymena thermophila was resuspended in water and added to culture flasks (500 μL in 20 mL) or 24-well plates (50 μL / well). Sterility of Tetrahymena thermophila was tested by plating and 16S rDNA PCR as described in the above section. (iii) Fine powder feeding. When indicated, axenic zebrafish were fed every 48 h with pre-irradiated fine powder feed (ZM-000 fish feed, ZM Ltd) suitable for the early first-feeding mouth gape size

[65] .

[0344] Re-conventionalization of axenic zebrafish. At 4 dpf, just after hatching, axenic zebrafish larvae were re-conventionalized with a single bacterial population or a mixture of several bacterial populations. Ten bacterial species that constitute the core protective microbiota were grown for 24 h at 28 °C in a suitable medium (TYES or LB). The bacteria were then pelleted, washed twice in sterile water, and all adjusted to the same cell density (OD 600 = 1 or 5 × 10 7 cfu / mL). (i) Re-conventionalization with a single species. The bacteria were resuspended and administered at 5 × 10 5The final concentration of cfu / mL was transferred to a culture flask containing sterile fish. (ii) Re-normalization was carried out with the bacterial mixture. To prepare Mix10, Mix9, Mix8 and all other mixtures used, equimolar mixtures were prepared by adding individual bacterial species with an initial concentration of 5×10 7 cfu / mL. Each bacterial mixture suspension was added to a culture flask containing sterile fish at a final concentration of 5×10 5 cfu / mL.

[0345] Infection challenge. Flavobacterium columnare strains (Table 8) were grown overnight in TYES broth at 28 °C. Then, 2 mL of the culture was pelleted (10,000 rpm, 5 min) and washed once in sterile water. At 6 dpf, sterile zebrafish were immersed in a culture flask with a bacterial dose of 5×10 2 to 5×10 7 cfu / mL to expose them to the test pathogen for 3 hours. Then the fish were transferred to individual wells of a 24-well plate, each well containing 2 mL of water and 50 μL of freshly prepared sterile Tetrahymena thermophila. Mortality was monitored daily as described in

[23] , and Flavobacterium columnare as low as 54 ± 9 cfu / larva was recovered from the infected larvae. All zebrafish experiments were terminated on day 9 post-infection, and zebrafish were euthanized with tricaine (MS-222) (Sigma-Aldrich #E10521). Each experiment was repeated at least 3 times, with 10 to 15 larvae per condition and per experiment.

[0346] Collection of eggs in other zebrafish facilities

[0347] Conventional zebrafish eggs were collected in 50 mL Falcon tubes from the following facilities: Facility 1: Nadia Soussi-Yanicostas Facility at Robert Debré Hospital, Paris; Facility 2: Jussieu A2, University of Paris VI; Facility 3: Jussieu-C8 (UMR7622), University of Paris VI; Facility 4: Commercial Amagen Facility, Gif-sur-Yvette. Larvae were treated with the same rearing conditions, sterilization and infection procedures as in the Pasteur Institute facilities.

[0348] The bacterial load of the fish was determined using cfu counting. Zebrafish were euthanized by treatment with 0.3 mg / mL tricaine (MS-222) (Sigma-Aldrich #E10521) for 10 minutes. Then they were washed in 3 different sterile PBS-0.1% Tween baths to remove bacteria loosely attached to the skin. Finally, they were transferred to a tube containing calibrated glass beads (acid-washed, 425 μm to 600 μm, SIGMA-ALDRICH #G8772) and 500 μL of autoclaved PBS. They were homogenized using a FastPrep cell disruptor (BIO101 / FP120Q BioGene) at maximum speed (6.5 m / s) for 45 seconds. Finally, serial dilutions of the recovered suspension were spotted onto TYES agar and cfu were counted after incubation at 28 °C for 48 hours.

[0349] Characterization of the zebrafish microbiota content. Over a period of 3 months, the experiment was conducted independently 3 times, and 3 different batches of eggs were collected from different fish couples in different aquariums. The larvae were reared as described above. Sterile and conventional larvae were collected at 4 dpf, 6 dpf, and 11 dpf for each batch. Infected conventional larvae were exposed to Flavobacterium columnare by immersion as described above. ALG For 3 hours. For each experimental group, 10 larval pools in triplicate (one for each experimental batch) were euthanized, washed, and lysed as described above. The lysate was divided into 3 equal parts, one for culturing and then 16S rDNA gene sequencing (A), for generating a 16S rDNA gene clone library and Sanger sequencing (B), and Illumina sequencing based on metabarcoding (C).

