# Investigation of the Genus Flavobacterium as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of Flavobacterium collinsii

Bo-Hyung Lee, Pierre Nicolas, Izzet Burcin Saticioglu, Benjamin Fradet, Jean-François Bernardet, Dimitri Rigaudeau, Tatiana Rochat, Éric Duchaud

► To cite this version:

Bo-Hyung Lee, Pierre Nicolas, Izzet Burcin Saticioglu, Benjamin Fradet, Jean-François Bernardet, et al. Investigation of the Genus Flavobacterium as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of Flavobacterium collinsii. Applied and Environmental Microbiology, 2023, 89 (4), 10.1128/aem.02162-22. hal-04107725

HAL Id: hal-04107725

https://hal.science/hal-04107725

Submitted on 5 Jun 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

# Investigation of the Genus *Flavobacterium* as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of *Flavobacterium collinsii*

Bo-Hyung Lee,ª Pierre Nicolas,ᵇ Izzet Burcin Satcioglu,ᶜ Benjamin Fradet,ª Jean-François Bernardet,ª Dimitri Rigaudeau,ᵈ Tatiana Rochat,ª Eric Duchaudª

**ABSTRACT** Bacteria of the genus *Flavobacterium* are recovered from a large variety of environments. Among the described species, *Flavobacterium psychrophilum* and *Flavobacterium columnare* cause considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a *Flavobacterium collinsii* isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 *Flavobacterium* species revealed that *F. collinsii* stands within a cluster of species associated with diseased fish, the closest one being *F. tructae*, which was recently confirmed as pathogenic. We evaluated the pathogenicity of *F. collinsii* TRV642 as well as of *Flavobacterium bernardetii* F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs or mortalities were observed with *F. bernardetii*. *F. collinsii* showed very low virulence but was isolated from the internal organs of survivors, indicating that the bacterium is able to survive inside the host and may provoke disease in fish under compromised conditions such as stress and/or wounds. Our results suggest that members of a phylogenetic cluster of fish-associated *Flavobacterium* species may be opportunistic fish pathogens causing disease under specific circumstances.

**IMPORTANCE** Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development, and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the *Flavobacterium* species. We also focused on *Flavobacterium collinsii*, which belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium survived inside the host, likely escaping clearance by the immune system but without provoking massive mortality, suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.

**KEYWORDS** *Flavobacterium*, *Flavobacterium collinsii*, aquaculture, experimental challenges, fish pathogens, opportunistic pathogens, rainbow trout, virulence

**Editor** Charles M. Dozois, INRS Armand-Frappier Sante Biotechnologie Research Centre

**Copyright** © 2023 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

Address correspondence to Eric Duchaud, eric.duchaud@inrae.fr, or Bo-Hyung Lee, bohyung.lee@inrae.fr.

The authors declare no conflict of interest.

**Received** 16 January 2023

**Accepted** 24 February 2023

**Published** 28 March 2023

<footnote>ªUniversité Paris-Saclay, INRAE, UVSQ, VIM, Jouy-en-Josas, France</footnote>

<footnote>ᵇUniversité Paris-Saclay, INRAE, MalAGE, Jouy-en-Josas, France</footnote>

<footnote>ᶜDepartment of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Bursa Uludag University, Bursa, Turkey</footnote>

<footnote>ᵈUniversité Paris-Saclay, IERP, INRAE, Jouy-en-Josas, France</footnote>
Members of the genus *Flavobacterium* (phylum *Bacteroidota*, family *Flavobacteriaceae*) are most frequently isolated from environmental sources. They are common in freshwater environments and in soil, especially in the rhizosphere, but strains have also been isolated from brackish water or seawater and glaciers in temperate, tropical, and polar areas (1). Following new molecular omics-based approaches, the taxonomy has been clarified and the number of formally described *Flavobacterium* species has rapidly expanded to include 268 species at the time of writing (https://lpsn.dsmz.de/genus/Flavobacterium [accessed August 2022]).

The vast majority of these species are considered harmless, consuming inert organic matter and thus playing an important role in biogeochemical cycles (2). Among them, *Flavobacterium johnsoniae* has emerged as a model organism for studying gliding motility and protein secretion (3). In addition, the genus encompasses two very important fish pathogens, *F. psychrophilum* and *F. columnare*, both reported to cause considerable losses in farmed and wild freshwater fish. To account for its genomic and phenotypic diversity, the latter has been recently divided into four distinct species, namely, *F. columnare*, *F. covaе*, *F. davisii*, and *F. oreochromis*, based on genomic comparisons as well as differences in host fish species and virulence (4). Another species, *F. branchiophilum*, is also known as a fish pathogen, but within more restricted geographical areas. In addition, the following species, often represented by a very small number of isolates, were recovered from diseased fish tissues and have been suspected to be pathogenic: *F. araucananum* from kidney and external lesions of Atlantic salmon (*Salmo salar*) (5); *F. bernardetii* from kidney and liver of rainbow trout (*Oncorhynchus mykiss*) (6); *F. turcicum* and *F. kayseriense* from rainbow trout kidney and spleen, respectively (7); *F. branchiarum* and *F. branchiicola* from rainbow trout gills (8); *F. chilense* from external lesions of rainbow trout (5); *F. collinsii* from the liver of rainbow trout (8); *F. hydatis* from the gills of diseased salmon (9, 10); *F. inkyongense* from diseased choco-late cichlids (*Hypselecara coryphaenoides*) (11); *F. johnsoniae*-like isolates from various diseased fish species (12); *F. oncorhynchi* from liver and gills of rainbow trout (13); *F. piscis* from liver, gills, and kidney of rainbow trout (14); *F. plurextorum* from liver and eggs of rainbow trout (15); and *F. succinicans* from gills of rainbow trout suffering bacterial gill disease (16). At least one species, *F. tructae*, which was isolated from liver, gills, and kidney of rainbow trout (14) and concurrently from kidney of feral spawning adult Chinook salmon (*Oncorhynchus tshawytscha*) under the alternative name of *F. spartansii* (17), may be considered a salmonid pathogen, as two isolates were able to induce pathological changes and mortality in experimentally infected Chinook salmon, though only using very high infectious doses (18). In contrast, most of the aforementioned fish-associated species have not been assessed for their level of virulence using experimental challenges.

The lack of information about the pathogenicity of many *Flavobacterium* species can lead to unnecessary and irrational use of antimicrobials and, conversely, to the lack of surveillance of bacteria that can cause disease outbreaks. Interestingly, the continuous increase in whole-genome sequencing generates data that may help identifying ecological niches and pathogenicity, by providing improved phylogenetic resolution and access to gene repertoires underpinning the phenotypes. As an initiative for this approach in the genus *Flavobacterium*, we exploited the large number of genomes available to investigate the relationship between phylogeny and environmental niches at the species level. In parallel and in attempt to satisfy Koch's postulates, we assessed the virulence of two recent isolates of *F. bernardetii* (6) and *F. collinsii*, a species closely related to *F. tructae*, using experimental infection in rainbow trout.

## RESULTS

**A new *F. collinsii* isolate retrieved from rainbow trout.** In order to survey the health status of rainbow trout raised at INRAE fish facilities (IERP), different organs of dead fish are regularly sampled to perform basic bacteriological quality controls. Organs (i.e., usually spleen and liver) are crushed in tryptone-yeast extract-salt (TYES) broth and
the lysates are spread on petri dishes to ensure the absence of any pathogenic *Flavobacterium* species. Surprisingly, during the summer of 2020, yellow-pigmented bacterial colonies with high spreading activity were observed after streaking the spleen of one rainbow trout fingerling (2 g) that had died without any disease symptom. The fish belonged to the highly *F. psychrophilum*-susceptible A36 isogenic line (19). A pure culture was obtained from one bacterial colony and 16S rRNA gene sequencing identified the bacterium as probably belonging to the species *F. collinsii*. For a more accurate characterization, the complete genome of this isolate, named TRV642, was resolved by high-throughput sequencing using the hybrid assembly of long Nanopore reads and short Illumina reads. The average nucleotide identity (ANI) between strain TRV642 and the *F. collinsii* type strain, CECT 7796, was 98.30%, far above the threshold delineation (cutoff, 95 to 96%) of a bacterial species (20), thus confirming the initial 16S rRNA gene taxonomic affiliation.

