The use of salmonid epithelial cells to characterize the toxicity of Tenacibaculum maritimum soluble extracellular products.

AIMS: This study assessed how the etiological agent of mouth rot in farmed Atlantic salmon, Tenacibaculum maritimum, induces toxicity in host salmonid barrier cells, and determined whether environmental changes are relevant for these effects. METHODS AND RESULTS: Tenacibaculum maritimum soluble extracellular products (ECPs) were collected and used to treat Atlantic salmon and rainbow trout intestinal barrier cell lines as a comparative model of bacterial-salmonid cell interactions. Cellular assays that examine cell membrane integrity, marker expression, and metabolic activity revealed that T. maritimum ECPs induced salmonid epithelial cell death through an apoptosis mechanism. Changes in salinity (25, 29, and 33 ppt) and temperature (12°C, 18°C, and 24°C) within the natural ranges observed in Pacific Northwest aquaculture facilities affected bacterial growth and cytotoxicity of T. maritimum ECPs. CONCLUSIONS: Our results suggest epithelial barriers as targets of T. maritimum-mediated toxicity in farmed mouth rot-infected Atlantic salmon. The induction of apoptosis by T. maritimum soluble ECPs may also help to explain the absence of overt inflammation typically reported for these fish.

Tenacibaculum dicentrarchi produce outer membrane vesicles (OMV) that are associated with the cytotoxic effect in rainbow trout head kidney macrophages.

Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

Tenacibaculum tangerinum sp. nov., isolated from a tidal flat sediment.

An orange-coloured bacterium, designated as strain GRR-S3-23T, was isolated from a tidal flat sediment collected from Garorim Bay, Chuncheongbuk-do, Republic of Korea. Cells of GRR-S3-23T were aerobic, Gram-stain-negative, rod-shaped and motile. GRR-S3-23T grew at 18-40 °C (optimum, 30 °C), pH 7.0-9.0 (optimum, pH 7.0) and with 2-4 % NaCl (optimum, 2-3 % w/v). Results of 16S rRNA gene sequence analysis indicated that GRR-S3-23T was closely related to Tenacibaculum aiptasiae a4T (97.6 %), followed by Tenacibaculum aestuarii SMK-4T (97.5 %), Tenacibaculum mesophilum MBIC 1140T (97.4 %), Tenacibaculum singaporense TLL-A2T (97.3 %), Tenacibaculum crassostreae JO-1T (97.2 %),and Tenacibaculum sediminilitoris YKTF-3T (97.1 %). The average amino acid identity values between GRR-S3-23T and the related strains were 86.8-72.8 %, the average nucleotide identity values were 83.3-74.1 %, and the digital DNA-DNA hybridization values were 27.0-19.6 %. GRR-S3-23T possessed menaquinone-6 (MK-6) as major respiratory quinone and had summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c, 20.6 %) and iso-C15 : 1G (10.8 %) as major fatty acids (>10.0 %). The polar lipid profiles of GRR-S3-23T contained phosphatidylethanolamine, one unidentified aminolipid, one unidentified aminophospholipid, three unidentified lipids, one unidentified glycolipid and four unidentified phospholipids. The DNA G+C content of GRR-S3-23T was 33.7%. On the basis of the results of the polyphasic analysis involving phylogenetic, phylogenomic, physiological and chemotaxonomic analyses described in this study, GRR-S3-23T is considered to represent a novel species within the genus Tenacibaculum, for which the name Tenacibaculum tangerinum is proposed. The type strain is GRR-S3-23T (=KCTC 102029T=KACC 23271T=JCM 36353T).

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.

The effect of a biofilm-forming bacterium Tenacibaculum mesophilum D-6 on the passive film of stainless steel in the marine environment.

The microbiologically influenced corrosion of 304 stainless steel in the presence of a marine biofilm-forming bacterium Tenacibaculum mesophilum D-6 was systematically investigated by means of electrochemical techniques and surface analyses to reveal the effect of the selective attachment and adsorption of the biofilms on the passivity breakdown of the stainless steel. It was found that the T. mesophilum D-6 was electroactive and could oxidize low valent cations and metal, facilitating the local dissolution of the passive film and the substrate in the film defects, nearly doubling the surface roughness. The biofilms of T. mesophilum D-6 with mucopolysaccharide secreta and chloride ions tended to preferentially adsorb at the defects of the passive film on the steel, yielding non-homogeneous microbial aggregates and local Cl- enrichment there. The adsorption of the bacteria and chloride ions reduced the thickness of passive film by 23.9%, and generate more active sites for pitting corrosion on the passive film and more semiconducting carrier acceptors in the film. The maximum current density of the 304 SS in the presence of T. mesophilum D-6 was over one order of magnitude higher than that in the sterile medium, and the largest pit was deepened 3 times.

