Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains.

Tenacibaculosis is a bacterial ulcerative disease affecting marine fish and represents a major threat to aquaculture worldwide. Its aetiological agents, bacteria belonging to the genus Tenacibaculum, have been present in Norway since at least the late 1980's and lead to regular ulcerative outbreaks and high mortalities in production of farmed salmonids. Studies have shown the presence of several Tenacibaculum species in Norway and a lack of clonality in outbreak-related strains, thus preventing the development of an effective vaccine. Hence, a thorough examination of the bacterial diversity in farmed fish presenting ulcers and the geographical distribution of the pathogens should provide important insights needed to strengthen preventive actions. In this study, we investigated the diversity of Tenacibaculum strains isolated in 28 outbreaks that occurred in Norwegian fish farms in the period 2017-2020. We found that 95% of the 66 strains isolated and characterized, using an existing MultiLocus Sequence Typing system, have not previously been identified, confirming the high diversity of this genus of bacteria in Norway. Several of these Tenacibaculum species seem to be present within restricted areas (e.g., Tenacibaculum dicentrarchi in western Norway), but phylogenetic analysis reveals that several of the strains responsible of ulcerative outbreaks were isolated from different localities (e.g., ST- 172 isolated from northern to southern parts of Norway) and/or from different hosts. Understanding their reservoirs and transmission pathways could help to address major challenges in connection with prophylactic measures and development of vaccines.

Tenacibaculum pelagium sp. nov., isolated from marine sediment.

A novel bright yellow pigmented, Gram-stain-negative, gliding, aerobic and rod-shaped marine bacterium, designated strain S7007T, was isolated from a marine sediment sample taken from Jingzi Wharf, Weihai, China. The bacterium was able to grow at 4-33 °C (optimum 28 °C), at pH 6.5-9.0 (optimum 7.0) and with 2.0-4.0% (w/v) NaCl (optimum 3.0%). According to the phylogenetic analysis based on the 16S rRNA gene sequences, strain S7007T was associated with the genus Tenacibaculum and showed highest similarity to Tenacibaculum adriaticum JCM 14633T (98.0%). The average nucleotide identity (ANI) scores of strain S7007T with T. adriaticum JCM 14633T and T. maritimum NBRC 110778T were 78.3% and 77.1%, respectively and the Genome-to-Genome Distance Calculator (dDDH) scores were 20.5% and 19.9%, respectively. The sole isoprenoid quinone was MK-6 and the major cellular fatty acids (> 10.0%) were iso-C15:0, iso-C15:0 3-OH, iso-C15: 1 G and summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c). The major polar lipids of strain S7007T were phosphatidylethanolamine, phosphatidyldimethylethanolamine, one unidentified lipid and two unidentified aminolipids. The genomic DNA G + C content was 30.9 mol %. The combined phenotypic data and phylogenetic inference that strain S7007T should be classified as a novel species in the genus Tenacibaculum, for which the name Tenacibaculum pelagium sp. nov. is proposed. The type strain is S7007T (= MCCC 1H00428T = KCTC 72941T).

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.

New salmonid hosts for Tenacibaculum species: Expansion of tenacibaculosis in Chilean aquaculture.

The success and sustainability of Chilean aquaculture largely depends on the control of endemic and emerging pathogens, including several species of the genus Tenacibaculum. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" have been detected and confirmed in Chilean Atlantic salmon (Salmo salar). However, no outbreaks of tenacibaculosis in rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch) have been reported, either in Chile or globally. The aims of this study were to determine whether the mortalities recorded for rainbow trout and coho salmon from five marine fish farms located in the Los Lagos, Aysén, and Magallanes Regions could be caused by Tenacibaculum spp. The diseased fish exhibited cutaneous haemorrhages, tail and peduncle rots, and damage on the mouth and tongue. Microbiological analysis of infected external tissues yielded 13 bacterial isolates. The isolates were identified as members of the genus Tenacibaculum through biochemical analysis (e.g. Gram-stain negative, straight rods, filamentous cells and motile by gliding), but differences existed in biochemical results, making species-level identification through biomolecular tools essential. The 16S rRNA analysis found that the majority of isolates were more closely related to "T. finnmarkense" than T. dicentrarchi, while the phylogenetic trees resulting from multilocus sequence data recovered the four main clades (clades I to IV) identified by Olsen et al. (2017, Veterinary Microbiology, 205, 39). This is the first documented occurrence of clinical tenacibaculosis in farmed rainbow trout and coho salmon globally, and it extends the known host distribution of this pathogen in Chile. Moreover, we confirm the presence of Tenacibaculum species in the Chilean Patagonia. These findings highlight the importance of establishing preventative measures to minimize the spread of this disease within the Chilean marine aquaculture industry, as well as the need for monitoring initiatives worldwide in these farmed fish species.