[0350] A) Bacterial culturing followed by identification based on the 16S rDNA gene

[0351] Serial-dilute the lysates and immediately plate them onto R2A, TYES, LB, MacConkey, BHI, BCYE, TCBS, and TSB agars and incubate at 28 °C for 24–72 h. For each agar, record the colony morphology type, pick colonies and streak them again on the same agar, in duplicate. To identify individual morphology types, pick single colonies for each identified morphology type from each agar, vortex in 200 μL of DNA-free water, and boil at 90 °C for 20 min. Use 5 μL of this bacterial suspension as a template for colony PCR, and amplify the 16S rDNA gene using the universal primer pair 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)) for the bacterial domain. In a 50 μL reaction system, the final concentration of each primer used is 0.2 μM. The PCR cycling conditions are as follows: initial denaturation at 94 °C for 2 min; then 32 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, and extension at 72 °C for 2 min; and finally extension at 72 °C for 10 min. Verify the PCR products of the 16S rDNA gene on a 1% agarose gel, purify them using a PCR purification kit, and send two PCR products of each morphology type for sequencing (Eurofins, Ebersberg, Germany). Manually proofread the 16S rDNA sequences and remove low-quality sequences from the analysis. Trim the primer sequences and compare the sequences with GenBank (NCBI) by BLAST and with the Ribosomal Database Project by SeqMatch. For genus determination, a similarity cutoff of 95% was used, and for operational taxonomic unit determination, a cutoff of 98% was used.

[0352] B) 16S rDNA gene clone library generation

[0353] According to the manufacturer's instructions, use Mobio kit to extract total DNA from the lysates. Also perform extractions with sterile larvae and DNA-free water as control samples. Verify the extracted genomic DNA by Tris-acetate-EDTA-agarose gel electrophoresis (1%) stained with GelRed, and quantify it by directly applying 2.5 μL to an ND-1000 spectrophotometer. Amplify the 16S rDNA gene by PCR using primers 8f and 1492r and check and purify the products as described in part A. Here, we added 25–50 ng of DNA as a template to a 50 μL reaction system. According to the manufacturer's instructions, use The Easy vector system (Promega) was used to generate clone libraries. Colony PCR was performed with the vector primers gemsp6 (5’-GCT GCG ACT TCA CTA GTG AT-3’ (SEQ ID NO: 9)) and gemt7 (5’-GTGGCA GCG GGAATT CGA T-3’ (SEQ ID NO: 10)) to determine the presence of the cloned insert fragments. Clones with the correct-sized insert fragments were purified as described above and sent for sequencing (Eurofins, Ebersberg, Germany). A blank using DNA-free water as a template was run as a control in all procedures. The clone library coverage was calculated using the formula [1-(n1 / N2)]×100, where n1 is the number of singletons detected in the clone library and N2 is the total number of clones generated for the sample. The minimum coverage of the generated clone library was 95%, and a minimum of 48 clones were generated for each sample. Sequence analysis and identification were performed as described in Part A.

[0354] C) By Illumina sequencing of the 16S rDNA gene

[0355] To identify the 16S rDNA gene diversity in the fish collected from our facility and four other zebrafish facilities, the fish were raised as described above. The axenic fish were sterilized as described above, and axenic and conventional fish that were not infected were collected at 6 dpf and 11 dpf. The fish were infected by immersion in Flavobacterium columnare ALG for 3 hours and then transferred to clean water. Infected conventional fish were collected at 6 dpf (6 hours after infection with Flavobacterium columnare) and at 11 dpf (as with the non-infected fish). Infected axenic larvae 6 hours after infection were collected only at 6 dpf, as all larvae died from the infection at 11 dpf. Ten larval pools in triplicate were euthanized, washed, and lysed as described above. Total DNA was extracted using the Mobio kit as described above, quantified using a ND-1000 spectrophotometer, and then sent to IMGM Laboratories GmbH (Germany) for Illumina sequencing. The primers Bakt_341F (5’-CCT ACG GGN GGC WGC AG-3’ (SEQ ID NO: 11)) and Bakt_805R (5’-GAC TAC HVG GGT ATC TAA TCC-3’ (SEQ ID NO: 12)), which amplify variable regions 3 and 4 of the 16S gene, were used for amplification

[63] .

[0356] According to the manufacturer's instructions, each amplicon was purified using a technology based on solid-phase reversible immobilization (SPRI) paramagnetic beads (AMPure XP beads, Beckman Coulter) with a bead:DNA ratio of 0.7:1 (v / v). The amplicons were normalized using the Sequal-Prep kit (Life Technologies) so that each sample contained approximately 1 ng / μL DNA. Samples, positive and negative controls were generated in one library. The High Sensitivity DNA LabChip kit was used for the 2100 Bioanalyzer system (both from Agilent Technologies) to examine the quality of the purified amplicon library. Using Kit Nano v2 (500 cycles) (Illumina Inc.) was used to generate clusters and sequencing. Before sequencing, cluster generation was performed by two-dimensional bridge amplification, followed by paired-end sequencing, generating 2×250 bp paired-end (PE) reads.

