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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., (© 2024 John Wiley & Sons Ltd.)

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl 3 had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2023. Published by Elsevier B.V.)

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., (© 2021 John Wiley & Sons Ltd.)

A novel bright yellow pigmented, Gram-stain-negative, gliding, aerobic and rod-shaped marine bacterium, designated strain S7007 T , 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 S7007 T was associated with the genus Tenacibaculum and showed highest similarity to Tenacibaculum adriaticum JCM 14633 T (98.0%). The average nucleotide identity (ANI) scores of strain S7007 T with T. adriaticum JCM 14633 T and T. maritimum NBRC 110778 T 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-C 15:0 , iso-C 15:0 3-OH, iso-C 15: 1 G and summed feature 3 (comprising C 16:1 ω7c and/or C 16:1 ω6c). The major polar lipids of strain S7007 T 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 S7007 T 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 S7007 T (= MCCC 1H00428 T  = KCTC 72941 T ).

Numerous Tenacibaculum species, including T. dicentrarchi, T. maritimum and T. finnmarkense, are associated with tenacibaculosis in finfish; however, quantitative identification techniques are limited. Quantitative PCR assays were developed to detect T. dicentrarchi and T. finnmarkense. TaqMan assays using 16S rDNA demonstrated low detection limits (0.07-269 bacteria), suitable amplification efficiencies (>86%) and moderate specificity. However, the amplification of isolates with 100% sequence similarity to T. finnmarkense AY7486TD using both the T. finnmarkense and T. dicentrarchi assays indicates that other genes should be investigated. Both assays may help describe the pathogenesis of tenacibaculosis and may aid management practices for the aquaculture industry., (© 2021 John Wiley & Sons Ltd.)

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 TNO020 T 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 TNO020 T , 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 TNO020 T (=CCUG 73833 T =NCIMB 15240 T ) 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 TNO006 T (=CCUG 73831 T =NCIMB 15238 T ) as the type strain. We further propose the subdivision of T. finnmarkense sp. nov. into two genomovars, T. finnmarkense genomovar finnmarkense with strain TNO006 T (=CCUG 73831 T =NCIMB 15238 T ) as the type strain and T. finnmarkense genomovar ulcerans with strain TNO010 T (=CCUG 73832 T =NCIMB 15239 T ) as the type strain.

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., (© 2020 John Wiley & Sons Ltd.)

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-A2 T , shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764 T . The genome of strain TLL-A2 T was sequenced and compared to that of T. mesophilum DSM 13764 T . Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764 T and strain TLL-A2 T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2 T and T. mesophilum DSM 13764 T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2 T (=DSM 106434 T , KCTC 62393 T ) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

An outbreak of disease characterized by skin ulcers, fin rot and mortality was observed a few days after the transfer of Atlantic salmon (Salmo salar) from a freshwater smolt production facility to a land-based seawater post-smolt site. Dead and moribund fish had severe skin and muscle ulcers, often 2-6 cm wide, particularly caudal to the pectoral fins. Microscopic examination of smears from ulcers and head kidney identified long, slender Gram-negative rods. Histopathological analysis revealed abundance of long, slender Tenacibaculum-like bacteria in ulcers and affected fins. Genetic characterization using multilocus sequence analysis (MLSA) of seven housekeeping genes, including atpA, dnaK, glyA, gyrB, infB, rlmN and tgt, revealed that the isolates obtained during the outbreak were all clustered with the Tenacibaculum dicentrarchi-type strain (USC39/09 T ) from Spain. Two bath challenge experiments with Atlantic salmon and an isolate of T. dicentrarchi from the outbreak were performed. No disease or mortality was observed in the first trial. In the second trial with a higher challenge dose of T. dicentrarchi and longer challenge time, we got 100% mortality within 48 hr. This is the first reported outbreak of disease caused by T. dicentrarchi in Norwegian farmed Atlantic salmon., (© 2019 John Wiley & Sons Ltd.)

A PCR protocol was optimised and validated for the detection of viable Tenacibaculum maritimum cells in salmon skin tissue. Viability conventional (vPCR) and quantitative PCR (v-qPCR) assays both had a limit of detection of 10 3  CFU mL -1 viable cells. The v-qPCR assay showed a linear quantification over 4 log units. Conventional vPCR showed complete signal suppression when only dead cells were present at concentrations lower than 10 6  CFU mL -1 . While the v-qPCR did not result in complete suppression when only dead cells were present, a method was developed to determine if viable cells were present based on the % Δ in cycle threshold (Ct) value. The procedure was validated for high-throughput processing and an enrichment protocol was validated to reliably detect low concentrations of viable cells both with and without a high background of dead cells. Performing this protocol on naturally infected tissues showed that vPCR and v-qPCR reduced the potential for false positives compared to using conventional PCR and qPCR. The optimised protocol developed for this study provides an efficient, reliable and robust alternative for the detection of viable T. maritimum in skin tissue., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

A Gram-stain-negative, rod-shaped, aerobic bacterial strain, designated LPB0136 T , was isolated from a squid Todarodes pacificus, caught in the East Sea, off Korea. LPB0136 T 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-4 T and Tenacibaculum caenipelagi HJ-26M T . 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 LPB0136 T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculum todarodis sp. nov. is proposed. The type strain is LPB0136 T (=KACC 18887 T =JCM 31564 T ).

