Flavobacterium psychraquaticum sp. nov., isolated from water system of Atlantic salmon (Salmo salar) smolts cultured in Chile.

Strain LB-N7T, a novel Gram-negative, orange, translucent, gliding, rod-shaped bacterium, was isolated from water samples collected from an open system of Atlantic salmon (Salmo salar) smolts in a fish farm in Chile during a flavobacterial infection outbreak in 2015. Phylogenetic analysis based on 16S rRNA sequences (1337 bp) revealed that strain LB-N7T belongs to the genus Flavobacterium and is closely related to the type strains Flavobacterium ardleyense A2-1T (98.8 %) and Flavobacterium cucumis R2A45-3T (96.75 %). The genome size of strain LB-N7T was 2.93 Mb with a DNA G+C content 32.6 mol%. Genome comparisons grouped strain LB-N7T with Flavobacterium cheniae NJ-26T, Flavobacterium odoriferum HXWNR29T, Flavobacterium lacisediminis TH16-21T and Flavobacterium celericrescens TWA-26T. The calculated digital DNA-DNA hybridization values between strain LB-N7T and the closest related Flavobacterium strains were 23.3 % and the average nucleotide identity values ranged from 71.52 to 79.39 %. Menaquinone MK-6 was the predominant respiratory quinone, followed by MK-7. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The primary polar lipids detected included nine unidentified lipids, two amounts of aminopospholipid and phospholipids, and a smaller amount of aminolipid. Phenotypic, genomic, and chemotaxonomic data suggest that strain LB-N7T (=CECT 30406T=RGM 3221T) represents as a novel bacterial species, for which the name Flavobacterium psychraquaticum sp. nov. is proposed.

Phenotypic and genomic characterization of a non-pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211-J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP-1 gene (87.85% aminoacidic sequence identity), encoding an extended-spectrum subclass B3 metallo-ß-lactamase and conferring carbapenem-hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline-class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Proteomic characterization of Tenacibaculum dicentrarchi under iron limitation reveals an upregulation of proteins related to iron oxidation and reduction metabolism, iron uptake systems and gliding motility.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Flavobacterium pygoscelis sp. nov., isolated from a chinstrap penguin chick (Pygoscelis antarcticus).

Strain I-SCBP12nT, a novel Gram-stain-negative, aerobic, non-spore-forming, motile-by-gliding and rod-shaped bacterium, was isolated from a chinstrap penguin chick (Pygoscelis antarcticus) during a 2015 expedition to the Chilean Antarctic territory. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain I-SCBP12nT belonged to the genus Flavobacterium, being closely related to strains Flavobacterium chryseum P3160T (98.52 %), Flavobacterium hercynium WB 4.2-33T (98.47 %) and Flavobacterium chilense LM-19-FpT (98.47 %). The genome size of strain I-SCBP12nT was 3.69 Mb with DNA G+C content 31.95 mol%. Genomic comparisons of strain I-SCBP12nT with type species in the genus Flavobacterium were performed, with obtained average values near 75.17 and 84.33 % for the blast and MUMer analyses of average nucleotide identity, respectively, and 0.86 for the tetranucleotides frequency analysis. These values are far from the accepted species cut-off values. Strain I-SCBP12nT contained MK-6 as the predominant menaquinone and the major polar lipids were aminophospholipid, an unidentified aminolipid and unidentified lipids. The predominant fatty acids (> 5 %) were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C16 : 1, iso-C16 : 0 3-OH, C15 : 1 ω6c and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Phenotypic, chemotaxonomic and genomic data supported the assignment of strain I-SCBP12nT (=CECT 30404T=RGM 3223T) to a novel species of Flavobacterium, for which the name Flavobacterium pygoscelis sp. nov.is proposed.

Tenacibaculum bernardetii sp. nov., isolated from Atlantic salmon (Salmo salar L.) cultured in Chile.