[0357] Reporter 2.5.1.3 software was used for raw data analysis (signal processing, demultiplexing, trimming of adapter sequences). CLC Genomics Workbench 8.5.1 (Qiagen) was used for read merging, quality trimming and QC reporting, and OTU definition was performed in the CLC plugin Microbial Genomics module.

[0358] Comparison of whole larvae with gut bacterial content

[0359] Re-normalized with Mix10 and at 6 dpf with Flavobacterium columnare ALG Larvae infected for 3 hours were euthanized and washed. DNA was extracted from 10 pooled whole larvae or 10 pooled intestinal tracts dissected with sterile surgical forceps for Illumina 16S rDNA gene sequencing. Sterile larvae and dissected sterile guts were sampled and used as controls. No statistically significant difference was found between whole fish and gut bacterial content (p = 0.99). Therefore, whole larvae were used for experiments monitoring bacterial establishment and recovery.

[0360] Whole genome sequencing

[0361] Chromosomal DNA of 10 species that constitute the core of the zebrafish larval microbiota was extracted using the DNeasy Blood & Tissue kit (QIAGEN), including RNase treatment. The quality and quantity of the DNA were evaluated on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific).

[0362] The DNA sequencing library was prepared using the Nextera DNA Library Preparation Kit (Illumina Inc.), and the library quality was examined on a Bioanalyzer 2100 (Agilent Technologies) using the High Sensitivity DNA LabChip Kit. Sequencing clusters were generated using the MiSeq Kit v2 (500 cycles) (Illumina Inc.) according to the manufacturer's instructions. DNA was sequenced by paired-end (PE) sequencing with 2×250 bp read length at the Helmholtz Centre for Infection Research. Between 1,108,578 and 2,914,480 reads were retrieved per sample, with a median of 1,528,402. The reads were quality-filtered, trimmed, and adapter-removed using fastq-mcf {Aronesty, 2011#29}, and the genome was assembled using SPAdes 2.5.1 {Bankevich, 2012#123}.

[0363] Bacterial species identification

[0364] Genome-based bacterial species identification was performed using the TrueBac ID system (v1.92, DB: 20190603) [https: / / www.truebacid.com / ]

[66] . When possible or when the 16S rDNA gene sequence similarity > 99%, species-level identification was performed based on an algorithm cutoff value set at 95% ANI.

[0365] Bacterial dynamics monitoring

[0366] Eggs collected from different fish mates in different fish tanks were used to run three independent experiments over 6 weeks to monitor establishment and recovery. The larvae were reared, disinfected, and infected as above, with the only difference being the use of 75 cm 3 culture flasks with breathable caps (containing 50 mL of sterile Volvic water) to accommodate the required large number of larvae, as the larvae used for time-course Illumina sequencing in each experiment were sequentially removed from the experiment monitoring larval survival. The animals were pooled (10 larvae per time point / condition), euthanized, washed, and lysed as described above, and stored at -20 °C until the end of survival rate monitoring and until all triplicates were collected.

[0367] A) Community establishment

[0368] To track the establishment of 10 core strains in the larvae, the axenic larvae were re-normalized as above with an equal ratio Mix10. At 4 dpf, immediately after adding the 10 core species, and 20 minutes, 2 hours, 4 hours, and 8 hours later, the re-normalized Mix10Larvae were sampled. Aseptic larvae, conventional larvae, and inocula were also sampled as controls.

[0369] B) Dysbiosis induction

[0370] For the re-normalization of 4 dpf zebrafish larvae, different doses of kanamycin (dose 1 = 200 μg / mL; dose 2 = 50 μg / mL; dose 3 = 25 μg / mL) and penicillin / streptomycin antibiotic mixture (dose 1 = 250 μg / mL; dose 2 = 15.6 μg / mL) were added to the water in the bottles to determine the antibiotic treatment (non-toxic to larvae but causing dysbiosis). Mix10 Zebrafish larvae were tested.

[0371] After 16 hours of treatment, the antibiotics were thoroughly rinsed off with sterile water, and the larvae were challenged with Flavobacterium columnare ALG resulting in the death of all larvae - for example, colonization resistance was successfully eliminated with optimal results in all replicates using 250 μg / mL penicillin / streptomycin and 50 μg / mL kanamycin as antibiotic treatments.

[0372] C) Community recovery

[0373] As in B), 4 dpf re-normalized larvae were treated with 250 μg / mL penicillin / streptomycin and 50 μg / mL kanamycin for 16 hours after 8 hours of hatching. The antibiotics were thoroughly washed off, and now the larvae were placed in sterile water for 24 hours to evaluate the resilience of the bacterial community. Samples (10 larval pools) were collected at 3 hours, 6 hours, 12 hours, 18 hours, and 24 hours during the recovery period and sent for 16S rDNA Illumina sequencing. Then at 6 dpf, the larvae were challenged with Flavobacterium columnare Mix10 for 3 hours, and the survival was monitored daily for 10 days after infection. ALG As described above, all time-course samples were sequenced by IMGM Laboratories GmbH.