Pathology of experimentally induced mouthrot caused by Tenacibaculum maritimum in Atlantic salmon smolts.

Mouthrot, caused by Tenacibaculum maritimum is a significant disease of farmed Atlantic salmon, Salmo salar on the West Coast of North America. Smolts recently transferred into saltwater are the most susceptible and affected fish die with little internal or external clinical signs other than the characteristic small (usually < 5 mm) yellow plaques that are present inside the mouth. The mechanism by which these smolts die is unknown. This study investigated the microscopic pathology (histology and scanning electron microscopy) of bath infected smolts with Western Canadian T. maritimum isolates TmarCan15-1, TmarCan16-1 and TmarCan16-5 and compared the findings to what is seen in a natural outbreak of mouthrot. A real-time RT-PCR assay based on the outer membrane protein A specific for T. maritimum was designed and used to investigate the tissue tropism of the bacteria. The results from this showed that T. maritimum is detectable internally by real-time RT-PCR. This combined with the fact that the bacteria can be isolated from the kidney suggests that T. maritimum becomes systemic. The pathology in the infected smolts is primarily mouth lesions, including damaged tissues surrounding the teeth; the disease is similar to periodontal disease in mammals. The pathological changes are focal, severe, and occur very rapidly with little associated inflammation. Skin lesions are more common in experimentally infected smolts than in natural outbreaks, but this could be an artefact of the challenge dose, handling and tank used during the experiments.

Genotyping of Tenacibaculum maritimum isolates from farmed Atlantic salmon in Western Canada.

Mouthrot infections (bacterial stomatitis) have a significant impact on the Atlantic salmon aquaculture industry in Western Canada due to economic losses and fish welfare. Bacteria isolated from lesions in the field have been identified as Tenacibaculum maritimum. Mouthrot is different to classical tenacibaculosis, which is most commonly associated with ulcerative lesions, frayed fins and tail rot. The marine fish pathogen T. maritimum is found worldwide; however, in Western Canada, the knowledge of the genetic profile of T. maritimum is limited. This study looked at increasing this knowledge by genotyping T. maritimum isolates collected from Atlantic salmon from farms in Western Canada. These genotypes were compared to other species of the genus Tenacibaculum, as well as other known sequence types within the species. The Western Canadian isolates belong to two new sequence types within the T. maritimum species. Phylogenetic analysis shows that the isolates form a distinct branch together with T. maritimum NCIMB 2154T separate from other Tenacibaculum type strains, and they are most closely related to strains from Norway and Chile.

Tenacibaculum insulae sp. nov., isolated from a tidal flat.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterial strain, designated JDTF-31T, was isolated from a tidal flat in Jindo, a South Korean island. Strain JDTF-31T grew optimally at 25 °C and in the presence of 2.0 % (w/v) NaCl. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain JDTF-31T fell within the cluster comprising the type strains of Tenacibaculum species, joining the type strain of Tenacibaculum soleae. The novel strain exhibited 16S rRNA gene sequence similarity values of 98.3, 97.8 and 97.1 % to the type strains of T. soleae, Tenacibaculum haliotisand Tenacibaculum ovolyticum, respectively, and of 94.2-96.8 % to the type strains of the other Tenacibaculum species. Strain JDTF-31T contained MK-6 as the predominant menaquinone and iso-C15 : 0 and iso-C15 : 0 3-OH as the major fatty acids. The major polar lipids of strain JDTF-31T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminophospholipid. The DNA G+C content of strain JDTF-31T was 31.3 mol% and its DNA-DNA relatedness values with the type strains of T. soleae, T. haliotis and T. ovolyticum were 16-27 %. The differential phenotypic properties, together with its phylogenetic and genetic data, revealed that strain JDTF-31T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain JDTF-31T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculuminsulae sp. nov. is proposed. The type strain is JDTF-31T (=KCTC 52749T=NBRC 112783T).