**Phylogenetic analysis and correlation with the ecological niche at the genus level.** To cover the diversity of the species with complete genome records available in public databases, we extracted from the RefSeq database all genomes belonging to genus *Flavobacterium* with the tag "representative," constituting a set of 195 genomes from distinct species. Gene repertoire comparisons delineated a core of 510 conserved single-copy genes. Their multiple-sequence alignment after gap removal represented 154,805 amino acids (98,363 polymorphic sites) and was used for phylogenetic reconstruction. A tentative phylogenetic tree for the 195 *Flavobacterium* species is shown in Fig. 1. The genome size and G+C content in comparison to the corresponding medians for the genus are also shown in Fig. 1.

To examine the links between position in the phylogenetic tree and ecological niche, we retrieved the environment from which the type strain was sampled from the original publication describing each species. The diversity of environments was summarized into 9 different categories (Fig. 1; see also Data Set S1 in the supplemental material), distinguishing 3 categories for the 29 species isolated from animal-associated samples, 5 categories for the 165 species isolated from other important natural environments, and 1 category, "other," to account for the single species isolated in a totally different and more artificial context (*F. supellecticarium* isolated from a "synthetic wooden board" (21)).

According to this classification, the 165 species (84.6% of the total) originating from non-animal-related environmental samples were composed of 60 species (30.8%) retrieved from freshwater, 45 species (23.1%) from cold/frozen environments (predominantly Antarctic soil or glacier), 38 species (19.5%) from soils, 11 species (5.6%) from marine environments (including sea sediments and algae), and 11 species (5.6%) from wastewater-related environments. Out of the 29 species (14.9% of the total) retrieved from, or in contact with, animals, only 7 of these animals were not fish. The 22 fish-associated species (11.3% of the total) were further divided into 7 confirmed pathogens (4 of which were previously considered genomovars of *F. columnare*) and 15 putative pathogens or fish commensals. Taken together, the classification of the environments and the phylogenetic tree shed light on the possible connections between evolutionary links and ecological niches in the genus *Flavobacterium*. Among the patterns that emerge from this picture, a group around *F. glauciei* is mostly composed of species retrieved from cold/frozen environments. Some links between phylogenetic position and genome characteristics were also observed. In terms of G+C content, 8 species with particularly high G+C contents form a phylogenetic group around *F. caeni*. In terms of genome size, a large phylogenetic group encompassing more than a quarter of all considered species (51 species [26.2%]), including *F. johnsoniae*, displays genomes up to 2 Mbp larger than the median value for the genus.

Whatever the specific category considered ("fish pathogen," "fish associated," or "animal other than fish"), species associated with animals are scattered across the phylogenetic tree. Exceptions are the 4 fish-pathogenic species resulting from the splitting of *F. columnare* (which group together), 2 pairs of closely related "fish-related species" (*F. plurextorum*-*F. oncorhynchi* and *F. erciyesense*-*F. turcicum*), and a more important cluster of species around *F. tructae* that includes *F. collinsii*.
<figure><img src="image_3.png" /><figcaption>**FIG 1** Tentative phylogenetic tree of 195 *Flavobacterium* species. The tree is based on the protein sequences of the 510 single-copy genes of all *Flavobacterium* species for which the complete genome is available in public databases. For clarity, the tree was midpoint rooted. Genome size and G+C contents are compared to the medians for the genus and shown as circular bar plots. Color and position on outer circle indicate the source of isolation of the type strain, with confirmed pathogen species in red and outermost position.</figcaption></figure>

**F. collinsii** belongs to a cluster of fish-associated species with large genomes.

Strikingly, *F. collinsii* is the closest relative of the confirmed pathogen *F. tructae* but the two species are well distinct, as confirmed by the ANI of only 87.56% between *F. collinsii* TRV642 and *F. tructae* MSU. These two species belong to a cluster of putative pathogenic species with *F. araucananum*, *F. bizetiae*, *F. piscis*, and *F. chilense*. All these species were isolated from diseased salmonid fish except for *F. bizetiae*, whose origin of isolation was more loosely described as "a diseased freshwater fish" (22). All these species are predicted to derive from a common ancestor, also shared with *F. aquidurense*, *F. psychroterrae*, and *F. hibernum*, 3 species not retrieved from animal-related material.
This cluster, enriched in fish-related species comprising *F. collinsii* and *F. tructae*, belongs to the above-mentioned large genome group comprising *F. johnsoniae*.

The complete genome of *F. collinsii* TRV642 consists of a circular chromosome of 5,554,530 bp and predicted to contain 4,285 coding DNA sequences (CDS), 69 tRNA genes, and 7 rRNA operons. The genome size is slightly smaller (by 181,002 bp) than that of the *F. collinsii* type strain, CECT 7796. All species belonging to the aforementioned cluster comprising *F. tructae* also possess large genomes (from 5.4 to 6.1 Mbp), comparable to the 6.1-Mbp genome of the well-studied environmental species *F. johnsoniae* (23). These genomes are about 2-fold larger than the 2.9-Mbp genome of *F. psychrophilum* (24) and the 3.2-Mbp genome of the *F. columnare* type strain, ATCC 23463 (4); these two species are well characterized and unquestionably fish pathogenic.

**Insights from the gene repertoire. (i) Protein secretion.** The type IX secretion system (T9SS) is responsible for protein secretion and required for gliding motility (3). This machinery is confined to the phylum *Bacteroidota*, and genes encoding the core components of the T9SS machinery, the attachment complex, and the *gld* genes needed for gliding motility were all identified in the *F. collinsii* TRV642 genome (Data Set S2).

Proteins secreted by the T9SS possess conserved C-terminal domains (CTDs). In the *F. collinsii* TRV642 genome, 45 genes encoding proteins with a type A (TIGR04183) CTD were identified (Data Set S3), some with predicted enzymatic functions, as well as 9 genes encoding proteins with a type B (TIGR04131) CTD, none with predicted enzymatic function. Enzymes with a type A CTD include 9 peptidases likely involved in protein/peptide degradation, all conserved in the genomes of *F. tructae* and *F. chilense* but absent, with a few exceptions, in those of *F. psychrophilum* and *F. columnare*. Among the other type A CTD enzymes, two are polysaccharide lyases and one is a glycoside hydrolase likely involved in carbohydrate degradation. This repertoire of T9SS-secreted enzymes supports the view of *F. collinsii* as a degrader of high-molecular-weight organic matter and suggests metabolic versatility, a functional trait shared by some members of the family *Flavobacteriaceae*, such as *F. johnsoniae* (23).

In addition to the *Bacteroidota*-specific T9SS, a B-type T4SS is also present in the *F. collinsii* TRV642 genome (Data Set S2). This versatile secretion system is utilized to mediate horizontal gene transfer and also allows Gram-negative pathogenic bacteria to translocate a wide variety of virulence factors into the host cell (25). Because the *F. collinsii* T4SS locus encompasses genes involved in DNA transfer and encoding relaxase/mobility family proteins (i.e., MobA/VirD2 and MobC), it is tempting to speculate that the *F. collinsii* T4SS is dedicated to the recruitment and delivery of DNA substrates.

**(ii) PUL and carbohydrate-active enzymes (CAZymes).** Members of the phylum *Bacteroidota* have developed multicomponent protein systems aimed at sensing, binding, transporting, and degrading specific glycans (26). Genes encoding these systems are often colocalized in regions referred to as polysaccharide utilization loci (PUL). Tandem *susD*-like and *susC*-like genes, which encode a carbohydrate-binding lipoprotein and a TonB-dependent transporter, respectively, are considered a hallmark of PUL, and these *susCD*-like gene pairs are generally used to identify PUL in *Bacteroidota* genomes (27). It has been suggested that *susCD*-like pairs could transport substrates other than carbohydrates (23), and this was recently confirmed by the finding that the BT2263-2264 pair of *Bacteroides thetaiotaomicron* and the *RagAB* transporter of *Porphyromonas gingivalis* import oligopeptides (28, 29).