Insights into the microbiome of farmed Asian sea bass (Lates calcarifer) with symptoms of tenacibaculosis and description of Tenacibaculum singaporense sp. nov.

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2T, shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764T. The genome of strain TLL-A2T was sequenced and compared to that of T. mesophilum DSM 13764T. Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764T and strain TLL-A2T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2T and T. mesophilum DSM 13764T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2T (=DSM 106434T, KCTC 62393T) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

Identification and typing of fish pathogenic species of the genus Tenacibaculum.

Tenacibaculosis is a major bacterial disease that causes severe fish outbreaks and losses and limits the culture of a variety of commercially valuable anadromous and marine fish species in Europe, America, Asia and Oceania. Fish affected by tenacibaculosis have external lesions and necrosis that affect different areas of the body surface, reducing their commercial value. Several species of Tenacibaculum have been identified as the causal agent of tenacibaculosis in fish, including Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum discolor, Tenacibaculum gallaicum, Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (quotations marks denote species that have not been validly published). Diagnosis of tenacibaculosis is usually based on culture-dependent detection and identification techniques which are time-consuming and do not allow to differentiate closely related species. The development of reliable techniques for studying the relationships between members of the genus Tenacibaculum and for distinguishing fish-pathogenic species of Tenacibaculum genus is, therefore, a key step in understanding the diversity and incidence of tenacibaculosis in global aquaculture, designing effective prevention strategies and early implementation of infection control measures. In this review, recent advances in molecular, serological, proteomic and chemotaxonomic techniques developed for the identification and differentiation of Tenacibaculum species, as well as for the analysis of their genetic and epidemiological relationships are discussed. Key features of current diagnostic methods likely to facilitate control and prevention of tenacibaculosis and to avoid the spread of its aetiological agents are also outlined.

Tenacibaculum todarodis sp. nov., isolated from a squid.

A Gram-stain-negative, rod-shaped, aerobic bacterial strain, designated LPB0136T, was isolated from a squid Todarodes pacificus, caught in the East Sea, off Korea. LPB0136T contained a circular chromosome of 3.02 Mb with a DNA G+C content of 30.7 mol%. The genome included 2726 protein-coding genes and three copies of rRNA operons. Phylogenetic analysis of the 16S rRNA gene sequence indicated that this isolate represents a member of the genus Tenacibaculum with an independent genomic species status, showing sequence similarities of 95.9 % to Tenacibaculum aestuarii SMK-4T and Tenacibaculum caenipelagi HJ-26MT. The detected respiratory quinone (MK-6) and major polar lipid (phosphatidylethanolamine) were similar to the chemotaxonomic profile of other species of the genus Tenacibaculum. The major cellular fatty acids profile (iso-C15 : 0, iso-C15 : 0 3-OH and iso-C15 : 0G) was also similar to those of members of genus Tenacibaculum, but the contents and amounts differed from those of closely related neighbours. Many biochemical and physiological characteristics also distinguished the isolate from other species within the genus Tenacibaculum. On the basis of the pholyphasic taxonomic data determined in this study, strain LPB0136T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculum todarodis sp. nov. is proposed. The type strain is LPB0136T (=KACC 18887T=JCM 31564T).

Comparison of serological and molecular typing methods for epidemiological investigation of Tenacibaculum species pathogenic for fish.