Strain PVT-9aT, a novel Gram-stain-negative, aerobic, non-spore-forming, motile-by-gliding and rod-shaped bacterium, was isolated from a skin lesion of Atlantic salmon (Salmo salar L.) during a tenacibaculosis outbreak that occurred in 2016 at a Chilean fish farm. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain PVT-9aT belonged to the genus Tenacibaculum, being related to the closest type strains Tenacibaculum haliotis KCTC 52419T (98.49 % sequence similarity), Tenacibaculum aestuariivivum JDTF-79T (97.36 %), Tenacibaculum insulae JDTF-31T (97.29 %) and Tenacibaculum ovolyticum IFO 15947T (97.15 %). The genome size of strain PVT-9aT was 2.73 Mb with a DNA G+C content 31.09 mol%. Average nucleotide identity analysis among 30 Tenacibaculum species rendered the most similar strains as follows: T. haliotis KCTC 52419T (87.91 %), T. ovolyticum IFO 15947T (82.47 %), Tenacibaculum dicentrarchi 35/09T (81.08 %), Tenacibaculum finnmarkense gv finnmarkense TNO006T (80.91 %) and T. finnmarkense gv ulcerans TNO010T (80.96 %). Menaquinone MK-6 was the predominant respiratory quinone. The predominant cell fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G and iso-C15 : 0 3-OH. Phenotypic, chemotaxonomic and genomic data supported the assignment of strain PVT-9aT (=DSM 115155T=RGM 3472T) as representing a novel species of Tenacibaculum, for which the name Tenacibaculum bernardetii sp. nov. is proposed.

Healthy and infected Atlantic salmon (Salmo salar) skin-mucus response to Tenacibaculum dicentrarchi under in vitro conditions.

Tenacibaculosis caused by Tenacibaculum dicentrarchi is the second most important bacterial disease that affects the Chilean salmon industry. The impacted fish show severe external gross skin lesions on different areas of the body. The external mucus layer that covers fish skin contains numerous immune substances that act as one of the main defense barriers against microbial colonization and invasions by potential pathogens. The present in vitro study aimed to evaluate and elucidate the role of the external mucus layer in the susceptibility of Atlantic salmon (Salmo salar) to three Chilean T. dicentrarchi strains and the type strain. For this, mucus collected from healthy and diseased (i.e., with T. dicentrarchi) Atlantic salmon were used, and various antibacterial and inflammatory parameters were analysed. The T. dicentrarchi strains were attracted to the mucus of Atlantic salmon regardless of health status. All four strains adhered to the skin mucus and very quickly grew using the mucus nutrients. Once infection was established, different mucosal defense components were activated in the fish, but the levels of bactericidal activity and of other enzymes were insufficient to eliminate T. dicentrarchi. Alternatively, this pathogen may be able to neutralize or evade these mechanisms. Therefore, the survival of T. dicentrarchi in fish skin mucus could be relevant to facilitate the colonization and subsequent invasion of hosts. The given in vitro results suggest that greater attention should be given to fish skin mucus as a primary defense against T. dicentrarchi.

Development of a PCR assay for the specific identification of the fish pathogen Tenacibaculum piscium.

Tenacibaculosis is an emerging disease that severely affects salmonid farming in Chile, producing high mortalities and causing great economic losses. This work describes a novel PCR assay for the specific detection of Tenacibaculum piscium, a species recently described and identified in tenacibaculosis outbreaks in Norway and Chile. The designed primers amplified a 678-bp fragment of the peptidase gene (peptidase M23 family) from T. piscium. This method is specific for T. piscium; no other chromosomal DNA amplification products were obtained for other Tenacibaculum species. In pure cultures, the PCR assay detected up to 500 pg of DNA, or the equivalent of 2.44 ± 0.06 × 104 CFU/ml. For seeded fish samples (i.e., gills, liver, kidney, and mucus), the sensitivity limit was 4.88 ± 0.11 × 106 CFU/g, sufficient to detect T. piscium in acute infections in fish. Notably, this sensitivity level was 100-fold lower for DNA extracted from mucus samples. As compared to other existing methodologies (e.g., gene sequencing), the PCR approach described in this work allowed for the easiest detection of T. piscium in mucus samples obtained from challenged fish, an important outcome considering that the identification of this bacterium is difficult. Our results indicate that the designed specific primers and PCR method provide a rapid and specific diagnosis of T. piscium.

In vitro phenotypic evidence for the utilization of iron from different sources and siderophores production in the fish pathogen Tenacibaculum dicentrarchi.