The *F. collinsii* TRV642 genome contains 29 *susCD*-like pairs, most of which are encompassed in bona fide PUL, namely, with adjacent genes encoding obvious additional polysaccharide utilization proteins (i.e., CAZymes) (Table 1; see also the figure in the supplemental material). About half of these PUL have been previously identified in the *F. johnsoniae* UW101 genome (23). For instance, PUL TRV642_1394 to -1405 are predicted to be dedicated to α-glucan/starch degradation (corresponding to PUL Fjoh_1398 to -1408), PUL TRV642_1594 to -1602 to β-glucan/xylan degradation (corresponding to PUL Fjoh_1559 to -1567), and PUL TRV642_4202 to -4209 to chitin degradation (corresponding to PUL Fjoh_4555 to -4564). In addition to these PUL shared with *F. johnsoniae*,
<table><caption>TABLE 1 susCD pairs identified in the *Flavobacterium collinsii* TRV642 genome</caption><thead><tr><th>Locus tag</th><th>Product</th><th>InterPro identifier(s)</th><th>In a bona fide PUL</th><th>Predicted substrate</th></tr></thead><tbody><tr><td>TRV642_0169</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_0168</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_0202</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligopeptide</td></tr><tr><td>TRV642_0201</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_0403</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_0404</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_0405</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_0406</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_0407</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_0408</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_0663</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_0664</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_0665</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_0666</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_1402</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>α-Glucans/starch</td></tr><tr><td>TRV642_1403</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_1595</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>β-Glucans/xylan</td></tr><tr><td>TRV642_1596</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_1932</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligosaccharides</td></tr><tr><td>TRV642_1933</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_1968</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_1969</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_2080</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_2081</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_2255</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_2254</td><td>SusD-like protein</td><td>IPR033985</td><td></td><td></td></tr><tr><td>TRV642_2325</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_2326</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_2567</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligosaccharides</td></tr><tr><td>TRV642_2566</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_2576</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_2575</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_2869</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligosaccharides</td></tr><tr><td>TRV642_2870</td><td>SusD-like protein</td><td>RagB/SusD domain</td><td></td><td></td></tr><tr><td>TRV642_3296</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_3295</td><td>SusD-like protein</td><td>IPR033985</td><td></td><td></td></tr><tr><td>TRV642_3433</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_3432</td><td>SusD-like protein</td><td>IPR041662</td><td></td><td></td></tr><tr><td>TRV642_3468</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>α-Glucans</td></tr><tr><td>TRV642_3467</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_3614</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_3613</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_3711</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligosaccharides</td></tr><tr><td>TRV642_3712</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_3733</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Pectins</td></tr><tr><td>TRV642_3732</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_3861</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligopeptide</td></tr><tr><td>TRV642_3860</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_4207</td><td>SusC-like protein</td><td>IPR023996</td><td>Yes</td><td>Chitin</td></tr><tr><td>TRV642_4206</td><td>SusD-like protein</td><td>IPR024302</td><td></td><td></td></tr><tr><td>TRV642_4321</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_4322</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_4333</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>No</td><td>Unknown</td></tr><tr><td>TRV642_4334</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_4524</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Oligopeptide</td></tr><tr><td>TRV642_4525</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr><tr><td>TRV642_4549</td><td>SusC-like protein</td><td>IPR023996 and IPR023997</td><td>Yes</td><td>Heparin/chondroitin sulfate</td></tr><tr><td>TRV642_4550</td><td>SusD-like protein</td><td>IPR012944 and IPR033985</td><td></td><td></td></tr></tbody></table>
some PUL are likely of functional importance. For instance, PUL TRV642_0198 to -0209 encompass 5 peptidases (belonging to families S9, S37, M24, and M43) and are likely involved in the uptake of oligopeptides. PUL TRV642_4544 to -4556 encompass 12 genes, among which are TRV642_4547, encoding a polysaccharide lyase (family PL8) protein precursor containing a type A T9SS C-terminal secretion signal likely anchored in the outer membrane, facing the extracellular milieu, and *hepC*, encoding a protein highly similar (63% amino acid identity) to heparinase III of the nonpathogenic soil bacterium *Pedobacter heparinus*, degrading heparan sulfate (30) and likely located in the periplasm. These two genes, organized in tandem, as well as other glycoside hydrolase (GH)-, polysaccharide lyase (PL)- and sulfatase-encoding genes, are likely involved in the degradation of complex sulfated carbohydrates belonging to the glycosaminoglycan family, such as heparan sulfate.

Genome analysis revealed a large gene repertoire (122) of carbohydrate-active enzyme (CAZymes) modules encompassed in 112 genes. Strikingly, this gene repertoire is approximately two times larger than in representative strains of pathogenic species *F. psychrophilum* (56) and *F. columnare* (63) (Table 2). Overrepresentation is even more obvious when CAZymes dedicated solely to carbohydrate degradation are taken into account; for example, the *F. collinsii* genome encodes 51 GHs and 9 PLs. In contrast, the *F. psychrophilum* JIP02/86 genome contains 8 GHs and 0 PLs and the *F. columnare* ATCC 49512 genome contains 13 PLs and 3 PLs. The high number of genes encoding CAZymes is a trait shared with the genomes of *F. tructae* and the cluster of putative pathogenic species (i.e., *F. collinsii*, *F. araucananum*, *F. bizetiae*, *F. piscis*, and *F. chilense*). These numbers range from 112 for *F. collinsii* to 238 for *F. piscis* (Table 2), the latter being comparable to the number found in the environmental bacterium *F. johnsoniae* (243). Therefore, data from T9SS-secreted proteins, PUL systems, and CAZymes indicate that *F. collinsii* evolved to take advantage of a large variety of both proteineous and carbohydrate substrates.

**(iii) Antibiotic biosynthesis.** The *F. collinsii* genome contains two large gene clusters (203 kb for TRV642_2161 to -2233 and 54 kb for TRV642_4252 to -4268) encoding nonribosomal peptide/polyketide synthase enzymes likely involved in antibiotic biosynthesis. The genomes of *F. tructae* and *F. johnsoniae* also contain antibiotic biosynthesis gene clusters, whereas those of *F. psychrophilum* and *F. columnare* are devoid of these loci, as well as of other antibiotic biosynthesis-encoding genes.

**Virulence assessment in rainbow trout.** With the increasing number of *Flavobacterium* species isolated from diseased fish worldwide (31), a better characterization of their lifestyle and their impact on fish health is needed. Within the time frame of this study, a novel *Flavobacterium* species named *F. bernardetii* was described after the recovery of two isolates from diseased rainbow trout exhibiting neurological symptoms (6). Together with *F. collinsii*, this species was proposed as a possible pathogen, although pathogenicity was not properly validated using experimental infection.

In an attempt to evaluate the virulence of *F. collinsii* TRV642 and *F. bernardetii* F-372T in rainbow trout, groups of fish from Sy (trial 1) and Aut (trial 2) INRAE reference lines were experimentally infected by intramuscular injection and maintained in flow water at 10°C for assessment of symptoms and mortality. In this experimental infection model, the positive-control strain FRGDSA 1881/11, belonging to the pathogenic species *F. psychrophilum*, was responsible for high mortality, with a 50% lethal dose (LD₅₀) of 4.9 × 10² CFU (trial 1 [Fig. 2A]).