[0374] Statistical analysis of the metataxonomic data

[0375]

[0376] ​16S rRNA analysis was performed using SHAMAN {Volant, 2019#125}. Library adapters, primer sequences, and base pairs that appeared at the 5′ and 3′ ends with a Phred quality score <20 were trimmed using Alientrimmer (v0.4.0). Reads that mapped exactly to the zebrafish genome (mm10) were removed. 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., and Mahé, F., “VSEARCH: a versatile open source tool for metagenomics”, PeerJ 2016, 4: e2584]. This process included several steps of 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}, completed using 16S sequences of 10 bacterial communities with VSEARCH, and filtered according to their identity to the reference {Yarza, 2014#127}. Annotations were retained when the identity between the OTU sequence and the reference sequence was ≥78.5% (for class), ≥82% (for order), ≥86.5% (for family), ≥94.5% (for genus), and ≥98% (for species). In this article, 73.2% of the OTU set was annotated, and 91.69% of them were annotated to the genus level.

[0377] The input amplicons were then aligned with the OTU set to obtain an OTU contingency table, which contains the number of amplicons associated with each OTU using global alignment with VSEARCH. The OTU count data matrix was normalized for library size at the OUT level using weighted non-empty count normalization. The normalized counts were then summed within each genus. Then, a generalized linear model (GLM) implemented in the DESeq2 R package 95 was applied to detect differences in genus abundance between each group. We defined a GLM that included treatment (condition) and time (variable) as main effects and the interaction between treatment and time. The resulting P values were adjusted according to the Benjamini and Hochberg procedure.

[0378] Statistical analysis can be reproduced on SHAMAN by loading the count table, the target classification results, and the contrast file obtained from figshare ( https: / / doi.org / 10.6084 / m9.figshare.11417082.v2 ).

[0379] Determination of cytokine levels

[0380] Total RNA was extracted from individual zebrafish larvae 18 hours after pathogen exposure (12 hours after washing) using the RNeasy kit (Qiagen). Oligonucleotide (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 thermal cycler (Applied Biosystems). Primers for ef1a (housekeeping gene, used for normalization of cDNA amount), il1b, il10, and il22 were described in (Rendueles 2012). Data were analyzed using the ΔΔCt method. Four larvae were analyzed for each condition. Zebrafish genes and proteins mentioned in the text: ef1a NM_131263; il1b BC098597; il22 NM_001020792; il10 NM_001020785; myd88 NM_212814.

[0381] Histological comparison of sterile, conventional, and re-conventionalized fish, and sterile and conventional fish infected or not infected with Flavobacterium columnare

[0382] Fish were collected 24 hours after infection (7 dpf) and fixed in Trump fixative (4% methanol-free formaldehyde, 1% glutaraldehyde in 0.1 M PBS, pH 7.2) at 4°C for 24 hours

[67] , and then sent to the PIBiSA microscopy facility of the University of Tours Medical School (France) (https: / / microscopies.med.univ-tours.fr / ), where the whole fixed animals were processed and embedded in epoxy resin. Semi-thin sections (1 μm) were cut using an ultramicrotome and then stained with toluidine blue for observation and imaging by light microscopy, or processed for transmission electron microscopy.

[0383] Pretreatment of adult zebrafish with Chryseobacterium indologenes

[0384] AB strain zebrafish were used. The fish were reared in dechlorinated circulating water at 28°C and then transferred to a continuous-flow aquarium at 3 - 4 months of age for infection experiments. Chryseobacterium indologenes was grown in TYES broth at 150 rpm and 28°C until the stationary phase. The bacterial culture was washed twice in sterile water and adjusted to OD 600nn = 1. The Chryseobacterium indologenes bacterial suspension was added at 2.10 6The final concentration of cfu / mL was directly added to the water (1 L) where the fish were located to re - normalize the adult fish. The contact between the bacteria and the fish was maintained for 24 hours by stopping the water flow, and then the bacteria were removed by resuming the water flow. After changing the water, Chryseobacterium massiliense was applied twice, and an equal volume of sterile water was added to the control group.

[0385] Adult zebrafish infection challenge

[0386] After re - normalizing the fish with Chryseobacterium massiliense, the infection with Flavobacterium columnare was carried out. The infection was performed as previously described by Li and his colleagues with little modification [Li et al., 2017]. Briefly, at 150 rpm and 28 °C, the Flavobacterium columnare strain ALG - 0530 was grown in TYES broth until the late exponential phase. Then, the bacterial culture was directly diluted into the water (200 mL) of the aquarium at a final concentration of 5×10 6 cfu / mL. The contact between the bacteria and the fish was maintained for 1 hour by stopping the water flow, and then the bacteria were removed by resuming the water flow. The control group used sterile TYES broth. At the start of the immersion challenge, the bacterial count was determined by plating serial dilutions of water samples on TYES agar. During the immersion, the water was maintained at 28 °C and continuously aerated. Each group consisted of 10 fish. The virulence was evaluated based on the fish mortality 10 days after infection.