In the present study, the potential of serological methods, the repetitive extragenic palindromic PCR (REP-PCR) and the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) for the typing of the species Tenacibaculum maritimum, Tenacibaculum soleae and Tenacibaculum discolor was evaluated. Moreover, molecular and proteomic techniques were used to assess variability among strains belonging to different serotypes, as well as isolated from different host species and geographical areas. Slide agglutination and dot-blot assays demonstrated the lack of immunological relationships among Tenacibaculum species analyzed. The serotype O1 was predominant within T. maritimum isolates regardless of the fish species or geographical area. Two serotypes were distinguished within T. soleae isolates and at least one within T. discolor strains. Species- and strain-specific profiles were obtained from the analysis of T. maritimum, T. soleae and T. discolor by REP-PCR and ERIC-PCR, demonstrating their potential as diagnostic tools. The genotyping analysis using both techniques showed genetic variability among the strains of each fish pathogenic Tenacibaculum species analysed. However, Tenacibaculum strains isolated from different host species or geographical areas or belonging to different serotypes produced REP and ERIC profiles with high similarity. Analysis by MALDI-TOF-MS of the T. maritimum strains could not detect any serotype-identifying biomarkers. Serotype-specific mass peaks were found for the serotypes O1 and O2 of T. soleae and for the serotype O1 of T. discolor. However, no relationships between the proteomic profiles and the source of isolation of the strains were obtained for any of the Tenacibaculum species analysed in this study.

First description of a mortality event in adult Pacific oysters in Italy associated with infection by a Tenacibaculum soleae strain.

Summer mortality episodes in adult Pacific oysters have been described since the 1950s in various farming areas. Starting in 2012, a recrudescence of mortalities in commercial-sized oysters was first observed in France and then in Italy, with seasonality extension and translation later in the year. Moribund individuals collected during an event in Italy in December 2014 showed yellowish lesions of the mantle and adductor muscle. Histological examination revealed filamentous bacteria associated with necrotic areas. Quantitative PCRs targeting OsHV-1 and Vibrio aestuarianus detected only high loads of the pathogenic bacteria in tissues of symptomatic individuals. A lower diversity of the hemolymph microbiota was also evidenced in moribund individuals, with a predominance of Vibrio and Arcobacter species. A strain of Flavobacteriaceae was isolated from all the symptomatic individuals. Sequence analysis of the 16S rRNA gene identified the strain as Tenacibaculum soleae. When strain pathogenicity was tested by injection in adult individuals, it induced mortality rates of up to 45%, even in the absence of V. aestuarianus. As mortality occurred only 11 days post-infection, further investigation is needed to determine its effective virulence in natural conditions. This is the first description of a Tenacibaculum strain associated with bivalve mortalities.

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).

MALDI-TOF mass spectrometry for rapid differentiation of Tenacibaculum species pathogenic for fish.

Tenacibaculosis is a fish disease that limits the culture of a variety of marine fish species of commercial value in the world. The genus Tenacibaculum includes several species, and their discrimination is of clinical interest in order to improve the management of an outbreak of the disease. In this study, a novel proteomic approach based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was evaluated for the identification and differentiation of Tenacibaculum species. The peak mass lists derived from MALDI-TOF-MS analysis were examined for the detection of potential biomarkers, similarity and cluster analysis and principal component analysis (PCA). Culture media used for bacterial growth did not affect the mass fingerprints. Eight genus-specific peaks were found in all the Tenacibaculum species analysed. Moreover, at least one species-specific peak was found in the species Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum dicentrarchi, Tenacibaculum litoreum and Tenacibaculum ovolyticum. These peaks could serve as biomarkers for the rapid identification of these bacterial fish pathogens. The cluster and PCA clearly separated the species T. maritimum, T. soleae, T. dicentrarchi and T. ovolyticum in different clusters. However, species of Tenacibaculum discolor and Tenacibaculum gallaicum were difficult to distinguish based on their protein fingerprints. To our knowledge, this is the first study that deals with the characterization and determination of biomarkers of Tenacibaculum species by MALDI-TOF mass spectrometry. This approach proved to be an effective and reliable technique for the discrimination of the Tenacibaculum species; therefore, it could be integrated as a routine diagnostic tool in microbiological laboratories.

Primary Isolation and Characterization of Tenacibaculum maritimum from Chilean Atlantic Salmon Mortalities Associated with a Pseudochattonella spp. Algal Bloom.