In trial 1, only one fish injected with the median infectious dose of *F. bernardetii* F-372T (7.9 × 10³ CFU; Sy line) died at 12 days postinfection (dpi) (Fig. 2B); the fish had no symptoms and internal organs, including the brain, showed no trace of *F. bernardetii*. In trial 2, the experiment was repeated by injecting a group of 10 fish with the highest dose (1.3 × 10⁷ CFU; Aut line), and no mortality occurred. At 20 dpi, internal organs (spleen, liver, kidney and brain) were sampled and streaked on TYES agar (TYESA). No bacterial growth occurred from any organ/fish, indicating that all fish had eliminated
<table><caption>TABLE 2 Number of CAZyme modules encoded by *Flavobacterium* species</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Strain</th><th rowspan="2">Environment(s)</th><th colspan="8">No. of:</th><th rowspan="2">Total modules</th><th rowspan="2">Total genes</th></tr><tr><th>Genome size (Mbp)</th><th>Glycoside hydrolases</th><th>Glycosyl transferases</th><th>Polysaccharide lyases modules</th><th>Carbohydrate esterases</th><th>Carbohydrate-binding modules</th><th>20</th><th>243</th></tr></thead><tbody><tr><td>Flavobacterium johnsoniae</td><td>UW101</td><td>Freshwater, soil</td><td>6.09</td><td>157</td><td>57</td><td>12</td><td>13</td><td>20</td><td>259</td><td>56</td><td></td></tr><tr><td>Flavobacterium psychrophilum</td><td>JIP02/86</td><td>Pathogenic essentially for Salmonidae</td><td>2.86</td><td>8</td><td>44</td><td>0</td><td>4</td><td>2</td><td>58</td><td></td><td></td></tr><tr><td>Flavobacterium branchiophilum</td><td>FL-15</td><td>Pathogenic essentially for Salmonidae</td><td>3.55</td><td>26</td><td>52</td><td>2</td><td>5</td><td>4</td><td>89</td><td>85</td><td></td></tr><tr><td>Flavobacterium columnare</td><td>ATCC 49512</td><td>Pathogenic for many freshwater fish species</td><td>3.16</td><td>13</td><td>42</td><td>3</td><td>4</td><td>3</td><td>65</td><td>63</td><td></td></tr><tr><td>Flavobacterium collinii</td><td>TRV642</td><td>Putative pathogen</td><td>5.55</td><td>51</td><td>48</td><td>9</td><td>7</td><td>7</td><td>122</td><td>112</td><td></td></tr><tr><td>Flavobacterium chilense</td><td>DSM 24724</td><td>Putative pathogen</td><td>6.11</td><td>124</td><td>45</td><td>14</td><td>11</td><td>22</td><td>216</td><td>198</td><td></td></tr><tr><td>Flavobacterium tructae</td><td>MSU</td><td>Putative pathogen</td><td>5.35</td><td>50</td><td>50</td><td>7</td><td>7</td><td>7</td><td>121</td><td>113</td><td></td></tr><tr><td>Flavobacterium piscis</td><td>CCUG 60099</td><td>Putative pathogen</td><td>5.9</td><td>162</td><td>50</td><td>10</td><td>12</td><td>17</td><td>251</td><td>238</td><td></td></tr><tr><td>Flavobacterium bizetiae</td><td>CIP 105534</td><td>Putative pathogen</td><td>5.87</td><td>149</td><td>50</td><td>23</td><td>13</td><td>17</td><td>252</td><td>222</td><td></td></tr><tr><td>Flavobacterium araucanum</td><td>DSM 24704</td><td>Putative pathogen</td><td>6.01</td><td>108</td><td>48</td><td>7</td><td>8</td><td>20</td><td>191</td><td>174</td><td></td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>D.</figcaption></figure>

<figure><img src="image_7.png" /></figure>

**FIG 2** Evaluation of the virulence in rainbow trout. (A and B) Kaplan-Meier survival curves of rainbow trout fingerling (trial 1; Sy line; 6 to 8 g) after intramuscular injection with *F. psychrophilum* FRGDSA 1882/11 (A) and *F. bernardetii* F-372T (B) bacterial suspensions with doses ranging over 6 orders of magnitude. TYES broth was used as a negative control. (C) Rainbow trout fingerling (trial 1; Sy line) infected with *F. collinsii* TRV642 and attacked by dominant fish display damaged caudal fin and tissue. (D) Bacterial loads in internal organs in rainbow trout fingerling (trial 2; Aut line; 5 g) after intramuscular injection with *F. collinsii* TRV642 using a high infectious dose (7 × 10⁶ CFU). Filled circles, diseased fish euthanized at 13 dpi; open circles, survivor fish sampled at 20 dpi. Colors indicate *F. collinsii*-positive samples retrieved from same fish. The dotted line indicates the bacterial detection threshold.

injected bacteria. These results suggest that this species is nonpathogenic for rainbow trout under the conditions tested.

In trial 1, one fish injected with the highest dose (1.9 × 10⁶ CFU; Sy line) of *F. collinsii* TRV642 died at 12 dpi and the bacterium was successfully recovered from internal organs of this fish, including spleen, kidney, and liver. We also examined at the end of the experiment internal organs of 13 other fish and observed striking differences linked to the fish status. Indeed, starting from 9 dpi, it was noticed in several tanks that dominant fish of bigger size were attacking smaller fish. This behavior can be observed when fish are maintained in small groups, independently of any challenge. The attacked fish then showed heavily damaged caudal fin and caudal tissue erosion (Fig. 2C), leading to swimming defects. For ethical reasons, the dominant fish was euthanized starting from 14 dpi and the monitoring of mortality was ceased. In some tanks, another fish rapidly became dominant and continued to attack weaker fish. Between 14 and 20 dpi, dominant and attacked fish were euthanized and internal organs were sampled and streaked on TYESA. Out of 8 attacked fish, *F. collinsii* was successfully recovered from internal organs of 7. In sharp contrast, none of the 5 examined dominant fish was positive for *F. collinsii*.

In order to evaluate the possible link between dominance/subordinate status and disease development, an additional experiment was conducted in trial 2. Three groups of 10 fish (Aut line) were infected by injection with a high infectious dose (7 × 10⁶ CFU; 2 groups) or with sterile TYES broth (control group; 1 group). One fish from the infected groups developed injection site necrosis from 7 dpi and was euthanized at 13 dpi to respect humane endpoints. Bacterial examination confirmed the presence of *F. collinsii* in the external lesion, and bacterial loads were >8 × 10² CFU in internal organs (Fig. 2D). No additional mortality occurred. Three dominant fish were observed in the control group, but not in the duplicated infected group. At 20 dpi, all fish of the
infected groups were euthanized and internal organs were sampled for bacterial load determination. *F. collinsii* was detected in at least one organ for 9 fish out of 19. Bacterial loads were lower than observed for the diseased fish (Fig. 2D). Taken together, these results indicate that under our experimental conditions, *F. collinsii* TRV642 has very low virulence but is able to survive at low level in internal organs and to produce disease in individuals with compromised conditions such as stress and/or wounds.

DISCUSSION

Since its thorough 1996 revision on the basis of 16S rDNA gene sequencing (10), the genus *Flavobacterium* has considerably expanded as a result of the growing number of worldwide sampling campaigns (https://lpsn.dsmz.de/search?word=flavobacterium). Most of the *Flavobacterium* species described so far are *a priori* nonpathogenic, environmental isolates, retrieved from a wide diversity of sources (e.g., freshwater and salt water, soil, and rhizosphere). However, a number of newly described species have also been described from fish or fish farm environments, which raised questions on whether some could cause disease (31) like *F. columnare* and *F. psychrophilum*: *F. columnare* is the causative agent of columnaris disease, which affects a large variety of freshwater fish species generally reared at relatively warm temperatures, such as catfish, tilapia, and ornamental fish (32); *F. psy- chrophilum* causes rainbow trout fry syndrome and bacterial cold-water disease, affecting predominantly salmonid reared in cold freshwater. To a lesser extent, bacterial gill disease elicited by *F. branchiophilum* also affects a number of cultured fish species throughout the world (33). However, the last species is considered by some authors an opportunistic pathogen, arising under suboptimal environmental conditions (16). In addition to these species, many *Flavobacterium* species retrieved from diseased fish have been described. A large number of uncharacterized fish-associated *Flavobacterium* spp. were also identified during an 8-year follow-up study of the Laurentian Great Lakes in Michigan (34). However, although these bacteria were indeed retrieved from diseased fish, it was unclear whether the symptoms could actually be attributed to the bacteria that were recovered from the infected fish.

There is a serious lack of studies aiming to demonstrate bacterial virulence using ex-
perimental infection models to fulfill Koch's postulates. It is also unclear which bacteria
are true pathogens and which are opportunistic pathogens or even only secondary colo-
nizers, i.e., primarily saprophytic or commensal bacteria able to invade the host only
when its defenses are compromised (e.g., by wounds, stress, or disease). This information
is crucial for appropriate management measures to mitigate disease emergence and
spreading. Here, we report the isolation of *F. collinsii* strain TRV642 from the spleen of a
dead rainbow trout raised in an INRAE experimental fish facility. The dead trout did not
display any disease symptoms and belonged to a highly *F. psychrophilum*-susceptible
A36 isogenic line that should be considered outstandingly fragile (19).

