Host specificity and virulence of Flavobacterium psychrophilum: a comparative study in ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss) hosts.

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.

Phylogenomic characterization of Flavobacterium psychrophilum isolates retrieved from Turkish rainbow trout farms.

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.

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

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.

Zobellia alginiliquefaciens sp. nov., a novel member of the flavobacteria isolated from the epibiota of the brown alga Ericaria zosteroides (C. Agardh) Molinari & Guiry 2020.

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).

High Genetic Diversity in Flavobacterium psychrophilum Isolates from Healthy Rainbow Trout (Oncorhynchus mykiss) Farmed in the Same Watershed, Revealed by Two Typing Methods.

Flavobacterium psychrophilum affects salmonid health worldwide and causes economic losses. The genetic diversity of the pathogen must be considered to develop control methods. However, previous studies have reported both high and low levels of genetic diversity. The present longitudinal study aimed at assessing the genetic diversity of F. psychrophilum at a small temporal and geographic scale. Four farms located on the same watershed in France were studied. Rainbow trout (Oncorhynchus mykiss) batches were monitored, and apparently healthy individuals were sampled over 1 year. A total of 288 isolates were recovered from fish organs (gills and spleen) and eggs. Pulsed field gel electrophoresis revealed high genetic diversity. Multilocus sequence typing performed on a selection of 31 isolates provided congruent results, as follows: 18 sequence types (STs) were found, of which 13 were novel. The mean gene diversity (H = 0.8413) was much higher than that previously reported for this host species, although the sampling was restricted to a single watershed and 1 year. Seven isolates out of 31 were assigned to clonal complex ST10 (CC-ST10), which is the predominant clonal complex in the main salmonid production areas. A split decomposition tree reflected a panmictic population. This finding is important for aquaculture veterinarians in their diagnostic procedure, as the choice of adequate antibiotic treatment is conditioned by the correct identification of the causative agent. Furthermore, this study expands our knowledge on genetic diversity required for the development of an effective vaccine against F. psychrophilumIMPORTANCE The bacterium Flavobacterium psychrophilum is a serious pathogen in many fish species, especially salmonids, that is responsible for considerable economic losses worldwide. In order to treat infections and to develop vaccines, the genetic diversity of this bacterium needs to be known. We assessed the genetic diversity of F. psychrophilum isolates from apparently healthy rainbow trout raised in several fish farms in the same watershed in France. Two different genotyping methods revealed high diversity. The majority of isolates were unrelated to clonal complex sequence type 10 (CC-ST10), the clonal complex that is predominant worldwide and associated with disease in rainbow trout. In addition, we found 13 novel sequence types. These results suggest that a diverse subpopulation of F. psychrophilum may be harbored by rainbow trout.

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum causes bacterial cold-water disease in wild and aquaculture-reared fish and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, Flavobacterium columnare, requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The F. psychrophilum genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the Bacteroidetes to delete a core F. psychrophilum T9SS gene, gldN The ΔgldN mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type gldN on a plasmid restored secretion. Compared to wild-type and complemented strains, the ΔgldN mutant was deficient in adhesion, gliding motility, and extracellular proteolytic and hemolytic activities. The ΔgldN mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease.IMPORTANCE Bacterial cold-water disease, caused by F. psychrophilum, is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to F. psychrophilum and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors and targets for the development of control measures.

Tenacibaculum piscium sp. nov., isolated from skin ulcers of sea-farmed fish, and description of Tenacibaculum finnmarkense sp. nov. with subdivision into genomovars finnmarkense and ulcerans.

Results of previous multilocus sequence and whole-genome-based analyses have suggested that a homogeneous group of isolates belonging to the genus Tenacibaculum, represented by strain TNO020T and associated with skin ulcer development in sea-farmed fish, represents an as-yet-undescribed species. Comparative whole-genome analysis performed in the present study clustered five isolates, including TNO020T, in a distinct lineage within the genus Tenacibaculum. Phenotypic differences, high intra-cluster average nucleotide identity (ANI) values and low ANI values with other Tenacibaculum species support the proposal of a novel species, for which we propose the name Tenacibaculum piscium sp. nov. with strain TNO020T (=CCUG 73833T=NCIMB 15240T) as the type strain. Further, large-scale genome analyses confirmed the existence of two different phylogenetic lineages within 'T. finnmarkense', a species effectively but not validly published previously. ANI values just above the species delineation threshold of 95-96 % confirmed that both lineages belong to the same species. This result was also supported by DNA-DNA hybridization values. Phenotypically, the two conspecific lineages are distinguishable by differences in growth temperature range and ability to degrade l-proline. For the group of isolates already commonly known as 'T. finnmarkense', we propose the name Tenacibaculum finnmarkense sp. nov., with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain. We further propose the subdivision of T. finnmarkense sp. nov. into two genomovars, T. finnmarkense genomovar finnmarkense with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain and T. finnmarkense genomovar ulcerans with strain TNO010T (=CCUG 73832T=NCIMB 15239T) as the type strain.

Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing.

Tenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fish disease. This filamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft-sequenced genomes from isolates selected based on available MLST data, geographical origin and host fish. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein-coding genes accounting for ~75% of the genes in each genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp-1) whose analysis reveals an important contribution of recombination (r/m ≥ 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends between these subgroups and specific geographical origin or ecological niche remains to be clarified. We also evaluated the potential of MALDI-TOF-MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribosomal proteins explaining the detected mass shifts were identified. By combining nine polymorphic biomarker ions, we identified combinations referred to as MALDI-Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identified twenty MALDI-Types as well as four MALDI-Groups (MGs). We propose this MALDI-TOF-MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large-scale epidemiological surveys.

Serological diversity in Flavobacterium psychrophilum: A critical update using isolates retrieved from Chilean salmon farms.

Chile is currently the second largest producer of farmed salmon worldwide, but Flavobacterium psychrophilum, as one of the most detrimental pathogens, is responsible for major losses during the freshwater culturing step in salmonid fish farms. An antigenic study conducted 10 years ago reported four serological groups using 20 F. psychrophilum Chilean strains. To reduce disease outbreaks and to develop vaccine candidates, antigenic knowledge needs to be regularly updated using a significant number of additional recent F. psychrophilum isolates. The present study aimed at investigating the serological diversity of 118 F. psychrophilum isolates collected between 2006 and 2018 from farmed Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch). The current study supports an expansion of the known antigenic groups in Chile from 4 to 14. However, the use of the slide-agglutination technique for serotyping is costly, is labour-intensive and requires significant technical expertise. Addressing these points, the mPCR-based procedure was a very useful tool for serotyping the collected Chilean F. psychrophilum isolates. This technique revealed the presence of diverse mPCR serotypes (i.e. types 0, 1, 2 and 4). Therefore, mPCR should be employed to select the bacterial strain(s) for vaccine development and to conduct follow-up, selective breeding or epidemiological surveillance in Chilean fish farms. Given the presented findings, changes to Chilean fish-farming practices are vital for ensuring the continued productivity and well-being of farmed salmonids.

Alteromonas fortis sp. nov., a non-flagellated bacterium specialized in the degradation of iota-carrageenan, and emended description of the genus Alteromonas.

Strain 1T, isolated in the 1970s from the thallus of the carrageenophytic red algae, Eucheuma spinosum, collected in Hawaii, USA, was characterized using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, ovoid or rod-shaped and grew optimally at 20-25 °C, at pH 6-9 and with 2-4 % NaCl. Strain 1T used the seaweed polysaccharides ι-carrageenan, laminarin and alginic acid as sole carbon sources. The major fatty acids were C16 : 0, C18 : 1 ω7c and summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2OH) with significant amounts (>6 %) of C16 : 0 N alcohol and 10 methyl C17 : 0. The respiratory quinone was Q-8 and major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and an unknown aminolipid. Phylogenetic analyses showed that the bacterium is affiliated to the genus Alteromonas (family Alteromonadaceae, class Gammaproteobacteria). Strain 1T exhibited 16S rRNA gene sequence similarity values of 98.8 and 99.2 % to the type strains of Alteromonas mediterranea and Alteromonas australica respectively, and of 95.2-98.6 % to other species of the genus Alteromonas. The DNA G+C content of strain 1T was determined to be 43.9 mol%. Digital DNA-DNA hybridization predictions by the ANI and GGDC methods between strain 1T and other members of the genus Alteromonas showed values below 83 % and 30 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain 1T is distinct from species of the genus Alteromonas with validly published names and that it represents a novel species of the genus Alteromonas, for which the name Alteromonasfortis sp. nov. is proposed. The type strain is 1T (=ATCC 43554T=RCC 5933T=CIP 111645T=DSM 106819T).