This study presents the first isolation of Tenacibaculum maritimum from farmed Atlantic Salmon Salmo salar in Chile. The isolate, designated T. maritimum Ch-2402, was isolated from gills of Atlantic Salmon at a farm located in region X, Los Lagos, Chile, during the harmful algal bloom caused by Pseudochattonella spp. in February 2016. The algal bloom is reported to have caused 40,000 metric tons of mortality in this salmon farming area. The bacterium T. maritimum, which causes tenacibaculosis, is recognized as an important pathogen of marine fish worldwide. Genetic, phylogenetic, and phenotypic characterizations were used to describe the T. maritimum Ch-2402 isolate. The isolate was similar to the type strain of T. maritimum but was genetically unique. Tenacibaculum dicentrarchi isolates were also recovered during sampling from the same farm. Based on the fact that T. maritimum has been shown to cause disease in Atlantic Salmon in other regions, the presence of this bacterium poses a potential risk of disease to fish in the Chilean aquaculture industry. Received November 6, 2016; accepted May 29, 2017.

The low diverse gastric microbiome of the jellyfish Cotylorhiza tuberculata is dominated by four novel taxa.

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial-animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial 'Candidatus Syngnamydia', and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: 'Candidatus Syngnamydia medusae', 'Candidatus Medusoplasma mediterranei' and 'Candidatus Tenacibaculum medusae'.

Pseudotenacibaculum haliotis gen. nov., sp. nov., a new member within the Tenacibaculum-Polaribacter clade of the family Flavobacteriaceae, isolated from the intestine of adult abalones, Haliotis diversicolor and H. discushannai.

Two rod-shaped marine bacterial strains named FDZSB0410T and FDZWPB0420, were isolated from the intestine of two adult abalone species, Haliotis diversicolor and Haliotis discushannai, respectively. The strains were non-flagellated, motile by gliding, oxidase-positive but catalase-negative, and agar-degrading. The two strains had identical 16S rRNA gene sequences with the highest sequence similarity less than 96.0 % with species of the genera Tenacibaculumand Polaribacter within the family Flavobacteriaceae. Phylogenetic analysis indicated the strains represented an independent cluster neighbouring with the species of the genera Tenacibaculum and Polaribacter. Strain FDZSB0410T can grow under conditions at 15 to 37 °C with optimum of 30 °C, salinity range from 3 to 4 % (NaCl, w/v) with optimum of 3 %, and pH range from 7 to 8 with optimum of pH 7. The predominant fatty acids of strain FDZSB0410T consisted of iso-C15 : 0, iso-C15 :1 G, summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c), iso-C13 : 0, iso-C17 : 0 3-OH, and iso-C15 : 0 3-OH. The ubiquinone system detected was MK-6. The flexirubin pigment was absent, but the carotenoid was present. The polar lipids were identified as aminolipid, glycolipid, phosphatidylglycerol, an unknown phospholipid and four unknown lipids. The DNA G+C content was 35.3 mol%. On the basis of the genotypic and phenotypic characterizations, strain FDZSB0410T and strain FDZWPB0420 represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Pseudotenacibaculum haliotis gen. nov., sp. nov. is proposed. The type strain of the type species is FDZSB0410T (=KCTC 52127T=MCCC 1A01897T).

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

A Gram-stain-negative, non-flagellated, non-gliding, non-spore-forming bacterial strain, designated YKTF-3T, was isolated from a tidal flat on the Yellow Sea, South Korea. Strain YKTF-3T grew optimally at 30 °C and in the presence of 2.0-4.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain YKTF-3T fell within the clade comprising the type strains of Tenacibaculumspecies. The novel strain exhibited 16S rRNA gene sequence similarity values of 97.22-98.34 % to the type strains of Tenacibaculum gallaicum, Tenacibaculum ascidiaceicola, Tenacibaculum litoreum, Tenacibaculum discolor, Tenacibaculum aestuarii and Tenacibaculum lutimaris, and of 95.08-96.95 % to the type strains of the other Tenacibaculum species. Strain YKTF-3T contained MK-6 as the predominant menaquinone and iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and iso-C17 : 0 3-OH as the major fatty acids. The major polar lipids of strain YKTF-3T were phosphatidylethanolamine, one unidentified lipid, one unidentified aminophospholipid, one unidentified glycolipid and one unidentified aminolipid. The DNA G+C content was 32.3 mol% and its mean DNA-DNA relatedness values with the type strains of the six phylogenetically closely relatedTenacibaculum species were 10-27 %. Differential phenotypic properties, together with phylogenetic and genetic distinctiveness, revealed that strain YKTF-3T is separate from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain YKTF-3T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum sediminilitoris sp. nov. is proposed. The type strain is YKTF-3T (=KCTC 52210T=NBRC 111991T).

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).

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)).