Iron uptake during infection is an essential pathogenicity factor of several bacteria, including Tenacibaculum dicentrarchi, an emerging pathogen for salmonid and red conger eel (Genypterus chilensis) farms in Chile. Iron-related protein families were recently found in eight T. dicentrarchi genomes, but biological studies have not yet confirmed functions. The investigation reported herein clearly demonstrated for the first time that T. dicentrarchi possesses different systems for iron acquisition-one involving the synthesis of siderophores and another allowing for the utilization of heme groups. Using 38 isolates of T. dicentrarchi and the type strain CECT 7612T , all strains grew in the presence of the chelating agent 2.2'-dipyridyl (from 50 to 150 µM) and produced siderophores on chrome azurol S plates. Furthermore, 37 of the 38 T. dicentrarchi isolates used at least four of the five iron sources (i.e. ammonium iron citrate, ferrous sulfate, iron chloride hexahydrate, haemoglobin and/or hemin) when added to iron-deficient media, although the cell yield was less when using hemin. Twelve isolates grew in the presence of hemin, and 10 of them used only 100 µM. Under iron-supplemented or iron-restricted conditions, whole cells of three isolates and the type strain showed at least one membrane protein induced in iron-limiting conditions (c.a. 37.9 kDa), regardless of the isolation host. All phenotypic results were confirmed by in-silico genomic T. dicentrarchi analysis. Future studies will aim to establish a relationship between iron uptake ability and virulence in T. dicentrarchi through in vivo assays.

Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

Evaluation of the in vitro susceptibility of Tenacibaculum dicentrarchi to tiamulin using minimum inhibitory concentration tests.

Tenacibaculosis caused by Tenacibaculum dicentrarchi is the third most important bacterial fish infection affecting the Chilean salmon industry. Losses to this disease are most frequently controlled by treatments with florfenicol and oxytetracycline. However, recent tenacibaculosis outbreaks were controlled through the extra-label, oral administration of tiamulin, resulting in high treatment efficiency. In this study, we present an analysis of susceptibility patterns of 32 T. dicentrarchi isolates and the type strain CECT 7612T to tiamulin by determining the minimum inhibitory concentrations (MICs) according to the procedures recommended by the Clinical and Laboratory Standard Institute, but fixing incubation temperature to the more appropriate for the growth of T. dicentrarchi (18 ºC). The MICs of the T. dicentrarchi isolates were unimodally distributed (0.06-1.0 µg/ml range), while the CECT 7612T strain presented an MIC of 0.5 µg/ml. Calculations using Normalized Resistance Interpretation provided epidemiological cut-off values of ≤1.0 µg/ml, with the 33 T. dicentrarchi classified as wild type. In Chile, tiamulin is authorized for use in other livestock species, but application in salmonids is extra-label. Our presented in vitro results suggest that tiamulin is a viable alternative to florfenicol, specifically as tiamulin requires comparatively lower concentrations to inhibit T. dicentrarchi. Considering that tiamulin is also exclusively for veterinary use, is classified as "least important" by the World Health Organization and has not resulted in the development of bacterial resistance, pharmaceutical companies should be requested to register tiamulin and provide alternative antimicrobial treatments for the salmonid industry.

Comparison between genome sequences of Chilean Tenacibaculum dicentrarchi isolated from red conger eel (Genypterus chilensis) and Atlantic salmon (Salmo salar) focusing on bacterial virulence determinants.

Tenacibaculum dicentrarchi is an emerging pathogen for salmonid cultures and red conger eel (Genypterus chilensis) in Chile, causing high economic losses not only in Chile but also to the global salmon industry. Infected fish show severe gross skin lesions that are sometimes accompanied by bone exposure. Despite pathogenicity demonstrated by Koch's postulates, no knowledge is currently available regarding the virulence machinery of T. dicentrarchi strains. Comparisons between the genome sequences of the eight T. dicentrarchi strains obtained from G. chilensis and Atlantic salmon (Salmo salar) provide insights on the existence of genomic diversity within this bacterium. The T. dicentrarchi type strain 3509T was used as a reference genome. Depending on the T. dicentrarchi strain, the discovered diversity included genes associated with iron acquisition mechanisms, copper homeostasis encoding, resistance to tetracycline and fluoroquinolones, pathogenic genomic islands and phages. Interestingly, genes encoding the T9SS membrane protein PorP/SprF were retrieved in all of the analysed T. dicentrarchi strains, regardless of the host fish (i.e. red conger eel or Atlantic salmon). However, the T6SS core component protein VgrG was identified in only one Atlantic salmon strain. Three types of peptidase genes and proteins associated with quorum sensing were detected in all of the T. dicentrarchi strains. In turn, all eight strains presented a total of 17 proteins associated with biofilm formation, which was previously confirmed through physiological studies. This comparative analysis will help elucidate and describe the genes and pathways that are likely involved in the virulence process of T. dicentrarchi. All or part of these predicted genes could aid the pathogen during the infective process in fish, making further physiological research necessary for clarification.

Evidence for the existence of extracellular vesicles in Renibacterium salmoninarum and related cytotoxic effects on SHK-1 cells.

