ABSTRACT Marine aquaculture sustainability is increasingly threatened by tenacibaculosis, a disease caused by Tenacibaculum species that are thought to persist through biofilm formation. However, the architectural diversity of biofilms across the genus and its contribution to persistence remain unclear. Here, we conducted a large-scale phenotypic analysis of 40 Tenacibaculum isolates including 19 T. maritimum strains, an aquaculture-relevant fish pathogen, and 21 type strains representing different species. Using three complementary growth models: planktonic free-cell cultures, macro-colony biofilms, and submerged biofilms, 17 quantitative parameters were measured. We observed substantial inter- and intra-species heterogeneity. Strains adopted distinct developmental phenotypes, ranging from a high-yield, slow-growth profile associated with thick, structured three-dimensional biofilms to fast-growing communities with limited architectural complexity. Confocal imaging revealed high structural variation across the genus, with Congo red-labeled extracellular materials exhibiting strain-specific differences in porosity and spatial distribution within the biofilm. Multivariate analysis partitioned the collection into four phenotypic clusters defined by distinct growth and structural signatures. Antimicrobial susceptibility assays using oxytetracycline hydrochloride in four representative strains showed that biofilm-associated tolerance was strain-dependent. Together, this work identifies biofilm architecture as a measurable and informative dimension of phenotypic diversification in Tenacibaculum and provides candidate structural markers to support future mechanistic studies and the development of strategies aimed at limiting the persistence of problematic Tenacibaculum species in aquaculture systems. IMPORTANCE The genus Tenacibaculum includes several fish-pathogenic species that pose major concerns in marine aquaculture, yet their biofilm biology remains poorly characterized. By analyzing 40 isolates across complementary growth models, we show that biofilm architecture represents a quantifiable source of phenotypic variation within the genus. Strains adopted various developmental profiles that uncouple the growth rate, biomass yield, and three-dimensional structural complexity. Importantly, experiments performed on four representative strains indicate that biofilm-associated antimicrobial survival is strain-dependent and may be influenced by biofilm structural organization. These findings emphasize the need to account for structure-based functional profiling in addition to species-based classification. By identifying candidate architectural markers and representative phenotypic clusters, this work provides a rational framework for future mechanistic studies and for evaluating whether specific biofilm traits are associated with persistence or risk in aquaculture systems.

ABSTRACT Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in salmonids and other fish, resulting in substantial economic losses in aquaculture worldwide. The mechanisms F. psychrophilum uses to cause disease are poorly understood. Despite considerable effort, most strains of F. psychrophilum have resisted attempts at genetic manipulation. F. psychrophilum restriction-modification (R-M) systems may contribute to this resistance. Restriction endonucleases (REases) rapidly degrade nonself DNA if it is not properly methylated by their cognate DNA methyltransferases (MTases). We used comparative genomics to show that R-M systems are abundant in F. psychrophilum and that strain-specific variations partially align with phylogeny. We identified two critical type II R-M systems, HpaII-like (FpsJI) and ScrFI-like (FpsJVI), that are conserved in most of the sequenced strains. Protection of foreign DNA against HpaII and ScrFI was achieved by expression of the MTases M.FpsJI and M.FpsJVI in Escherichia coli . Furthermore, deleting the two REase genes from F. psychrophilum resulted in efficient conjugative DNA transfer from E. coli into the otherwise genetically intractable F. psychrophilum strain CSF259-93. This allowed us to construct a CSF259-93 mutant lacking gldN , a core component of the type IX protein secretion system. The pre-methylation system developed in this study functions in all tested F. psychrophilum strains harboring HpaII-like and ScrFI-like REases. These newly developed genetic tools may allow the identification of key virulence factors and facilitate the development of live attenuated vaccines or other measures to control BCWD. IMPORTANCE Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is a problem for salmonid aquaculture worldwide, and current control measures are inadequate. An obstacle in understanding and controlling BCWD is that most F. psychrophilum strains resist DNA transfer, thus limiting genetic studies of their virulence mechanisms. F. psychrophilum restriction enzymes that destroy foreign DNA were suspected to contribute to this problem. Here, we used F. psychrophilum DNA methyltransferases to modify and protect foreign DNA from digestion. This allowed efficient conjugative DNA transfer into nine diverse F. psychrophilum strains that had previously resisted DNA transfer. Using this approach, we constructed a gene deletion mutant that failed to cause disease in rainbow trout. Further genetic studies could help determine the molecular factors involved in pathogenesis and may aid development of innovative BCWD control strategies.