[0387] Statistical methods

[0388] Statistical analysis was performed using the unpaired, non - parametric Mann - Whitney test. The analysis was carried out using Prism v8.2 (GraphPad software).

[0389] Evenness: The Shannon diversity index was calculated using the formula (Hs = -∑[P(ln(P))]), where P is the relative species abundance. The total evenness of the Shannon index was calculated as E = H s / H max . The lower the evenness of the community among species (and the presence of dominant species), the lower this index.

[0390] Management of rainbow trout larvae

[0391] Fertilized rainbow trout eggs were purchased from the French Aqualande Group. Upon arrival, the eggs were acclimated to the environment at 16 °C before operation. All procedures were carried out in a laminar flow microbiological cabinet using disposable plasticware. The eggs were kept in 145×20 mm Petri dishes until hatching in 75 mL of autoclaved dechlorinated water. After hatching, the fish were transferred and kept in 100 mL of sterile water in 250 mL vented - cap culture flasks at 16 °C. The fish were fed irradiated powdered food 21 days after hatching. To avoid waste accumulation and oxygen limitation, we renewed half of the water volume every two days to maintain the health of the rainbow trout larvae.

[0392] Sterilization and Rearing of Sterile Rainbow Trout

[0393] First, transfer rainbow trout eggs to sterile Petri dishes (140 mm, 150 eggs / dish) and wash them twice with sterile methylene blue solution (0.05 mg / mL). Next, we keep the freshly fertilized eggs in 75 mL of methylene blue solution and expose them to the antibiotic mixture

[136] described previously for 5 hours (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 drug amphotericin B solution (250 μg / mL)), with stirring at room temperature. Then wash the eggs 3 times with fresh sterile water. After that, bleach them (0.005%) for 15 minutes. Wash the eggs 3 times again with sterile water. After that, treat the eggs with iodophor disinfectant Romeiod for 10 minutes. Finally, wash the eggs 3 times and we keep them in 75 mL of sterile water supplemented with antibiotics at 16 °C until hatching. 5 to 7 days after treatment, the eggs hatch spontaneously. After hatching, immediately transfer the fish to 75 cm 3 vented-cap culture flasks containing 100 mL of fresh sterile water without antibiotics (12 larvae / flask). Determine the hatching rate by counting the hatched larvae in the Petri dish against the total number of eggs.

[0394] We monitor the sterility at different times during the experiment by dropping 50 μL of the rearing water from each flask onto LB agar plates, YPD agar, and TYES agar, all incubated under aerobic conditions at 16 °C. We also check for bacterial contamination of the fish larvae weekly. Randomly selected fish are euthanized with an excess of filtered tricaine methanesulfonate solution (MS222, 300 mg / L). The whole fish are mechanically disrupted in Lysing Matrix tubes containing 1 mL of sterile water and 425 - 600 μm glass beads (Sigma). The samples are homogenized on a FastPrep-24 instrument (XXX) at 6.0 ms -1 for 45 seconds. Serial dilutions of the homogenate are plated on TYES agar, YPD agar, and LB agar. When the water samples or the collected euthanized and homogenized fish show any bacterial CFUs on any of the different media used, these animals (or flasks) are removed from the experiment. The absence of any contamination in the fish larvae is further confirmed by PCR using primers specific for the 16S region of the chromosome (27F: 5'-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 13); 1492R 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO: 14))

[177] .

[0395] Bacterial Strains and Growth Conditions

[0396] The bacterial strains used in this study are listed in Table 3. Flavobacterium columnare strains Fc7 and IA-S-4, and Chryseobacterium massiliense 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 FRGDSA1882 / 11 were grown in TYES broth at 150 rpm and 28 °C. Yersinia ruckeri strain JIP 27 / 88 was 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) broth at 150 rpm and 28 °C. If needed, 15 g / L of agar was added for solid media. Stock cultures were stored at -80 °C in their respective broths containing 20% (vol / vol) glycerol.

[0397] Fish Infection Challenge

[0398] At different temperatures, the pathogenic bacteria were grown in appropriate media until the stationary phase. Then, each culture was pelleted (10,000 rpm, 5 min) and washed once in sterile water. The bacteria were resuspended and added to the culture flasks at a final concentration of 10 7 cfu / mL. After co-culturing with the pathogenic bacteria at 16 °C for 24 h, the fish were washed 3 times by changing the water. Ten to twelve larvae were used per experiment per condition. Bacterial counts were determined at the start and end of the immersion challenge by plating serial dilutions of the water samples on specific media for each pathogen. Each experiment was repeated at least 2 times. Virulence was evaluated based on the fish mortality 10 days post-infection.