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.

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., (© 2017 John Wiley & Sons Ltd.)

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterial strain, designated JDTF-31 T , was isolated from a tidal flat in Jindo, a South Korean island. Strain JDTF-31 T 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-31 T 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-31 T 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-31 T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminophospholipid. The DNA G+C content of strain JDTF-31 T 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-31 T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain JDTF-31 T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculuminsulae sp. nov. is proposed. The type strain is JDTF-31 T (=KCTC 52749 T =NBRC 112783 T ).

A novel Gram-stain-negative, aerobic, rod-shaped, non-flagellated and agar-digesting marine bacterium, designated as HZ1 T , 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 HZ1 T 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%. HZ1 T 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 HZ1 T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculumagarivorans sp. nov. is proposed. The type strain is HZ1 T (=MCCC 1H00174 T =KCTC 52476 T ).

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterial strain, designated JDTF-79 T , 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-79 T 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-79 T 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-79 T 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-79 T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminophospholipid. The DNA G+C content of strain JDTF-79 T was 30.3 mol%. Strain JDTF-79 T 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-79 T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain JDTF-79 T 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-79 T (=KCTC 52980 T =NBRC 112903 T ).

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.

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).

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'., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

Skin ulcer development in sea-reared salmonids, commonly associated with Tenacibaculum spp., is a significant fish welfare- and economical problem in Norwegian aquaculture. A collection of 89 Tenacibaculum isolates was subjected to multilocus sequence analysis (MLSA). The isolates were retrieved from outbreaks of clinical disease in farms spread along the Norwegian coast line from seven different fish species over a period of 19 years. MLSA analysis reveals considerable genetic diversity, but allows identification of four main clades. One clade encompasses isolates belonging to the species T. dicentrarchi, whereas three clades encompass bacteria that likely represent novel, as yet undescribed species. The study identified T. maritimum in lumpsucker, T. ovolyticum in halibut, and has extended the host and geographic range for T. soleae, isolated from wrasse. The overall lack of clonality and host specificity, with some indication of geographical range restriction argue for local epidemics involving multiple strains. The diversity of Tenacibaculum isolates from fish displaying ulcerative disease may complicate vaccine development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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).

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).

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., (© 2016 Blackwell Verlag GmbH.)

This report describes a case of the first isolation of Tenacibaculum maritimum from a captive-bred adult female sand tiger shark (Carcharias taurus) housed at the Cattolica Aquarium (Italy). The animal showed, between the second dorsal fin and the precaudal pit, skin lesions characterized by the presence of abundant whitish necrotic tissue. Through routine bacteriological examination, a bacterium was isolated from a skin lesion and subsequently identified as T. maritimum by phenotypic characters and species-specific polymerase chain reaction. The antimicrobial sensitivity of the isolated strain was evaluated for 11 antimicrobial agents by disk diffusion method. Antibiotic therapy was conducted with enrofloxacin at 10 mg kg(-1) i.m. on alternate days for 10 days. One month after the end of treatment skin lesions showed complete resolution and the shark recovered completely. The case presented here represents the first report of infection by T. maritimum in a sand tiger shark and highlights the potential pathogenic role of this microorganism in elasmobranchs kept in an aquarium.

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).

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)).

A novel bacterial strain, designated S2-2T, was isolated from the intestine of a sea cucumber Apostichopus japonicus in Xiapu, Fujian province, China. Strain S2-2T was found to be aerobic, Gram-stain-negative, pale yellow, rod-shaped, oxidase- and catalase-positive. Growth occurred at 15-36 °C (optimum, 25-32 °C), in the presence of 2-7% sea salt (w/v, optimum, 3-5%) and at pH 6-9 (optimum, pH 7.0). The isolate was able to hydrolyse gelatin, casein and DNA, but unable to degrade Tween 20, 40 and 80, starch and cellulose. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain S2-2T represented a member of the genus Tenacibaculum, with highest sequence similarity to Tenacibaculum aiptasiae a4T (96.9% similarity), followed by Tenacibaculum xiamenense WJ-1T (96.5% similarity) and showed lower similarities (93.3-95.9%) with other members of the genus Tenacibaculum. The major cellular fatty acids were summed feature 3 (comprising C16 : 1ω6c/C16 : 1ω7c, 33.8%), iso-C15 : 0 (13.7%), iso-C15 : 1G (8.6%), iso-C15 : 0 3-OH (5.3%) and C15 : 0 3-OH (5.1%). The DNA G+C content of the chromosomal DNA was determined to be 31.8 mol%. The respiratory quinone was determined to be MK-6. Phosphatidylethanolamine, phosphatidylglycerol, one unknown aminophospholipid, one unknown phospholipid, one unknown glycolipid and five unknown lipids were detected as major polar lipids. Hence, the combined genotypic and phenotypic data indicated that strain S2-2T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculum holothuriorum sp. nov. is proposed. The type strain is S2-2T ( = MCCC 1A09872T = LMG 27758T).