Extracellular vesicles (EVs) in bacteria have been implicated in invasive and, through enzymes, infective processes. One Gram-positive bacterium lacking any EV research, despite having commercial impacts on the aquaculture industry, is Renibacterium salmoninarum. We addressed this gap in knowledge by utilizing scanning electron microscopy to provide the first reported evidence for the production of EVs by R. salmoninarum strain H-2. Dispersive light scattering detected that the EVs were heterogeneous in size, and the protein compositions were similar to the bacterial membrane and contained the virulent protein factors p22 and p57. The EVs additionally had a concentrated negative charge compared with R. salmoninarum H-2, as determined by Z potential. Finally, these particles seemed to play a role in host invasion in vitro in the salmon head kidney cell line, as demonstrated by the occurrence of a cytotoxic effect within the first 48 hr post-infection. Higher EV concentrations (i.e. 52.6 µg/ml) were more toxic than R. salmoninarum H-2. This information serves as a foundation to develop and test possible uses for R. salmoninarum EVs in salmon aquaculture, inspiring future advances against bacterial kidney disease.

Proposed protocol for performing MIC testing to determine the antimicrobial susceptibility of Renibacterium salmoninarum in Chilean salmon farms.

Surveillance of antibiotic resistance is of paramount importance for animal welfare and production. Despite aquaculture being a main source of animal protein, studies on antibiotic susceptibility in fish pathogens are scarce. Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD), is one of the most common bacterial pathogens affecting salmon farming. In this work, we present an analysis of susceptibility patterns using determinations of minimum inhibitory concentration (MIC) for 65 field isolates, which were collected over seven years (2013-2019) from Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) farms across southern Chile. The MIC protocol described by the Clinical Laboratory Standards Institute (CLSI) was used, but with microdilution instead of macrodilution and eight instead of four days of incubation. Two laboratories independently conducted analyses to provide data on the epidemiological cut-off values for R. salmoninarum to florfenicol, oxytetracycline and erythromycin. By using two calculation methods, our results provide evidence for an evolving subpopulation of non-wild-type isolates for the macrolide erythromycin, which is consistent with the respective treatment frequencies prescribed against BKD. Contrasting with what was expected, R. salmoninarum isolates were most susceptible to florfenicol and oxytetracycline, both of which are widely used antibiotics currently used in the Chilean salmon industry. The presented findings can serve as a reference for national or international antibiotic surveillance programmes, for both MIC interpretation and to identify emerging resistance to the conventional drugs used in BKD management. Finally, our results indicate that an 8-day incubation period for establishing MIC values of R. salmoninarum should be considered in a future revision of the CLSI guidelines.

First report and characterization of Tenacibaculum maritimum isolates recovered from rainbow trout (Oncorhynchus mykiss) farmed in Chile.

The present study reports on the first isolation of Tenacibaculum maritimum in rainbow trout (Oncorhynchus mykiss) farmed in Chile. In March 2020, two cages raising rainbow trout (~250 g) in the Los Lagos Region suffered a disease outbreak. In total, 17,554 fish died (3.5%-4.8% accumulated mortality). Microbiological analysis of the diseased fish obtained two representative isolates (i.e. Tm-035 and Tm-036). These were obtained from the external gross skin lesions-typical of tenacibaculosis-of two fish. Phenotyping, PCR tests and sequencing of the 16S rRNA and housekeeping genes confirmed the isolates as T. maritimum. The pathogenic potential of Tm-035 was further assessed by bath challenging Atlantic salmon (Salmo salar), which killed 70 ± 15% of fish within 11 days. Dead fish presented the same external clinical signs as did the farmed rainbow trout specimens. This research further broadens the known host distribution of this pathogen. Furthermore, the virulence experiments demonstrated that T. maritimum does not have a specific host. Additional studies are needed to evaluate the risk of T. maritimum for the O. mykiss farming industry.

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.

Flavobacterium salmonis sp. nov. isolated from Atlantic salmon (Salmo salar) and formal proposal to reclassify Flavobacterium spartansii as a later heterotypic synonym of Flavobacterium tructae.