Whole-genome assembly revealed limited differences in genome size between iso-
late TRV642 of *F. collinsii* and the type strain, CECT 7796. Strikingly, these genome sizes
are far above the mean (4.05 Mbp) and median (3.80 Mbp) genome sizes in the genus
*Flavobacterium*. With 5.74 Mbp, strain CECT 7796T possesses the 15th largest genome
out of 195 available for the genus (Data Set S1). On the other hand, the two unquestion-
ably fish-pathogenic species *F. psychrophilum* and *F. columnare* (as well as its newly
described sister species *F. covaë*, *F. davisii*, and *F. oreochromis*) possess rather compact,
about half smaller, genomes. The presumptively opportunistic pathogen *F. branchiophi-
lum* also harbors a relatively small genome of about 3.6 Mbp. The genome of *F. collinsii*
is in the same range of size as environmental members of the genus, which suggests an
extended ecological niche. Indeed, bacteria living in habitats with diversified nutrient
supply tend to have larger genomes and target more complex substrates (35), whereas
pathogenicity is generally associated with reduced genome size (36).

The vast majority of the *Flavobacterium* species harboring large genomes are
grouped in the phylogenetic tree reconstructed on the alignment of the core genome
(Fig. 1), suggesting a common ancestor with a large genome. Since these species were
retrieved from a variety of ecological niches (e.g., seawater, freshwater, and soil), it is
difficult to formulate a hypothesis for the habitat of this ancestor. Nevertheless, inside or
outside this group some subgroups of bacteria isolated from the same type of habitat
were identified. In particular, *F. collinsii* tightly clusters with *F. tructae* and several other
species (i.e., *F. chilense*, *F. piscis*, *F. bizetiae*, and *F. araucananum*) considered putative fish
pathogens because of their isolation from diseased fish. This common source of isolation
suggests evolutionarily conserved characteristics. Similarly, a number of species from
cold environments, including *F. glaciei*, grouped together elsewhere in the tree.

It is noteworthy that the picture for the habitat of the different species may be
blurred by the very limited number of isolates reported for the vast majority of the
considered species, which leads us to base our classification essentially on the origin of
the type strain. Confining a species to a single habitat may be an oversimplifying hy-
pothesis, especially in the case of the heterotrophic bacteria of the *Flavobacterium* ge-
nus. For instance, in 2013, Loch et al. (34) described 32 clusters of isolates belonging to
the genus *Flavobacterium* recovered from diseased as well as apparently healthy wild,
feral, and farmed fish in Michigan, many of which—most likely representing novel spe-
cies—displayed significant similarities to environmental species such as *F. hercynium*,
*F. pectinovorum*, and *F. frigidimaris*. Furthermore, a 2015 study reported that farmed
freshwater fish carried *F. suncheonense*, *F. indicum*, *F. aquaticum*, *F. granuli*, *F. hercy-
nium*, and *F. terrae*, all previously described as environmental species (37). These stud-
ies highlight the extreme diversity of *Flavobacterium* species retrieved from fish and
the difficulty-to-identify pathogens.

In line with its large genome, *F. collinsii* TRV642 possesses a broad gene repertoire encompassing 29 PUL likely dedicated to the harvesting of nutrients from the extracellular environment. In addition, the diversity of T9SS cargo proteins, including peptidases and CAZymes, together with other enzymes dedicated to carbohydrate gathering and breakdown, suggests metabolic versatility allowing the bacterium to utilize a large spectrum of carbon sources. Indeed, numerous copies of PUL were identified in environmental species of the family *Flavobacteriaceae*, especially those that are able to utilize a large variety of nutrients sources (e.g., 20 *susCD*-like pairs in *Gramella forsetii* KT0803, 42 in *F. johnsoniae* UW101, and 71 in *Zobellia galactanivorans* Dsij), whereas very few pairs were identified in the bona fide fish-pathogenic species (e.g., only 1 *susCD*-like pair in *F. psychrophilum* JIP02/86, 2 in *F. columnare* ATCC 23463T, and 6 in *Tenacibaculum maritimum* NCIMB 2154T). With reference to the Brillat-Savarin's aphorism "Tell me what you eat, and I will tell you what you are," one should consider that *F. collinsii* and probably most, if not all, of the aforementioned species occasionally retrieved from fish and harboring large genomes are very likely versatile in their diets. As a result, one might suggest that, as reported for others *Bacteroidota*, these bacteria are associated with niches containing diverse nutrient sources, including eukaryotic organisms, with whom they may have evolved a range of relationships potentially from symbiotic, mutualistic, or commensal to pathogenic interactions. The presence of antibiotic biosynthesis gene clusters in the *F. collinsii* genome was also reported for some mutualistic or commensal bacteria, where they are used to fight other competitors occupying the same niche (38), a phenomenon participating in competitive exclusion.

Using a rainbow trout experimental infection model based on intramuscular injec-
tion, which is generally much more efficient than the immersion model since it
bypasses natural protection barriers of mucus and skin, neither mortality nor symp-
toms were observed using the highest dose (1.3 × 10⁷ CFU) of *F. bernardetii* F-372T. For
*F. collinsii* TRV642, the bacterium produced disease in only 2 fish out of 30 at a high
dose (>10⁶ CFU) but was recovered from internal organs (spleen, kidney, and liver) for
half of the sampled survivors, including asymptomatic and attacked fish. It is also note-
worthy that *F. collinsii* TRV642 was originally isolated from a highly *F. psychrophilum*
susceptible A36 isogenic rainbow trout line. These results suggest that when *F. collinsii*
is able to invade internal tissues, likely through skin lesions, bacterial cells partly resist
clearance by the immune system and persist inside the host at a low level. Some fish
may be at higher risk of *F. collinsii* infection development due to unknown factors such as host genotype, environmental parameters, coinfections, stresses, and wounds.

In contrast to *F. bernardetii* and *F. collinsii*, experimental infection with *F. psychrophilum* of the Sy rainbow trout line resulted in high mortality: the median lethal doses (LD₅₀s) ranged from ~10⁵ CFU for strains with moderate virulence, such as OSU THCO2-90 (39), to <10³ CFU for highly virulent strains as documented for FRGDSA 1882/11 in this study. However, we cannot exclude that different rainbow trout genetic backgrounds, rearing conditions, bacterial growth conditions, or even other *F. bernardetii* and *F. collinsii* isolates could actually produce severe disease.

Many members of the family *Flavobacteriaceae*, and more generally of the phylum *Bacteroidota*, are normal constituents of the host microbiota. However, some of their traits could be considered “dual-use” virulence traits because, while serving important functions for survival in the environment, they can also function as virulence factors (40). Bacteria encompassing these dual-use virulence traits can rapidly proliferate to degrade and metabolize host macromolecules under certain conditions, as recently reported for *Bacteroidota* causing opportunistic diseases in marine eukaryotes (41).

Taken together, our results suggest that some recently described *Flavobacterium* species isolated from fish are likely saprophytic or commensal bacteria that may behave as opportunistic pathogens able to proliferate on decaying tissue to exploit the available nutrients, causing disease under specific circumstances. Indeed, commercially reared fish are subjected to intensive farming practices resulting in welfare issues associated with high stocking density and handling processes that can result in trauma, providing favorable conditions for bacteria to thrive.

## MATERIALS AND METHODS

**Bacterial strains and growth conditions.** Bacteria were grown on tryptone-yeast extract-salt (TYES) broth (0.4% [wt/vol] tryptone, 0.04% [wt/vol] yeast extract, 0.05% [wt/vol] MgSO₄·7H₂O, 0.02% [wt/vol] CaCl₂·2H₂O, 0.05% [wt/vol] D-glucose [pH 7.2]) or on TYES agar (TYESA) supplemented with 5% fetal bovine serum for 4 days at 18°C. Precultures were prepared using a single colony in TYES broth and grown overnight at 18°C and 200 rpm. Cultures were prepared by dilution of stationary preculture and grown overnight to the early stationary phase (optical density at 600 nm [OD₆₀₀] of 1.0 ± 0.1) for infection challenges. Cultures were 10-fold serially diluted in 1% [wt/vol] peptone water, and CFU were counted *a posteriori* after 48 to 72 h of incubation at 18°C on TYESA. For long-term storage, stationary-phase bacterial cultures were stored at -80°C with 20% (vol/vol) glycerol.