With the aim of finding new chemical entities selective for fish pathogens to avoid drug resistance in humans, a series of coumarin-chalcone hybrid compounds with different patterns of substitution were designed and synthesized. Their antibacterial activity was evaluated against important types of human bacteria strains (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) and against a fourteen strains of the marine pathogen Tenacibaculum maritimum, responsible for tenacibaculosis in fish, which is an important disease that causes great economical loss in the aquaculture industry. All the amino derivatives 5-12 presented high activity against different strains of T. maritimum, no activity against any of the three human pathogenic bacteria strains and no toxicity. Compounds 6, 7 and 11 were the most promising molecules. The most sensitive strains to these compounds were LL01 8.3.8 and LL01 8.3.1, being compound 11 up to 20 times more active than enrofloxacin. Therefore these scaffolds are good candidates for aquaculture treatments, avoiding possible drug resistance problems in humans., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Tenacibaculum maritimum is a Gram-negative bacterium that causes large losses in farmed turbot, Psetta maxima (L.). Main lesions included erosive and ulcerative skin injuries in different locations in the body of the fish. This study was set up to gain insights into the immune response of the turbot against this bacterial infection. To determine the variations in the peripheral blood leukocytes a minimum of 200 leukocytes in blood smears were counted. The presence of immunoglobulin positive (Ig(+)) cells in spleen, kidney, intestine, thymus and skin from turbot experimentally infected with T. maritimum were studied using an immunohistochemical method. In fish challenged with the bacterium the percentage of circulating granulocytes, lymphocytes and trombocytes showed significant differences at different points of sampling. Moreover, the number of Ig(+) cells significantly increased in the spleen, kidney, intestine and thymus. In the skin a strong immunoreactivity was observed in the interstitial liquid in damaged areas. The results obtained in this study indicated that the infection by T. maritimum in turbot induced an immune response that involved changes in the number of peripheral blood leukocytes and in the number and distribution of Ig(+) cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A specific and sensitive multiplex PCR (mPCR) method was developed as a useful tool for the simultaneous detection of two important flatfish pathogens in marine aquaculture, Tenacibaculum maritimum and Edwardsiella tarda. In fish tissues, the average detection limit for these mPCR-amplified organisms was 2 × 10 ⁵ ± 0.2 CFU/g and 4 × 10 ⁵ ± 0.3 CFU/g, respectively. These values are similar or even lower than those previously obtained using the corresponding single PCR. Moreover, mPCR did not produce any nonspecific amplification products when tested against 36 taxonomically related and unrelated strains belonging to 33 different bacterial species. Large amounts of DNA from one of the target bacterial species in the presence of low amounts from the other did not have a significant effect on the amplification sensitivity of the latter., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)

Tenacibaculum maritimum is the causative agent of tenacibaculosis, a bacterial disease with a worldwide distribution, which causes important losses in the turbot aquaculture. Despite the importance of this bacterium, little is known about pathogenesis of the tenacibaculosis, pattern of lesions and the portal of entry of T. maritimum. Turbots (Psetta maxima) were experimentally infected with T. maritimum using subcutaneous and intraperitoneal routes of inoculation and samples of skin and internal organs were taken throughout the assay. Fish inoculated by both infection routes suffered a septicaemia but only the subcutaneous inoculation reproduces the disease signs described in natural outbreaks. Bacterial antigen was detected by immunohistochemistry in the internal organs 3 h after infection in fish inoculated by subcutaneous route and 6 h after the inoculation of fish by intraperitoneal route. In summary, both routes of inoculation are able to cause an infection and bacteraemia in the fish. However, subcutaneous inoculation route reproduces the disease in a faster and more reliable way than the intraperitoneal route. Moreover, bacterium spreads along the internal organs easily, but needs a gateway to penetrate in the organism and this portal of entry could be skin., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A Gram-stain-negative, elongated rod-shaped, yellow-pigmented, aerobic bacterial strain, designated WJ-1(T), was isolated from coastal seawater in Xiamen, Fujian province, China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain WJ-1(T) fell within the genus Tenacibaculum and was most closely associated with Tenacibaculum aestuarii SMK-4(T) (96.7% 16S rRNA gene sequence similarity); lower similarities were shown to other members of the genus Tenacibaculum (<96.2%). The strain formed a distinct lineage with Tenacibaculum litopenaei B-I(T) (96.0%), Tenacibaculum geojense YCS-6(T) (94.5%) and Tenacibaculum jejuense CNURIC 013(T) (95.4%). Growth was observed at temperatures from 16 to 38 °C, at salinities from 2 to 4% and at pH 6-9. The major fatty acids were summed feature 3 (C(16:1)ω6c and/or C(16 1)ω7c), iso-C(17:0) 3-OH, iso-C(15:0) and iso-C(15:0) 3-OH. The DNA G+C content of strain WJ-1(T) was 33.2 mol% and the major respiratory quinone was menaquinone-6 (MK-6). Differential phenotypic properties and phylogenetic distinctiveness in this study distinguished strain WJ-1(T) from all other members of the genus Tenacibaculum. According to the morphology, physiology, fatty acid composition and 16S rRNA gene sequence data, strain WJ-1(T) represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculum xiamenense sp. nov. is proposed. The type strain is WJ-1(T) ( =CGMCC 1.12378(T) =LMG 27422(T)).