BACKGROUND:Bacterial cold-water disease (BCWD), caused by Flavobacterium psychrophilum, remains a major challenge for rainbow trout (Oncorhynchus mykiss) aquaculture, due to the absence of effective vaccines and increasing concerns over antibiotic use. Genetic selection for disease resistance offers a sustainable alternative. In this study, we investigated the genetic architecture of BCWD resistance in two French commercial rainbow trout populations using a standardized waterborne infection model and high-density SNP genotyping. RESULTS:Survival following experimental infection varied significantly between populations, with population B showing higher resistance (71.3% vs. 50.7% of survival at 29 days post-infection). Genome-wide association studies (GWAS) were performed using a Bayesian sparse linear mixed model (BSLMM), separately in each population and in a combined dataset. Eleven quantitative trait loci (QTLs) were identified across the analyses, with limited overlap between populations, highlighting the complexity and partial divergence of resistance architectures. Several candidate genes located within QTL regions were involved in immune signalling, inflammation, macrophages/neutrophils biology, and soluble factors important for antibacterial defences. Notably, two QTLs contained genes from the complement system (e.g., C3, Cfb), highlighting their central role in resistance to F. psychrophilum. CONCLUSIONS:Our findings underscore the polygenic nature of BCWD resistance, the influence of host genetic background, and provide valuable targets for selection for BCWD resistance in rainbow trout breeding programs.

Ten novel Gram-negative, aerobic, non-sporulating, yellow-pigmented rod-shaped bacterial strains motile by gliding were isolated from marine organisms/environments in French Polynesia. Three of them designated as 190524A05cT, 190524A02bT and 190130A14aT were retrieved from orbicular batfish (Platax orbicularis) mucus. Online database comparisons using 16S rRNA amplicons resulted in over 95% similarity to the genus Tenacibaculum. Phylogenetic analyses based on 679 concatenated core protein sequences revealed that strains 190524A05cT, 190524A02bT and 190130A14aT showed the highest similarity to Tenacibaculum skagerrakense DSM 14836T, Tenacibaculum xiamenense LMG 27422T and Tenacibaculum holothuriorum S2-2T, respectively. Digital DNA–DNA hybridization and average nt identity values between strains 190524A05cT, 190524A02bT and 190130A14aT and other type strains were less than 76.25 and 24.1%, respectively. The DNA G+C content was 31.48, 30.66 and 31.98 mol% for strains 190524A05cT, 190524A02bT and 190130A14aT, respectively. Menaquinone-6 was detected as the major isoprenoid quinone in these three strains. The major polar lipids (phosphatidylethanolamine and aminophospholipid) were similar to the chemotaxonomic profile of other species of the genus Tenacibaculum. Strain 190524A05cT contained summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH), iso-C15:1 G, iso-C15:0 and iso-C17:0 3-OH as the major cellular fatty acids. Strain 190524A02bT contained summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH), iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH as the major cellular fatty acids. Strain 190130A14aT contained iso-C15:1 G, summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH), iso-C15:0 and iso-C17:0 3-OH as the major cellular fatty acids. Based on the phenotypic and molecular features, these three strains represent novel species of the genus Tenacibaculum for which the names Tenacibaculum platacis sp. nov., with 190524A05cT (= CIP 112470T = DSM 118113T) as the type strain; Tenacibaculum vairaonense sp. nov., with 190524A02bT (= CIP 112469T = DSM 118112T) as the type strain; and Tenacibaculum polynesiense sp. nov., with 190130A14aT (= CIP 112468T = DSM 118111T) as the type strain, are proposed.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species.