[0399] Characterization of the Cultivable Conventional Rainbow Trout Microbiota

[0400] To identify the species that constitute the culturable conventional microbiota, three conventional rainbow trout larvae were sacrificed at 31 dph with an overdose of MS222. These fish were homogenized according to the above protocol, and serial dilutions of the homogenate suspension were plated on different media: TYES agar, LB agar, R2A agar, and TSA. The plates were incubated at 16 °C for 48 to 72 hours. After incubation, each morphologically distinct colony (based on form, size, color, texture, height, and margin) was isolated and stored at -80 °C in its respective broth containing 15% (vol / vol) glycerol. Individual identification based on 16S was performed by amplification and sequencing of the 16S chromosomal region using the universal oligonucleotides 27F and 1492R. Subsequently, the 16S rRNA gene sequences were compared with sequences available from the EzBioCloud database

[178] .

[0401] Reregularization of axenic rainbow trout microbiota

[0402] Each isolated bacterial species was grown in a suitable medium for 24 hours at 150 rpm and 28 °C. The bacteria were then pelleted and washed twice in sterile water. They were diluted to a final concentration of 5×10 7 cfu / mL. At 22 dph, axenic rainbow trout were reregularized by adding 1 mL of each bacterial suspension to the bottles (5×10 5 cfu / mL, final concentration). In the case of reregularizing the fish with a bacterial consortium, after washing the bacteria, all the isolated species were mixed in a water suspension at a concentration of 5×10 7 cfu / mL. Subsequently, this mixed bacterial suspension was added to the bottles containing axenic rainbow trout as described. In all cases, the reregularization of the fish was carried out for 48 hours, and then the fish were challenged with infection by Flavobacterium columnare. The bacterial suspension was added immediately after changing the water. Each experiment was repeated at least 2 times.

[0403] Histological examination

[0404] Tissue sections were used to compare the microscopic lesions in axenic and conventional fish after infection with Flavobacterium columnare. The sacrificed animals were fixed in Trump's fixative (4% methanol-free formaldehyde, 1% glutaraldehyde, in 0.1 M PBS, pH 7.2) at 4 °C for 24 hours

[179] . The whole fixed animals were processed and then embedded in epoxy resin. Semi-thin sections (1 μm) were cut using an ultramicrotome and stained with toluidine blue for observation and imaging by light microscopy.

[0405] 3D imaging of cleared fish by optical projection tomography (iDISCO)

[0406] For 3D imaging of whole cleared fish, the fish were fixed overnight at 4 °C with 4% formaldehyde dissolved in PBS. The fixed samples were rinsed with PBS. To make the tissue transparent, the fish were first decolorized twice by pretreatment in 0.5X SSC for 1 h at room temperature, and then incubated in 0.5X SSC + 0.5% KOH + 3% H2O2 at room temperature for 2 h. Decolorization was stopped by incubating twice in PBS for 15 min. The fish were then post-fixed with 2% formaldehyde dissolved in PBS for 2 h at room temperature and then rinsed twice with PBS for 30 min. The decolorized fish were cleared using the iDISCO+ protocol [Renier, 2016] (Renier et al., 2016, PMID 27238021). Briefly, during 1 h for each step, the samples were gradually dehydrated in an increasing methanol series (20%, 40%, 60%, 80%, and 100% in water, and 100% twice). The dehydrated samples were bleached by incubating overnight at 4 °C in methanol + 5% H2O2 and then incubated twice in 100% methanol for 1 h. Then they were incubated successively in 67% dichloromethane + 33% methanol for 3 h, in dichloromethane for 1 h, and finally in dibenzyl ether until the fish became completely transparent. Whole sample acquisition was performed on a light sheet ultramicroscope (LaVision Biotec, Bielefeld, Germany) with a 2x objective using 0.63x zoom parameters. Spontaneous fluorescence was obtained by illuminating both sides of the sample with a 488 nm laser. Z-stack scans were obtained with a Z-axis step of 2 μm.

[0407] Whole genome sequencing

[0408] Chromosomal DNA of Flavobacterium sp. strain 4466 isolated from the microbiota of juvenile rainbow trout was extracted using the DNeasy Blood & Tissue Kit (QIAGEN), including RNase treatment. The quality and quantity of DNA were evaluated on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). A DNA sequencing library was prepared using the Nextera DNA Library Preparation Kit (Illumina Inc.), and the library quality was examined using the high-sensitivity DNA LabChip kit on a Bioanalyzer 2100 (Agilent Technologies). Sequencing clusters were generated using the MiSeq reagents kit v2 (500 cycles) (Illumina Inc.) according to the manufacturer's instructions. DNA sequencing was performed by paired-end (PE) sequencing in both directions on the Microbiology Interplatform of the Pasteur Institute, generating 2×150 bp paired-end reads. The reads were quality-filtered, trimmed, and adapter-removed using fastq-mcf

[218] , and the genome was assembled using SPAdes 3.9.0

[219] .