**PCR amplification and 16S rRNA gene sequencing.** PCR amplification was performed using a universal bacterial 16S rRNA gene primer set composed of forward (27F; 5'-AGAGTTTGATCMTGGCTCAG-3') and reverse (1492R; 5'-TACGGYTACCTGTTACGACTT-3') primers. The PCR mixture contained final concentrations of 0.3 µM for each primer, 1.25 U of Dream Taq DNA polymerase, 1× buffer (Thermo Fisher Scientific), 0.3 mM deoxynucleoside triphosphates (dNTPs), and 1.5 µL of bacterial DNA prepared by heat lysis (at 99°C for 10 min) of stationary-phase culture. The volume was adjusted to 50 µL with double-distilled water (ddH₂O), and PCR was performed with initial denaturation at 95°C for 5 min followed by 30 cycles of amplification (95°C for 20 s for denaturation, 48°C for 20 s for annealing, and 72°C for 2 min for extension) and finished by extension at 72°C for 10 min. PCR amplicons were sequenced by Sanger sequencing. A specific *F. collinsii* PCR detection, targeting FLACOL7796_04448 CDS (UniProt no. A0A6J4GVB1), was developed using primers TRO1016 (5'-TTTCATGACGCATTGCTGCC-3') and TRO1017 (5'-AAAATTTTCCCGGCACACG-3'). The PCR mixture was prepared as for the 16S rRNA gene, and PCR was performed with initial denaturation at 95°C for 5 min, 30 cycles of amplification (95°C for 30 s for denaturation, 52°C for 30 s for annealing, and 72°C for 1 min for extension), and final extension at 72°C for 10 min. A positive *F. collinsii* PCR amplification provided a 479-bp band.

**Rainbow trout experimental infections.** Two trials were conducted using rainbow trout reference INRAE lines (42): Sy line (trial 1) and Aut line (trial 2). Fish were reared at 10°C in a recirculating aquaculture system (RAS) in 30-L tanks with dechlorinated tap water. Two days before infection, fish were transferred to the biosafety level 2 (BSL2) zone in 15-L tanks with flow water (1 renewal per hour). Experiments were carried out for *F. bernardetii* F-372T, *F. collinsii* TRV642, and *F. psychrophilum* FRGDSA 1882/11 using rainbow trout fingerlings (weight, 5 to 8 g).

**(i) Trial 1.** Experimental infections with *F. bernardetii* F-372T and *F. collinsii* TRV642 were performed using 7 groups of 10 fish (Sy line) for each bacterial species. In each challenge, one group of 10 fish was injected with sterile TYES broth as a negative control. Six groups were challenged at different doses, as follows: 6 suspensions were prepared by 10-fold serial dilutions (10⁻¹ to 10⁻⁶) of a bacterial culture, and then 50 µL of each suspension was administered per fish by intramuscular injection at a point midway between the insertion of the dorsal fin and the lateral line following anesthesia (50 mg/L; MS222; Sigma-Aldrich). For *F. psychrophilum*, only 5 groups corresponding to 10⁻³ to 10⁻⁷ dilutions were used and the
median lethal dose was estimated using the moving average and interpolation method described by
Thompson (43), with 3 dose groups used to calculate each moving average.

(ii) **Trial 2.** Experimental infections with *F. bernardetii* and *F. collinsii* were repeated independently using the rainbow trout Aut line and the highest dose using 4 groups of 10 fish injected with sterile TYES broth (1 replicate; negative control), *F. collinsii* TRV642 at 10⁻¹ dilution (2 replicates), or *F. bernardetii* F-372T at 10⁻¹ dilution (1 replicate). All fish were maintained using flow water at 10°C. Mortality was recorded twice a day, and fish were monitored for clinical signs and behavioral changes using a scoring grid throughout the challenge. For each tank, 5-min periods of direct observations performed before feeding were used to detect the presence of dominant and subordinate fish and to record individuals exhibiting external signs, including skin lesions and fin erosion. Dominance was recorded based on aggressive behavior, such as unidirectional sharp movement directed toward another fish, associated with biting.

Euthanasia was carried out by bathing fish in tricaine at concentration of 300 mg/L (MS222; Sigma-Aldrich). Dead or euthanized fish were examined for the presence of bacteria in organs including the spleen, kidney, and liver. Organs were homogenized in peptone water containing silica spheres using a FastPrep instrument (MP Biomedicals) at 6 m/s for 20 s. Tissue homogenates were streaked on TYESA and kept for 3 to 5 days at 18°C before examination for bacterial growth. The identification of *F. collinsii* colonies was performed by specific PCR detection.

**Ethics statement.** Animal experiments and sampling were performed in accordance with European Directive 2010/2063/UE. All animal work was approved by the Direction of the Veterinary Services of Versailles, France (authorization number C78-720), and by the ethics committee of the INRAE Center in Jouy-en-Josas (COMETHEA no. 45), France (authorization no. 2015100215242446).

**Sequencing and annotation of the *F. collinsii* genome.** Genomic DNA (gDNA) was extracted from stationary-phase broth culture using a genomic DNA-Tip 100/G system and buffer set (Qiagen) following the manufacturer's instruction. For short-read sequencing, the library was constructed using a TruSeq genomic kit (Illumina) and paired-end sequenced on a NextSeq instrument using a NextSeq 500/550 mid-output kit v2 (Illumina). In parallel, gDNA was sequenced on GridION (Oxford Nanopore) using a FLO-MIN106 flow cell for long-read sequencing. Hybrid genome assembly was performed using Unicycler v0.4.8, available on the PATRIC platform, with default parameters (44). Genome annotation and comparison were performed using the MicroScope platform (45). InterProScan was used to identify SusCD pairs using IPR023996 and IPR023997 for SusC and IPR012944, IPR024302, IPR041662, and IPR033985 for SusD with colocalization criteria. Proteins secreted by the T9SS harboring conserved C-terminal domains (CTDs) were identified using IPR026444 and IPR026341 for type A and B CTDs (46), respectively. Carbohydrate-active enzymes (CAZymes) were identified using dbCAN, which combines three tools; hits found by only one tool were removed to improve accuracy (47). Peptidases were identified using the MEROPS database (https://www.ebi.ac.uk/merops/) (48).

**Phylogenetic analysis.** Average nucleotide identities (ANIs) were computed using OrthoANIu and the calculator available at http://www.ezbiocloud.net/tools/ani (49).

Genome records were retrieved from the RefSeq database (https://www.ncbi.nlm.nih.gov/datasets/genomes/?taxon=237&utm_source=data-hub [accessed April 2022]) (50).

Proteomes were compared pairwise with BLASTP (v2.12, low-complexity filter disactivated, E value cut-off of 1e-5, otherwise default settings) (51) and the results served for a single linkage clustering of the genes based on a criterion of 45% amino acid sequence identity over 70% of the gene length. Conserved single-copy genes were identified as clusters with a single representative in each genome and their sequences were subjected to multiple-sequence alignment using Muscle (v3.8.31, default setting) (52).

Neighbor-joining phylogenetic tree reconstruction was conducted using FastTree (v2.1.10, default settings) (53) on the concatenated multiple-sequence alignments, after alignment gap removal. Statistical support of the tree topology was assessed by comparison with trees reconstructed on 400 bootstrap replicates of the original alignment.

**Data availability.** The annotated TRV642 genome sequence has been deposited at ENA under accession number OX336425.1.

## SUPPLEMENTAL MATERIAL

Supplemental material is available online only.

**SUPPLEMENTAL FILE 1**, PDF file, 0.1 MB.

**SUPPLEMENTAL FILE 2**, XLSX file, 0.1 MB.

**SUPPLEMENTAL FILE 3**, XLSX file, 0.04 MB.

**SUPPLEMENTAL FILE 4**, XLSX file, 0.04 MB.

## ACKNOWLEDGMENTS

This work was financially supported by the Agence Nationale de la Recherche (grant ANR-17-CE20-0020-01 FlavoPatho) and by institutional support from INRAE.

We are grateful to the staff of the fish facilities (IERP, INRAE, Infectiology of Fishes and Rodent Facility [https://www6.jouy.inrae.fr/ierp/) and PEIMA, INRAE, Fish Farming Systems Experimental Facility [https://www6.reennes.inrae.fr/peima/]) for supplying fish and for technical assistance and advice. We are grateful to the facilities and for the expertise of the high-throughput-sequencing platform of IMAGIF (http://www.i2bc.paris-saclay.fr/), the
INRAE MIGALE bioinformatics facility (https://migale.inrae.fr/), and the MicroScope annotation platform, and we are grateful to the National Infrastructure "France Génomique" for providing computational resources.