Flavobacterium psychrophilum, a devastating fish pathogen, is responsible for bacterial cold-water disease (BCWD), also known as rainbow trout fry syndrome. F. psychrophilum is the main causative agent of outbreaks in rainbow trout farms, especially at early live stages. In the present study, we aimed to characterize F. psychrophilum Turkish isolates. Eighteen isolates were retrieved from BCWD outbreaks between 2014 and 2021. In vitro phenotypic characterization showed gelatin and casein hydrolysis capacities and in vitro adhesion for all isolates, whereas elastinolytic activity was present for 16 of 18 isolates. We used complete genome sequencing to infer MLST-type, serotype and phylogenetic reconstruction. Strikingly, one strain isolated from Coruh trout (FP-369) belongs to ST393, a previously undescribed ST, and is phylogenetically distant from the other isolates. However, all strains retrieved from rainbow trout belong to the well-characterized clonal complex CC-ST10, 12 of 17 were tightly connected in a single cluster. Several serotypes (Types -1, -2 and -3) were represented among isolates, but no correlation was observed with geographic origins. This analysis suggests a regional dissemination of an epidemic, disease-producing bacterial population. This study provides a basis for epidemiological surveillance of isolates circulating in Turkey and phenotypic data for future molecular studies of virulence traits of this important fish pathogen.

Abstract Te.na.ci.ba'cu.lum. L. adj. tenax ‐acis , holding fast, tenacious; L. neut. n. baculum , stick; N.L. neut. n. Tenacibaculum , rod‐shaped bacterium that adheres to the surface of marine organisms. Bacteroidetes / Flavobacteriia / Flavobacteriales / Flavobacteriaceae / Tenacibaculum Members of the genus Tenacibaculum are exclusively found in marine environments. Most of them are attached to or associated with the surface of marine organisms such as fish, macroalgae, sponges, and invertebrates. However, some strains were isolated from seawater or marine sediments. Several species are important fish pathogens while others are able to degrade complex polysaccharides such as carrageenans or alginate. Rods are 0.1–0.7 μm in diameter and 0.4–40 μm in length. Filamentous cells >100 μm long and degenerative spherical cells may occur in older cultures. Ring‐shaped cells, spores, and gas vesicles are not formed. Cells are nonflagellated. Cells of most, if not all, species are motile by gliding. Gram‐stain‐negative. Cells produce a yellow pigment that is mainly zeaxanthin. Flexirubin‐type pigments are absent. Chemo‐organotrophic and heterotrophic. Aerobic. Catalase‐ and oxidase‐positive. Grow well on media containing seawater. The major respiratory quinone is menaquinone‐6. DNA G + C content (mol%) from the draft or complete genome sequence analysis : 29.6–32.2 (38.4 for Tenacibaculum litopenaei ). Type species : Tenacibaculum maritimum Suzuki et al. 2001 VP (basonyms: “ Flexibacter marinus ” Hikida et al. 1979, Flexibacter maritimus Wakabayashi et al. 1986 VP , “ Cytophaga marina ” Reichenbach 1989).

Strain LLG6346-3.1 T , isolated from the thallus of the brown alga Ericaria zosteroides collected in Mediterranean Sea near Bastia in Corsica, France, was characterized using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, motile by gliding, rod-shaped and grew optimally at 30-33 °C, at pH 8-8.5 and with 4-5 % NaCl. Strain LLG6346-3.1 T used the seaweed polysaccharide alginic acid as sole carbon source which was vigorously liquefied. Phylogenetic analyses showed that the bacterium is affiliated to the genus Zobellia (family Flavobacteriaceae , class Flavobacteriia ). Strain LLG6346-3.1 T exhibited 16S rRNA gene sequence similarity values of 98.5 and 98.3 % to the type strains of Zobellia russellii and Zobellia roscoffensis respectively, and of 97.4-98.2 % to other species of the genus Zobellia . The DNA G+C content of strain LLG6346-3.1 T was determined to be 38.28 mol%. Digital DNA-DNA hybridization predictions by the ANI and GGDC methods between strain LLG6346-3.1 T and other members of the genus Zobellia showed values of 76-88 %, and below 37 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain LLG6346-3.1 T is distinct from species of the genus Zobellia with validly published names and that it represents a novel species of the genus Zobellia , for which the name Zobellia alginoliquefaciens sp. nov. is proposed. The type strain is LLG6346-3.1 T (RCC 7657 T = LLG 32918 T ).