Tenacibaculum aestuariivivum sp. nov., isolated from a tidal flat.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterial strain, designated JDTF-79T, was isolated from a tidal flat in Jindo, an island of South Korea, and subjected to a taxonomic study using a polyphasic approach. Strain JDTF-79T grew optimally at 25 °C and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain JDTF-79T fell within the clade comprising the type strains of species of the genus Tenacibaculum, clustering with the type strains of Tenacibaculum dicentrarchi, Tenacibaculum ovolyticum, 'Tenacibaculum haliotis' and Tenacibaculum soleae. The novel strain exhibited highest 16S rRNA gene sequence similarity (98.3 %) to the type strain of T. dicentrarchi and sequence similarities of 93.5-96.9 % to the type strains of the other species of the genus Tenacibaculum. Strain JDTF-79T contained MK-6 as the predominant menaquinone and anteiso-C15 : 0, iso-C15 : 0 3-OH and iso-C15 : 0 as the major fatty acids. The major polar lipids of strain JDTF-79T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminophospholipid. The DNA G+C content of strain JDTF-79T was 30.3 mol%. Strain JDTF-79T had a mean DNA-DNA relatedness value of 19 % with the type strain of T. dicentrarchi. The differential phenotypic properties, together with the phylogenetic and genetic data, revealed that strain JDTF-79T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain JDTF-79T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum aestuariivivum sp. nov. is proposed. The type strain is JDTF-79T (=KCTC 52980T=NBRC 112903T).

Tenacibaculum agarivorans sp. nov., an agar-degrading bacterium isolated from marine alga Porphyra yezoensis Ueda.

A novel Gram-stain-negative, aerobic, rod-shaped, non-flagellated and agar-digesting marine bacterium, designated as HZ1T, was isolated from the marine alga Porphyra yezoensis Ueda (AST58-103) collected from the coastal area of Weihai, PR China. Phylogenetic analysis based on 16S rRNA gene sequences placed HZ1T in the genus Tenacibaculum, and it formed a distinct clade in the phylogenetic tree with the type strains of Tenacibaculum amylolyticum and Tenacibaculum skagerrakense, with 97.0 % and 96.7 % 16S rRNA gene sequence similarities, respectively. The DNA G+C content of the isolate was 31.8 mol%. HZ1T contained MK-6 as the predominant menaquinone and iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH and iso-C15 : 1G as the major fatty acids. The major polar lipids were phosphatidylethanolamine, four unidentified lipids and five unidentified aminolipids. On the basis of the results of the phylogenetic analysis and phenotypic properties, it is concluded that HZ1T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculumagarivorans sp. nov. is proposed. The type strain is HZ1T (=MCCC 1H00174T=KCTC 52476T).

Tenacibaculum haliotis sp. nov., isolated from the gut of an abalone Haliotis discus hannai.

A Gram-stain-negative, non-flagellated, gliding, non-spore-forming bacterial strain, designated RA3-2T, was isolated from the gut of an abalone (Haliotis discus hannai) collected from the sea around Jeju island, South Korea, and subjected to a polyphasic taxonomic study. RA3-2T grew optimally at 20 °C and in the presence of 2.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences indicated that RA3-2T fell within the clade comprising the type strains of species of the genus Tenacibaculum, clustering with the type strains of Tenacibaculum soleae, Tenacibaculum ovolyticum and Tenacibaculum dicentrarchi; showing 16S rRNA gene sequence similarity values of 96.2-96.8 %. The novel strain exhibited 16S rRNA gene sequence similarity values of 93.5-96.9 % to the type strains of the other species of the genus Tenacibaculum. RA3-2T contained MK-6 as the predominant menaquinone and iso-C15 : 0 and iso-C15 : 0 3-OH as the major fatty acids. The major polar lipids of RA3-2T were phosphatidylethanolamine, two unidentified lipids, one unidentified aminophospholipid and one unidentified glycolipid. The DNA G+C content of RA3-2T was 31.7 mol%. The differential phenotypic properties, together with the phylogenetic data, revealed that RA3-2T is separated from other species of the genus Tenacibaculum with validly published names. On the basis of the data presented, RA3-2T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum haliotis sp. nov. is proposed. The type strain is RA3-2T (=KCTC 52419T=NBRC 112382T).

Isolation, Characterization and Virulence Potential of Tenacibaculum dicentrarchi in Salmonid Cultures in Chile.