Correction to: Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

After publication of this work [1], we noted that there was an error in Table 3 Line 4.

Quantitative trait loci for resistance to Flavobacterium psychrophilum in rainbow trout: effect of the mode of infection and evidence of epistatic interactions.

BACKGROUND: Bacterial cold-water disease, which is caused by Flavobacterium psychrophilum, is one of the major diseases that affect rainbow trout (Oncorhynchus mykiss) and a primary concern for trout farming. Better knowledge of the genetic basis of resistance to F. psychrophilum would help to implement this trait in selection schemes and to investigate the immune mechanisms associated with resistance. Various studies have revealed that skin and mucus may contribute to response to infection. However, previous quantitative trait loci (QTL) studies were conducted by using injection as the route of infection. Immersion challenge, which is assumed to mimic natural infection by F. psychrophilum more closely, may reveal different defence mechanisms. RESULTS: Two isogenic lines of rainbow trout with contrasting susceptibilities to F. psychrophilum were crossed to produce doubled haploid F2 progeny. Fish were infected with F. psychrophilum either by intramuscular injection (115 individuals) or by immersion (195 individuals), and genotyped for 9654 markers using RAD-sequencing. Fifteen QTL associated with resistance traits were detected and only three QTL were common between the injection and immersion. Using a model that accounted for epistatic interactions between QTL, two main types of interactions were revealed. A "compensation-like" effect was detected between several pairs of QTL for the two modes of infection. An "enhancing-like" interaction effect was detected between four pairs of QTL. Integration of the QTL results with results of a previous transcriptomic analysis of response to F. psychrophilum infection resulted in a list of potential candidate immune genes that belong to four relevant functional categories (bacterial sensors, effectors of antibacterial immunity, inflammatory factors and interferon-stimulated genes). CONCLUSIONS: These results provide new insights into the genetic determinism of rainbow trout resistance to F. psychrophilum and confirm that some QTL with large effects are involved in this trait. For the first time, the role of epistatic interactions between resistance-associated QTL was evidenced. We found that the infection protocol used had an effect on the modulation of defence mechanisms and also identified relevant immune functional candidate genes.

Multilocus sequence typing identifies epidemic clones of Flavobacterium psychrophilum in Nordic countries.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.

Multilocus sequence analysis of the marine bacterial genus Tenacibaculum suggests parallel evolution of fish pathogenicity and endemic colonization of aquaculture systems.

The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species.

Population structure of the fish pathogen Flavobacterium psychrophilum at whole-country and model river levels in Japan.

The bacterium Flavobacterium psychrophilum is a serious problem for salmonid farming worldwide. This study investigates by multilocus sequence typing (MLST) the population structure of this pathogen in Japan where it is also a major concern for ayu, a popular game fish related to salmoniforms. A total of 34 isolates collected across the country and 80 isolates sampled in a single model river by electrofishing were genotyped. The data accounting for 15 fish species allowed identifying 35 distinct sequence types (ST) in Japan. These ST are distinct from those reported elsewhere, except for some ST found in rainbow trout and coho salmon, two fish that have been the subject of intensive international trade. The pattern of polymorphism is, however, strikingly similar across geographical scales (model river, Japan, world) in terms of the fraction of molecular variance linked to the fish host (~50%) and of pairwise nucleotide diversity between ST (~5 Kbp(-1)). These observations go against the hypothesis of a recent introduction of F. psychrophilum in Japan. Two findings were made that are important for disease control: 1) at least two independent F. psychrophilum lineages infect ayu and 2) co-infections of the same individual fish by different strains occur.

Interplay between a bacterial pathogen and its host in rainbow trout isogenic lines with contrasted susceptibility to cold water disease.

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.

Complete genome sequence of Flavobacterium indicum GPSTA100-9T, isolated from warm spring water.

We report here the complete annotated genome sequence of Flavobacterium indicum CIP 109464(T) (= GPTSA100-9(T)), isolated from warm spring water in Assam, India. The genome sequence of F. indicum revealed a number of interesting features and genes in relation to its environmental lifestyle.

Isolation, identification, virulence potential and genomic features of Tenacibaculum piscium isolates recovered from Chilean salmonids.

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. > 107 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.

Two functionally distinct heme/iron transport systems are virulence determinants of the fish pathogen Flavobacterium psychrophilum.

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.

Genomic characterization of Tenacibaculum maritimum O-antigen gene cluster and development of a multiplex PCR-based serotyping scheme.

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.