Strain LLG6346-3.1T, isolated from the thallus of the brown alga Ericaria zosteroides collected from the Mediterranean Sea near Bastia in Corsica, France, was characterised using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, motile by gliding, rod-shaped and grew optimally at 30-33 °C, at pH 8-8.5 and with 4-5 % NaCl. LLG6346-3.1T used the seaweed polysaccharide alginic acid as a sole carbon source which was vigorously liquefied. The results of phylogenetic analyses indicated that the bacterium is affiliated to the genus Zobellia (family Flavobacteriaceae, class Flavobacteriia). LLG6346-3.1T exhibited 16S rRNA gene sequence similarity values of 98.6 and 98.3 % to the type strains of Zobellia russellii and Zobellia roscoffensis, respectively, and of 97.4-98.5 % to members of other species of the genus Zobellia. The DNA G+C content of LLG6346-3.1T was determined to be 38.3 mol%. Digital DNA-DNA hybridisation predictions by the average nucleotide identity (ANI) and genome to genome distance calculator (GGDC) methods between LLG6346-3.1T and other members of the genus Zobellia showed values of 76-88 % and below 37 %, respectively. The results of phenotypic, phylogenetic and genomic analyses indicate that LLG6346-3.1T is distinct from species of the genus Zobellia with validly published names and that it represents a novel species of the genus Zobellia, for which the name Zobellia alginiliquefaciens sp. nov. is proposed. The type strain is LLG6346-3.1T (= RCC7657T = LMG 32918T).

Infectious diseases are a major constraint on aquaculture. Genetic lines with different susceptibilities to diseases are useful models to identify resistance mechanisms to pathogens and to improve prophylaxis. Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum represents a major threat for freshwater salmonid farming worldwide. A collection of rainbow trout ( Oncorhynchus mykiss ) isogenic lines was previously produced from a French domestic population. Here, we compared BCWD resistance phenotypes using a subset of isogenic lines chosen for their contrasted susceptibilities to F. psychrophilum . We applied individual monitoring to document the infection process, including time-course quantification of bacteremia and innate immune response. Strikingly, BCWD resistance was correlated with a lower bacterial growth rate in blood. Several immune genes were expressed at higher levels in resistant fish regardless of infection: the Type II arginase ( arg2 ), a marker for M2 macrophages involved in anti-inflammatory responses and tissue repair, and two Toll-like receptors ( tlr2 / tlr7 ), responsible for pathogen detection and inflammatory responses. This study highlights the importance of innate and intrinsic defense mechanisms in determining the outcome of F. psychrophilum infections, and illustrates that non-lethal time-course blood sampling for individual monitoring of bacteremia is a powerful tool to resolve within-host pathogen behavior in bacterial fish diseases.

Abstract Te.na.ci.ba'cu.lum. L. adj. tenax ‐acis , holding fast, tenacious; L. neut. n. baculum , stick; N.L. neut. n. Tenacibaculum , rod‐shaped bacterium that adheres to the surface of marine organisms. Bacteroidetes / Flavobacteriia / Flavobacteriales / Flavobacteriaceae / Tenacibaculum Members of the genus Tenacibaculum are exclusively found in marine environments. Most of them are attached to or associated with the surface of marine organisms such as fish, macroalgae, sponges, and invertebrates. However, some strains were isolated from seawater or marine sediments. Several species are important fish pathogens while others are able to degrade complex polysaccharides such as carrageenans or alginate. Rods are 0.1–0.7 μm in diameter and 0.4–40 μm in length. Filamentous cells >100 μm long and degenerative spherical cells may occur in older cultures. Ring‐shaped cells, spores, and gas vesicles are not formed. Cells are nonflagellated. Cells of most, if not all, species are motile by gliding. Gram‐stain‐negative. Cells produce a yellow pigment that is mainly zeaxanthin. Flexirubin‐type pigments are absent. Chemo‐organotrophic and heterotrophic. Aerobic. Catalase‐ and oxidase‐positive. Grow well on media containing seawater. The major respiratory quinone is menaquinone‐6. DNA G + C content (mol%) from the draft or complete genome sequence analysis : 29.6–32.2 (38.4 for Tenacibaculum litopenaei ). Type species : Tenacibaculum maritimum Suzuki et al. 2001 VP (basonyms: “ Flexibacter marinus ” Hikida et al. 1979, Flexibacter maritimus Wakabayashi et al. 1986 VP , “ Cytophaga marina ” Reichenbach 1989).