[0409] Phylogenetic analysis

[0410] The proteomes of 15 most closely related Flavobacterium strains identified by ANI analysis were retrieved from the NCBI RefSeq database (table below).

[0411]

[0412] These sequences were analyzed with the proteome of Flavobacterium sp. strain UGB 4466 using Phylophlan (version 0.43, March 2020)

[220] . This method uses the 400 most conserved proteins in the proteome and constructs a maximum-likelihood phylogenetic tree using RAxML (version 8.2.8)

[221] . The maximum-likelihood tree was bootstrapped with 1000 replicates.

[0413] Agar overlay assay for growth inhibition detection

[0414] The growth inhibition of Flavobacterium sp. 4466 has been evaluated using the agar spot test. Briefly, 125 μL of overnight cultures from different Flavobacterium columnare strains adjusted to OD1 were mixed into 5 ml of top agar (0.7% agar) and overlaid on TYES agar plates. 5 μL of the overnight culture of Flavobacterium sp. 4466 was dropped on the overlay of the target bacteria. The plates were incubated at 28 °C for 24 h. Growth inhibition of Flavobacterium columnare was recorded by observing a clear halo around the colonies of Flavobacterium sp. Sterile TYES broth was used as a mock, and the experiment was performed in triplicate.

[0415] Whole genome sequencing for taxonomic identification, antibiotic resistance, and virulence factor prediction based on whole genome sequence analysis

[0416] Chromosomal DNA of Chryseobacterium spp., 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 80866, scientific name Delftia acidovorans)), and Flavobacterium sp. strain 4466 was extracted using the DNeasy Blood & Tissue Kit (QIAGEN), including RNase treatment. The quality and quantity of DNA were evaluated on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). DNA sequencing libraries were prepared using the Nextera DNA Library Preparation Kit (Illumina Inc.), and the 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 v2 chemistry (500 cycles) (Illumina Inc.) according to the manufacturer's instructions. DNA sequencing was performed by paired-end (PE) sequencing in both directions on the Microbiology Interplatform at the Pasteur Institute, generating 2×150 bp paired-end reads. The reads were quality filtered, trimmed, and adapter removed using fastq-mcf

[201] , and the genomes were assembled using SPAdes 3.9.0

[202] .

[0417] Whole genome analysis for taxonomic identification, antibiotic resistance, and virulence factor prediction based on whole genome sequence analysis

[0418] Using the TrueBac ID system (v1.92, DB: 20190603)( https: / / www.truebacid.com / ), whole genome analysis

[203] was performed on Chryseobacterium spp., 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 80866, scientific name Delftia acidovorans)), and Flavobacterium sp. strain 4466. Identification at the species level was based on an algorithm cutoff set at 95% average nucleotide identity (ANI) or when the 16S rRNA gene sequence similarity > 99%. Using the Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / )Identify virulence factors. Use AMRFinderPlus, a tool that identifies AMR genes using protein annotation or nucleotide sequences through the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / pathogens / antimicrobialresistance / AMRFinder / ), to discover antibiotic resistance (AMR) genes.

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Claims

1. Use of a bacterial strain or a combination of bacterial strains in the preparation of a probiotic for fish, wherein, At least one of the bacterial strains or the combination of bacterial strains is selected from the following: Chryseobacterium massiliense identified by accession number No. I-5479 and deposited at the CNCM on January 24, 2020 Chryseobacterium massiliae strain and Flavobacterium sp. strain identified by accession number No. I-5481 and deposited at the CNCM on January 24, 2020 Flavobacterium sp. strain.

2. The application according to claim 1, wherein, The combination further comprises at least one other bacterial strain of the indigenous microbiota of the treated fish species.

3. The use according to claim 1, wherein the use is for use in the preparation of a probiotic for: a. preventing or minimizing infection by Flavobacterium columnare in a host fish or a group of treated host fish, or enhancing the resistance of a treated host fish or a group of treated host fish to Flavobacterium columnare; and / or b. preventing or controlling a disease in a treated fish species or a group of treated host fish, said disease being a disease caused by an infection with a Flavobacterium columnare pathogen.

4. The application according to claim 3, wherein The probiotic resists infection caused by a Flavobacterium columnare pathogen or a disease caused by an infection with a Flavobacterium columnare pathogen.

5. The application according to claim 4, wherein, The disease is columnaris disease.

6. The application according to claim 3, wherein, The at least one bacterial strain is administered to a host, which is an osteichthyan.