We declare no conflict of interest.

## REFERENCES

1. McBride MJ. 2014. The family *Flavobacteriaceae*, p 643–676. In Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (ed), The prokaryotes: other major lineages of bacteria and the archaea. Springer, Berlin, Germany.

2. Thomas F, Hehemann J-H, Rebuffet E, Czjzek M, Michel G. 2011. Environmental and gut Bacteroidetes: the food connection. Front Microbiol 2:93. https://doi.org/10.3389/fmicb.2011.00093.

3. McBride MJ. 2019. Bacteroidetes gliding motility and the type IX secretion system. Microbiol Spectr 7:7.1.15. https://doi.org/10.1128/microbiolspec.PSIB-0002-2018.

4. LaFrentz BR, Králová S, Burbick CR, Alexander TL, Phillips CW, Griffin MJ, Waldbieser GC, García JC, de Alexandre Sebastião F, Soto E, Loch TP, Liles MR, Snevik KR. 2022. The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare. Syst Appl Microbiol 45:126293. https://doi.org/10.1016/j.syapm.2021.126293.

5. Kämpfer P, Lodders N, Martin K, Avendaño-Herrera R. 2012. Flavobacterium chilense sp. nov. and Flavobacterium araucanum sp. nov., isolated from farmed salmonid fish. Int J Syst Evol Microbiol 62:1402–1408. https://doi.org/10.1099/ijs.0.033431-0.

6. Saticioglu IB, Ay H, Altun S, Sahin N, Duman M. 2021. Flavobacterium bernardotii sp. nov., a possible emerging pathogen of farmed rainbow trout (Oncorhynchus mykiss) in cold water. Aquaculture 540:736717. https://doi.org/10.1016/j.aquaculture.2021.736717.

7. Saticioglu IB, Ay H, Altun S, Duman M, Sahin N. 2021. Flavobacterium turcicum sp. nov. and Flavobacterium kayseriense sp. nov. isolated from farmed rainbow trout in Turkey. Syst Appl Microbiol 44:126186. https://doi.org/10.1016/j.syapm.2021.126186.

8. Zamora L, Vela AI, Sánchez-Porro C, Palacios MA, Domínguez L, Moore ERB, Ventosa A, Fernández-Garayzábal JF. 2013. Characterization of flavobacteria possibly associated with fish and fish farm environment. Description of three novel flavobacterium species: Flavobacterium collinsii sp. nov., Flavobacterium branchiarium sp. nov., and Flavobacterium branchicola sp. nov. Aquaculture 416–417:346–353.

9. Strohl WR, Tait LR. 1978. Cytophaga aquatilis sp. nov., a facultative anaerobe isolated from the gills of freshwater fish. Int J Syst Evol Microbiol 28: 293–303.

10. Bernardet J-F, Segers P, Vancanneyt M, Berthe F, Kersters K, Vandamme P. 1996. Cutting a Gordian Knot: emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (basonym, Cytophaga aquatilis Strohl and Tait 1978). Int J Syst Evol Microbiol 46: 128–148.

11. de Alexandre Sebastião F, LaFrentz BR, Shelley JP, Stevens B, Marancik D, Dunker F, Reavill D, Soto E. 2019. Flavobacterium inkyongense isolated from ornamental cichlids. J Fish Dis 42:1309–1313. https://doi.org/10.1111/jfd.13043.

12. Flemming L, Rawlings D, Chenia H. 2007. Phenotypic and molecular characterization of fish-borne Flavobacterium johnsoniae-like isolates from aquaculture systems in South Africa. Res Microbiol 158:18–30. https://doi.org/10.1016/j.resmic.2006.09.001.

13. Zamora L, Fernández-Garayzábal JF, Svensson-Stadler LA, Palacios MA, Domínguez L, Moore ERB, Vela AI. 2012. Flavobacterium oncorhynchus sp. nov., a new species isolated from rainbow trout (Oncorhynchus mykiss). Syst Appl Microbiol 35:86–91. https://doi.org/10.1016/j.syapm.2011.11.007.

14. Zamora L, Vela AI, Sánchez-Porro C, Palacios MA, Moore ERB, Domínguez L, Ventosa A, Fernández-Garayzábal JF. 2014. Flavobacterium tructae sp. nov. and Flavobacterium piscis sp. nov., isolated from farmed rainbow trout (Oncorhynchus mykiss). Int J Syst Evol Microbiol 64:392–399.

15. Zamora L, Fernández-Garayzábal JF, Sánchez-Porro C, Palacios MA, Moore ERB, Domínguez L, Ventosa A, Vela AI. 2013. Flavobacterium plurextorum sp. nov. isolated from farmed rainbow trout (Oncorhynchus mykiss). PLoS One 8:e67741. https://doi.org/10.1371/journal.pone.0067741.

16. Good C, Davidson J, Wiens GD, Welch TJ, Summerfelt S. 2015. Flavobacterium branchiophilum and F. succinicans associated with bacterial gill disease in rainbow trout Oncorhynchus mykiss (Walbaum) in water recirculation aquaculture systems. J Fish Dis 38:409–413. https://doi.org/10.1111/jfd.12249.

17. Loch TP, Faisal M. 2014. Flavobacterium spartansii sp. nov., a pathogen of fishes, and emended descriptions of Flavobacterium aquidurense and Flavobacterium araucanum. Int J Syst Evol Microbiol 64:406–412. https://doi.org/10.1099/ijs.0.051433-0.

18. Loch TP, Faisal M. 2016. Flavobacterium spartansii induces pathological changes and mortality in experimentally challenged Chinook salmon Oncorhynchus tshawytscha (Walbaum). J Fish Dis 39:483–488. https://doi.org/10.1111/jfd.12363.

19. Fraslin C, Quillet E, Rochat T, Dechamp N, Bernardet J-F, Collet B, Lallias D, Boudinot P. 2020. Combining multiple approaches and models to dissect the genetic architecture of resistance to infections in fish. Front Genet 11: 677. https://doi.org/10.3389/fgene.2020.00677.

20. Richter M, Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci U S A 106:19126–19131. https://doi.org/10.1073/pnas.0906412106.

21. Lin SY, Chen WM, Huang GH, Hameed A, Chang CT, Tsai CF, Young CC. 2020. Flavobacterium supellecticarium sp. nov., isolated from an abandoned construction timber. Int J Syst Evol Microbiol 70:3731–3739. https://doi.org/10.1099/ijsem.0.004227.

22. Mühle E, Abry C, Leclerc P, Goly G-M, Criscuolo A, Busse H-J, Kämpfer P, Bernardet J-F, Clermont D, Chesneau O. 2021. Flavobacterium bizetiae sp. nov., isolated from diseased freshwater fish in Canada at the end of the 1970s. Int J Syst Evol Microbiol 71:e004576. https://doi.org/10.1099/ijsem.0004576.

23. McBride MJ, Xie G, Martens EC, Lapidus A, Henrissat B, Rhodes RG, Goltsman E, Wang W, Xu J, Hunnicutt DW, Staroscik AM, Hoover TR, Cheng Y-Q, Stein JL. 2009. Novel features of the polysaccharide-digesting gliding bacterium Flavobacterium johnsoniae as revealed by genome sequence analysis. Appl Environ Microbiol 75:6864–6875. https://doi.org/10.1128/AEM.01495-09.

24. Duchaud E, Rochat T, Habib C, Barbier P, Loux V, Guérin C, Dalsgaard I, Madsen L, Nilsen H, Sundell K, Wiklund T, Strepparava N, Wahli T, Caburlotto G, Manfrin A, Wiens GD, Fujiwara-Nagata E, Avendaño-Herrera R, Bernardet J-F, Nicolas P. 2018. Genomic diversity and evolution of the fish pathogen Flavobacterium psychrophilum. Front Microbiol 9:138. https://doi.org/10.3389/fmicb.2018.00138.

25. Li YG, Hu B, Christie PJ. 2019. Biological and structural diversity of type IV secretion systems. Microbiol Spectr 7:7.2.30. https://doi.org/10.1128/microbiolspec.PSIB-0012-2018.