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.

Bacterial pathogens have a critical impact on aquaculture, a sector that accounts for half of the human fish consumption. Flavobacterium psychrophilum (phylum Bacteroidetes) is responsible for bacterial cold-water disease in salmonids worldwide. The molecular factors involved in host invasion, colonization and haemorrhagic septicaemia are mostly unknown. In this study, we identified two new TonB-dependent receptors, HfpR and BfpR, that are required for adaptation to iron conditions encountered during infection and for virulence in rainbow trout. Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of deletion mutants showed that they act without redundancy: BfpR is required for optimal growth in the presence of high haemoglobin level, while HfpR confers the capacity to acquire nutrient iron from haem or haemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein related to the HmuY family. We demonstrated that HfpY binds haem and contributes significantly to host colonization and disease severity. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote haem uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of pathogenicity of a serious pathogen belonging to the understudied family Flavobacteriaceae and point to the newly identified haem receptors as promising targets for antibacterial development.

Tenacibaculum maritimum is a devastating bacterial pathogen affecting a large variety of marine fish species. It is responsible for significant economic losses in aquaculture farms worldwide. Different typing methods have been proposed to analyse bacterial diversity and population structure. Serological heterogeneity has been observed and up to four different serotypes have been described so far. However, the underlying molecular factors remain unknown. By combining conventional serotyping and genome-wide association study, we identified the genomic loci likely involved in the O-antigen biosynthesis. This finding allowed the development of a robust multiplex PCR-based serotyping scheme able to detect subgroups within each serotype and therefore performs better than conventional serotyping. This scheme was successfully applied to a large number of isolates from worldwide origin and retrieved from a large variety of fish species. No obvious correlations were observed between the mPCR-based serotype and the host species or the geographic origin of the isolates. Strikingly, the distribution of mPCR-based serotypes does not follow the core genome phylogeny. Nevertheless, this simple and cost-effective mPCR-based serotyping method could be useful for different applications such as population structure analysis, disease surveillance, vaccine formulation and efficacy follow-up.

The orbicular batfish (Platax orbicularis), also called 'Paraha peue' in Tahitian, is the most important marine fish species reared in French Polynesia. Sudden and widespread outbreaks of severe 'white-patch disease' have occurred since 2011 in batfish farms one to three weeks after the transfer of juveniles from bio-secured hatcheries to lagoon cages. With cumulative mortality ranging from 20 to 90%, the sustainability of aquaculture of this species is severely threatened. In this study, we report for the first time the isolation from diseased batfish of several isolates belonging to the species Tenacibaculum maritimum, a major pathogen of many marine fish species. Histopathological analysis, an experimental bath challenge and a field monitoring study showed that T. maritimum is associated with 'white-patch disease'. Moreover, molecular and serological analyses performed on representative isolates revealed some degree of genetic diversity among the isolates, a finding of primary importance for epidemiological studies and the development of management and control strategies such as vaccination.