A Gram-staining-negative non endospore-forming strain, T13(2019)T was isolated from water samples from Atlantic salmon (Salmo salar) fry culture in Chile and studied in detail for its taxonomic position. The isolate shared highest 16S rRNA gene sequence similarities with the type strains of Flavobacterium chungangense (98.44 %) followed by Flavobacterium tructae and Flavobacterium spartansii (both 98.22 %). Menaquinone MK-6 was the predominant respiratory quinone in T13(2019)T. Major polar lipids were phosphatidylethanolamine, an ornithine lipid and the unidentified polar lipids L1, L3 and L4 lacking a functional group. The major polyamine was sym-homospermidine. The fatty acid profile contained major amounts of iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 3-OH, C15 : 0, summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH) and various hydroxylated fatty acids in smaller amounts, among them iso-C16 : 0 3-OH, and C15 : 0 3-OH, which supported the grouping of the isolate into the genus Flavobacterium. Physiological/biochemical characterisation and ANI calculations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation. In addition it became obvious, that the type strains of F. tructae and F. spartansii showed 100 % 16S rRNA gene sequence similarities and ANI values of 97.21%/ 97.59 % and DDH values of 80.40 % [77.5 and 83%]. These data indicate that F. tructae and F. spartansii belong to the same species and it is proposed that F. spartansii is a later heterotypic synonym of F. tructae. For strain T13(2019)T (=CIP 111411T=LMG 30298T=CCM 8798T) a new species with the name Flavobacterium salmonis sp. nov. is proposed.

Arthrobacter ulcerisalmonis sp. nov., isolated from an ulcer of a farmed Atlantic salmon (Salmo salar), and emended description of the genus Arthrobacter sensu lato.

A Gram-stain positive, pleomorphic, oxidase-negative, non-motile isolate from the ulcer of a farmed Atlantic salmon (Salmo salar), designated strain T11bT, was subjected to a comprehensive taxonomic investigation. A comparative analysis of the 16S rRNA gene sequence showed highest similarities to the type strains of Pseudarthrobacter siccitolerans (98.1 %) and Arthrobacter methylotrophus and Pseudarthrobacter phenanthrenivorans (both 98.0 %). The highest ANI value observed between the assembled genome of T11bT and the publicly available Pseudarthrobacter and Arthrobacter type strain genomes were 81.15 and 80.99 %, respectively. The major respiratory quinone was menaquinone MK-9(H2). The polyamine pattern contained predominantly spermidine. The polar lipid profile consisted of the major lipids diphosphatidylglycerol, phosphatidylglycerol, monogalactosyl-diacylglycerol and dimannosylglyceride. Minor amouts of trimannosyldiacylglycerol and phosphatidylinositol were also detected. The peptidoglycan was of the type A3α l-Lys-l-Ser-l-Thr-l-Ala (A11.23). In the fatty acid profile, anteiso and iso branched fatty acids predominated (anteiso C15 : 0, iso C16 : 0, anteiso C17 : 0). Moderate to low DNA-DNA similarities, physiological traits as well as unique traits in the fatty acid pattern distinguished strain T11bT from the next related species. All these data point to the fact that strain T11bT represents a novel species of the genus Arthrobacter for which we propose the name Arthrobacter ulcerisalmonis sp. nov. The type strain is T11bT (=CIP 111621T=CCM 8854T=LMG 30632T=DSM 107127T).

Psychrobacter pygoscelis sp. nov. isolated from the penguin Pygoscelis papua.

One slightly beige-white pigmented, Gram-stain-negative, rod-shaped bacterium, strain I-STPP5bT, was isolated from the trachea of a Gentoo penguin chick individual (Pygoscelin papua) investigated in Fildes Bay, Chilean Antarctic (62° 12' S, 58° 57' W). I-STPP5bT consists of a 3.4 Mb chromosome with a DNA G+C content of 44.4 mol%. Of the 3056 predicted genes, 1206 were annotated as hypothetical proteins and 51 were tRNAs. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the isolate shared a 16S rRNA gene sequence identity to the type strains of Psychrobacter phenylpyruvicus (98.8 %), Psychrobacter arenosus and Psychrobacter pasteurii (both 98.3 %), Psychrobacter piechaudii (98.2 %) and Psychrobacter sanguinis (98.1 %), but 16S rRNA gene sequence similarities to all other Psychrobacter species were ≤98.0 %. Partial gyrB nucleotide and amino acid sequence similarities among strain STPP5bT and the next related type strains were all below 81.8 and 92.9%, respectively. DNA-DNA hybridisation (DDH) with P. phenylpyruvicus LMG 5372T, P. arenosus DSM 15389T and P. sanguinis DSM 23635T also showed low values (all below 30 %). The main cellular fatty acids of the strain were C18 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c. Based on phylogenetic, chemotaxonomic, genomic and phenotypic analyses we propose a new species of the genus Psychrobacter, with the name Psychrobacter pygoscelis sp. nov. and strain I-STPP5bT (=CIP 111410T= CCM 8799T=LMG 30301T) as type strain.