26. Pollet RM, Martin LM, Koropatkin NM. 2021. TonB-dependent transporters in the Bacteroidetes: unique domain structures and potential functions. Mol Microbiol 115:490–501. https://doi.org/10.1111/mmi.14683.

27. Terrapon N, Lombard V, Gilbert HJ, Henrissat B. 2015. Automatic prediction of polysaccharide utilization loci in Bacteroidetes species. Bioinformatics 31:647–655. https://doi.org/10.1093/bioinformatics/btu716.

28. Glenwright AJ, Pothula KR, Bhamidimarri SP, Chorev DS, Baslé A, Firbank SJ, Zheng H, Robinson CV, Winterhalter M, Kleinekathöfer U, Bolam DN, van den Berg B. 2017. Structural basis for nutrient acquisition by dominant members of the human gut microbiota. Nature 541:407–411. https://doi.org/10.1038/nature20828.

29. Madej M, White JBR, Nowakowska Z, Rawson S, Scavenius C, Enghild JJ, Bereta GP, Pothula K, Kleinekathoefer U, Baslé A, Ranson NA, Potempa J, van den Berg B. 2020. Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis. Nat Microbiol 5:1016–1025. https://doi.org/10.1038/s41564-020-0716-y.

30. Su H, Blain F, Musil RA, Zimmermann JJ, Gu K, Bennett DC. 1996. Isolation and expression in Escherichia coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum. Appl Environ Microbiol 62:2723–2734. https://doi.org/10.1128/aem.62.8.2723-2734.1996.
31. Loch TP, Faisal M. 2015. Emerging flavobacterial infections in fish: a review. J Adv Res 6:283-300. https://doi.org/10.1016/j.jare.2014.10.009.

32. Declercq AM, Haesebrouck F, Van den Broeck W, Bossier P, Decostere A. 2013. Columnaris disease in fish: a review with emphasis on bacterium-host interactions. Vet Res 44:27. https://doi.org/10.1186/1297-9716-44-27.

33. Ostland VE, MacPHEE DD, Lumsden JS, Ferguson HW. 1995. Virulence of Flavobacterium branchiophilum in experimentally infected salmonids. J Fish Dis 18:249-262. https://doi.org/10.1111/j.1365-2761.1995.tb00300.x.

34. Loch TP, Fujimoto M, Woodiga SA, Walker ED, Marsh TL, Faisal M. 2013. Diversity of fish-associated flavobacteria of Michigan. J Aquat Anim Health 25:149-164. https://doi.org/10.1080/08997659.2012.758189.

35. Grondin JM, Tamura K, Déjean G, Abbott DW, Brumer H. 2017. Polysaccharide utilization loci: fueling microbial communities. J Bacteriol 199: e00860-16. https://doi.org/10.1128/JB.00860-16.

36. Murray GGR, Charlesworth J, Miller EL, Casey MJ, Lloyd CT, Gottschalk M, Tucker AWD, Welch JJ, Weinert LA. 2021. Genome reduction is associated with bacterial pathogenicity across different scales of temporal and ecological divergence. Mol Biol Evol 38:1570-1579. https://doi.org/10.1093/molbev/msaa323.

37. Verma DK, Rathore G. 2015. New host record of five Flavobacterium species associated with tropical fresh water farmed fishes from North India. Braz J Microbiol 46:969-976. https://doi.org/10.1590/S1517-838246420131081.

38. van der Meij A, Worsley SF, Hutchings MI, van Wezel GP. 2017. Chemical ecology of antibiotic production by actinomycetes. FEMS Microbiol Rev 41:392-416. https://doi.org/10.1093/femsre/fux005.

39. Zhu Y, Lechardeur D, Bernardet J-F, Kerouault B, Guérin C, Rigaudeau D, Nicolas P, Duchaud E, Rochat T. 2022. Two functionally distinct heme/iron transport systems are virulence determinants of the fish pathogen Flavobacterium psychrophilum. Virulence 13:1221-1241. https://doi.org/10.1080/21505594.2022.2101197.

40. Casadevall A, Steenbergen JN, Nosanchuk JD. 2003. 'Ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi—the Cryptococcus neoformans paradigm. Curr Opin Microbiol 6:332-337. https://doi.org/10.1016/S1369-5274(03)00082-1.

41. Hudson J, Egan S. 2022. Opportunistic diseases in marine eukaryotes: could Bacteroidota be the next threat to ocean life? Environ Microbiol 24: 4505-4518. https://doi.org/10.1111/1462-2920.16094.

42. D'Ambrosio J, Phocas F, Haffray P, Bestin A, Brard-Fudulea S, Poncet C, Quillet E, Dechamp N, Fraslin C, Charles M, Dupont-Nivet M. 2019. Genome-wide estimates of genetic diversity, inbreeding and effective size of experimental and commercial rainbow trout lines undergoing selective breeding. Genet Sel Evol 51:26. https://doi.org/10.1186/s12711-019-0468-4.

43. Thompson WR. 1947. Use of moving averages and interpolation to estimate median-effective dose. Bacteriol Rev 11:115-145.

44. Davis JJ, Wattam AR, Aziz RK, Brettin T, Butler R, Butler RM, Chlenski P, Conrad N, Dickerman A, Dietrich EM, Gabbard JL, Gerdes S, Guard A, Kenyon RW, Machi D, Mao C, Murphy-Olson D, Nguyen M, Nordberg EK, Olsen GJ, Olson RD, Overbeek JC, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomas C, VanOeffelen M, Vonstein V, Warren AS, Xia F, Xie D, Yoo H, Stevens R. 2020. The PATRIC Bioinformatics Resource Center: expanding data and analysis capabilities. Nucleic Acids Res 48:D606-D612. https://doi.org/10.1093/nar/gkz943.

45. Vallenet D, Calteau A, Dubois M, Amours P, Bazin A, Beuvin M, Burlot L, Bussell X, Fouteau S, Gautreau G, Lajus A, Langlois J, Planel R, Roche D, Rollin J, Rouy Z, Sabatet V, Médigue C. 2020. MicroScope: an integrated platform for the annotation and exploration of microbial gene functions through genomic, pangenomic and metabolic comparative analysis. Nucleic Acids Res 48:D579-D589. https://doi.org/10.1093/nar/gkz926.

46. Haft DH, Selengut JD, White O. 2003. The TIGRFAMs database of protein families. Nucleic Acids Res 31:371-373. https://doi.org/10.1093/nar/gk128.

47. Huang L, Zhang H, Wu P, Entwistle S, Li X, Yohe T, Yi H, Yang Z, Yin Y. 2018. dbCAN-seq: a database of carbohydrate-active enzyme (CAZyme) sequence and annotation. Nucleic Acids Res 46:D516-D521. https://doi.org/10.1093/nar/gkx894.

48. Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. 2018. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46:D624-D632. https://doi.org/10.1093/nar/gk1134.

49. Yoon S-H, Ha S, Lim J, Kwon S, Chun J. 2017. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281-1286. https://doi.org/10.1007/s10482-017-0844-4.

50. O'Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D, McVeigh R, Rajput B, Robbertse B, Smith-White B, Ako-Adjei D, Astashyn A, Badretdin A, Bao Y, Blinkova O, Brover V, Chetvernin V, Choi J, Cox E, Ermolaeva O, Farrell CM, Goldfarb T, Gupta T, Haft D, Hatcher E, Hlavina W, Joardar VS, Kodali VK, Li W, Maglott D, Masterson P, McGarvey KM, Murphy MR, O'Neill K, Pujar S, Rangwala SH, Rausch D, Riddick LD, Schoch C, Shkeda A, Storz SS, Sun H, Thibaud-Nissen F, Tolstoy I, Tully RE, Vatsan AR, Wallin C, Webb D, Wu W, Landrum MJ, Kimchi A, Tatusova T, DiCuccio M, Kitts P, Murphy TD, Pruitt KD. 2016. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 44:D733-D745. https://doi.org/10.1093/nar/gkv1189.

51. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009. BLAST+: architecture and applications. BMC Bioinformatics 10:421. https://doi.org/10.1186/1471-2105-10-421.

52. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792-1797. https://doi.org/10.1093/nar/gkh340.

53. Price MN, Dehal PS, Arkin AP. 2009. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 26: 1641-1650. https://doi.org/10.1093/molbev/msp077.