7. The application according to claim 3, wherein, The at least one bacterial strain is administered to a host, which comprises a mixed fish population.

8. The application according to claim 6, wherein The host is rainbow trout.

9. The application according to claim 6, wherein The host is a juvenile rainbow trout.

10. The application according to claim 7, wherein, The host consists of a mixed fish population.

11. The application according to claim 7, wherein, The mixed fish population comprises groups of fish at different stages of their development or growth.

12. The application according to claim 6, wherein The at least one bacterial strain is administered to an osteichthyan selected from the group consisting of: eel ( Anguilla sp.), salmon, tilapia ( Oreochromis sp.), hybrid striped bass, walleye ( Stitzostedion vitreum ), channel catfish, sunfish, Pimephales promelas , goldfish ( Carassisu auratus ), carp ( Cyprinus carplo ), and swordtail ( Xiphophorus maculatus ).

13. The application according to any one of claims 1 to 5, wherein, The at least one bacterial strain is administered to a population comprising osteichthyans.

14. The application according to any one of claims 1 to 4, 6-11, wherein, The at least one bacterial strain is administered in an aquaculture environment.

15. The application according to any one of claims 1 to 4, 6 - 11, wherein, The at least one bacterial strain is administered in a fish rearing environment.

16. The application according to claim 12, wherein, The salmon is Oncorhynchus sp. or salmo sp.

17. The application according to claim 12, wherein, The hybrid striped bass is Morone chrysops crossed with M. saxatilis .

18. The application according to claim 12, wherein, The sunfish is the largemouth bass ( Micropterus salmoides ).

19. The application according to claim 12, wherein, The osteichthyan is an ornamental fish.

20. The application according to claim 19, wherein The ornamental fish is a tropical fish species.

21. The application according to claim 19, wherein The ornamental fish is Poecilia sphenops Poecilia sphenops ).

22. The application according to claim 1 or 2, wherein The at least one bacterial strain is administered to rainbow trout.

23. The application according to any one of claims 1 to 5, wherein The at least one bacterial strain is administered to a population comprising rainbow trout.

24. The application according to any one of claims 1 to 11, wherein The probiotic is for preventing or alleviating rainbow trout diseases in an aquaculture environment, said rainbow trout diseases being columnaris disease.

25. The application according to any one of claims 1 to 11, wherein The probiotic is for preventing or alleviating rainbow trout diseases in a fish rearing environment, said rainbow trout diseases being columnaris disease.

26. The application according to any one of claims 1 to 11, wherein The bacterial strain or combination of bacterial strains is introduced into the environment of the fish.

27. The application according to any one of claims 1 to 11, wherein The bacterial strain or combination of bacterial strains is introduced into the symbiotic microbiota of the fish by administering an encapsulated bacterial strain or combination of bacterial strains.

28. The application according to any one of claims 1 to 11, wherein The bacterial strain or combination of bacterial strains is administered to a host in need thereof at a dose of 5×10 4 cfu / mL to 5×10 6 cfu / mL of the bacterial strain.

29. The application according to claim 28, wherein, The dose is 5×10 5 cfu / mL bacterial strain.

30. A probiotic composition for fish, said probiotic composition comprising at least one bacterial strain and other acceptable carriers, said at least one bacterial strain being selected from the group consisting of Chryseobacterium massiliense identified by accession number No. I-5479 deposited at the CNCM on January 24, 2020 Chryseobacterium massiliae strain and Flavobacterium sp. strain identified by accession number No. I-5481 deposited at the CNCM on January 24, 2020 Flavobacterium sp. strain.

31. The probiotic composition according to claim 30, wherein, The probiotic composition comprises an adjuvant component.

32. The probiotic composition according to claim 30, wherein, The at least one bacterial strain is an encapsulated bacterial strain.

33. Bacterial strains selected from the following: Chryseobacterium massiliense identified by the accession number No. I-5479 and deposited at the CNCM on January 24, 2020 Chryseobacterium massiliae strain and Flavobacterium sp. strain identified by the accession number No. I-5481 and deposited at the CNCM on January 24, 2020 Flavobacterium sp. strain.

34. Kit, the kit comprising the bacterial strain according to claim 33, or the probiotic composition according to any one of claims 30 to 32.

35. The kit according to claim 34, the kit comprising an instruction booklet for administering to a fish host in need thereof.

36. Use of a bacterial strain or a combination of bacterial strains in the preparation of a fish food product for fish, wherein, At least one of the bacterial strains or the combination of bacterial strains is selected from the following: Chryseobacterium massiliense identified by accession number No. I-5479 and deposited at the CNCM on January 24, 2020 Chryseobacterium massiliae strain and Flavobacterium sp. strain identified by accession number No. I-5481 and deposited at the CNCM on January 24, 2020 Flavobacterium sp. strain.

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