Flavobacterium psychrophilum, a member of the phylum Bacteroidetes, causes bacterial cold-water disease in salmonids worldwide. Infected fish exhibit tissue damages and develop hemorrhagic septicemia. The molecular factors involved in host colonization are mostly unknown. In this study, we identified two TonB-dependent receptors that are required for iron homeostasis and virulence in rainbow trout (Oncorhynchus mykiss). Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of single and double deletion mutants showed that the two receptors act without redundancy: BfpR is required for optimal growth in the presence of high hemoglobin level, while HfpR confers the capacity to acquire nutrient iron from heme or hemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein belonging to the HmuY family but with low sequence conservation compared to other members of this family. We demonstrated that HfpY is able to bind heme in vitro and contributes significantly to host colonization and disease severity in experimentally infected rainbow trout. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote heme uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of F. psychrophilum pathogenicity and point to the newly identified heme outer membrane receptors as promising targets for antibacterial development. Author summary Infectious diseases are a major threat for human and animal health. Bacterial pathogens impact aquaculture, a sector that accounts for half of the human fish consumption, and mechanistic insights into pathogenesis allow for improved control strategies. Pathogens have to retrieve essential nutrients to multiply. Host-derived heme constitutes the largest iron source during infection and pathogens have evolved fine-tuned iron acquisition systems to overcome host scavenging mechanisms while preventing iron toxicity. These mechanisms are poorly understood in pathogens of the family Flavobacteriaceae that have a critical impact on the fish farming, animal welfare and environment. Here, we report the identification and characterization of two heme uptake systems important for adaptation under distinct iron sources conditions encountered during infection and for virulence and host colonization in Flavobacterium psychrophilum, a serious pathogen of salmonids.

Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare are causing 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 F. 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 is standing within a cluster of species associated to 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 F. bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs nor mortalities were observed. However, F. collinsii was isolated from the internal organs of wounded fish, suggesting that the bacterium could invade fish under compromised conditions such as stress and/or wounds. Our results suggest that some fish-associated Flavobacterium species should be considered as 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 that 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 colonized only oppressed fish facing stressful conditions suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.

Tenacibaculum piscium, a gram-negative bacterium isolated from the skin ulcers of sea-farmed fish, has only been described in Norway. In the present study, we examined 16 Chilean Tenacibaculum isolates recovered from different organs in moribund and dead Atlantic salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch) cultured at different fish farms between 2014 and 2018. The present study applied biochemical, phenotypic, fatty acid and whole-genome sequence-based analyses to confirm the taxonomic status of the Chilean isolates. The obtained results are the first to confirm the presence of T. piscium in Chile and in Coho salmon, thus extending the recognized geographical and species distribution of this bacterium. Subsequent bath-challenge assays in Atlantic salmon utilizing three T. piscium isolates obtained from different hosts resulted in low cumulative mortality (i.e. 0-35%), even after exposure to an unnaturally high concentration of bacterial cells (i.e. > 10(7) cells/ml). However, scale loss and frayed fins were observed in dead fish. In silico whole-genome analysis detected various genes associated with iron acquisition, encoding of the type IX secretion system and cargo proteins, resistance to tetracycline and fluoroquinolones and stress responses. These data represent an important milestone towards a better understanding on the genomic repertoire of T. piscium.

The family Flavobacteriaceae (phylum Bacteroidetes) is a major component of soil, marine and freshwater ecosystems. In this understudied family, Flavobacterium psychrophilum is a freshwater pathogen that infects salmonid fish worldwide, with critical environmental and economic impact. Here, we report an extensive transcriptome analysis that established the genome map of transcription start sites and transcribed regions, predicted alternative sigma factor regulons and regulatory RNAs, and documented gene expression profiles across 32 biological conditions mimicking the pathogen life cycle. The results link genes to environmental conditions and phenotypic traits and provide insights into gene regulation, highlighting similarities with better known bacteria and original characteristics linked to the phylogenetic position and the ecological niche of the bacterium. In particular, osmolarity appears as a signal for transition between free-living and within-host programs and expression patterns of secreted proteins shed light on probable virulence factors. Further investigations showed that a newly discovered sRNA widely conserved in the genus, Rfp18, is required for precise expression of proteases. By pointing proteins and regulatory elements probably involved in host-pathogen interactions, metabolic pathways, and molecular machineries, the results suggest many directions for future research; a website is made available to facilitate their use to fill knowledge gaps on flavobacteria.