In this study, we isolated, identified and characterized isolates of Tenacibaculum dicentrarchi in Atlantic salmon (Salmo salar) farmed in Chile for the first time. In 2010 and 2014, mortalities were observed in Atlantic salmon (average weight 25-30 and 480-520 g, respectively) at an aquaculture centre in Puerto Montt, Chile. Severe tail rots, frayed fins and, in some cases, damaged gills were detected. Wet smear analyses of these lesions revealed a high occurrence of Gram-negative, filamentous bacteria. Microbiological analysis of infected gill and tail tissues yielded six bacterial isolates. All were identified as T. dicentrarchi through polyphasic taxonomy, which included phenotypic characterization, 16S rRNA sequencing and multilocus sequence typing. The latter method revealed a close relationship of the Chilean genotype with the T. dicentrarchi type strain and two Norwegian Atlantic cod (Gadus morhua) isolates. The pathogenic potential of the TdChD05 isolate was assessed by challenging Atlantic salmon and rainbow trout (Oncorhynchus mykiss) for one hour, which resulted in mean cumulative mortality rates of 65% and 93%, respectively, as well as clinical signs 14 days post-challenge. However, challenged Coho salmon (Oncorhynchus kisutch) presented no mortalities or clinical signs of infection. These findings indicate that the geographical and host distribution of T. dicentrarchi is wider than previously established and that this bacterium may have negative impacts on salmonid cultures.

Tenacibaculum finnmarkense sp. nov., a fish pathogenic bacterium of the family Flavobacteriaceae isolated from Atlantic salmon.

A novel Gram-stain negative, aerobic, non-flagellated, rod-shaped gliding bacterial strain, designated HFJ(T), was isolated from a skin lesion of a diseased Atlantic salmon (Salmo salar L.) in Finnmark, Norway. Colonies were observed to be yellow pigmented with entire and/or undulating margins and did not adhere to the agar. The 16S rRNA gene sequence showed that the strain belongs to the genus Tenacibaculum (family Flavobacteriaceae, phylum 'Bacteroidetes'). Strain HFJ(T) exhibits high 16S rRNA gene sequence similarity values to Tenacibaculum dicentrarchi NCIMB 14598(T) (97.2 %). The strain was found to grow at 2-20 °C and only in the presence of sea salts. The respiratory quinone was identified as menaquinone 6 and the major fatty acids were identified as summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH), iso-C15:0, anteiso-C15:0, iso-C15:1 and iso-C15:0 3-OH. The DNA G+C content was determined to be 34.1 mol%. DNA-DNA hybridization and comparative phenotypic and genetic tests were performed with the phylogenetically closely related type strains, T. dicentrarchi NCIMB 14598(T) and Tenacibaculum ovolyticum NCIMB 13127(T). These data, as well as phylogenetic analyses, suggest that strain HFJ(T) should be classified as a representative of a novel species in the genus Tenacibaculum, for which the name Tenacibaculum finnmarkense sp. nov. is proposed; the type strain is HFJ (T) = (DSM 28541(T) = NCIMB 42386(T)).

Tenacibaculum ascidiaceicola sp. nov., isolated from the golden sea squirt Halocynthia aurantium.

A Gram-stain-negative, non-flagellated, non-spore-forming bacterial strain motile by gliding, designated RSS1-6T, was isolated from a golden sea squirt Halocynthia aurantium and its taxonomic position was investigated by using a polyphasic approach. Strain RSS1-6T grew optimally at 30-37 °C and in the presence of 1.0-4.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain RSS1-6T fell within the clade comprising species of the genus Tenacibaculum, clustering with the type strains of Tenacibaculum discolor, Tenacibaculum litoreum and Tenacibaculum gallaicum with which it exhibited 16S rRNA gene sequence similarity values of 98.5-99.5 %. Strain RSS1-6T contained MK-6 as the predominant menaquinone and iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) as the major fatty acids. The major polar lipids of strain RSS1-6T were phosphatidylethanolamine, two unidentified lipids, one unidentified aminophospholipid and one unidentified glycolipid. The DNA G+C content was 32.5 mol% and the mean DNA-DNA relatedness values with the type strains of T. discolor, T. litoreum and T. gallaicum were 17.3-25.2 %. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain RSS1-6T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain RSS1-6T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum ascidiaceicola sp. nov. is proposed. The type strain is RSS1-6T ( = KCTC 42702T = NBRC 111225T).