A Gram-stain-negative, non-spore-forming, aerobic, non-flagellated, gliding and rod-shaped bacterial strain, designated HJ-26M(T), was isolated from a tidal flat sediment in the Korean peninsula. It grew optimally at 25-30 °C, at pH 7.0-8.0 and in the presence of 2 % (w/v) NaCl. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that the strain fell within the clade comprising Tenacibaculum species, clustering coherently with the type strains of Tenacibaculum lutimaris and Tenacibaculum aestuarii. Strain HJ-26M(T) exhibited the highest 16S rRNA gene sequence similarity values of 98.4 and 98.2 % to T. lutimaris TF-26(T) and T. aestuarii SMK-4(T), respectively, and of 94.9-97.4 % to the type strains of the other Tenacibaculum species. Strain HJ-26M(T) contained MK-6 as the predominant menaquinone and iso-C15:0 and iso-C17:0 3-OH as the major fatty acids. The DNA G+C content of strain HJ-26M(T) was 34.5 mol% and its mean DNA-DNA relatedness values with the type strains of T. lutimaris and T. aestuarii were 19 and 23 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain HJ-26M(T) is separate from other Tenacibaculum species. On the basis of the data presented, strain HJ-26M(T) is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum caenipelagi sp. nov. is proposed. The type strain is HJ-26M(T) (= KCTC 32323(T) = CECT 8283(T)).

A Gram-negative, non-spore-forming, aerobic, non-flagellated, non-gliding and rod-shaped bacterial strain, designated P-R2A1-2(T), was isolated from sea squirt (Halocynthia roretzi) collected from the South Sea, Korea. It grew optimally at 25-28 °C, at pH 7.0-8.0 and in the presence of 2 % (w/v) NaCl. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that the strain fell within the clade comprising Tenacibaculum species. Strain P-R2A1-2(T) exhibited the highest 16S rRNA gene sequence similarity values of 97.6, 97.2 and 97.0 % to Tenacibaculum aestuarii SMK-4(T), T. lutimaris TF-26(T) and T. aiptasiae a4(T), respectively, and of 94.5-96.8 % to the type strains of the other Tenacibaculum species. Strain P-R2A1-2(T) contained MK-6 as the predominant menaquinone and C16:1 ω7c and/or iso-C15:0 2-OH, iso-C15:0 3-OH and iso-C15:0 as the major fatty acids. The DNA G + C content of strain P-R2A1-2(T) was 30.7 mol % and its DNA-DNA relatedness values with the type strains of T. aestuarii, T. lutimaris and T. aiptasiae were 17 ± 4.2, 21 ± 6.1 and 16 ± 5.2 %, respectively. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that the novel strain is separate from other Tenacibaculum species. On the basis of the data presented, strain P-R2A1-2(T) is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum halocynthiae sp. nov. is proposed. The type strain is P-R2A1-2(T) (=KCTC 32262(T )= CCUG 63681(T)).

A Gram-staining-negative, strictly aerobic and rod-shaped bacterium, designated strain CNURIC013(T), was isolated from seawater collected on the coast of Jeju Island, South Korea. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CNURIC013(T) belonged to the genus Tenacibaculum, within the family Flavobacteriaceae. Sequence similarities between the novel strain and the type strains of recognized species of the genus Tenacibaculum were 93.6-96.0 %, the highest value being with Tenacibaculum litopenaei B-I(T) (96 %). The DNA G+C content of the novel strain was 34.5 mol% and the major respiratory quinone was menaquinone-6. The major fatty acids were summed feature 3 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH; 26.0 %), iso-C(15 : 0) (24.4 %), iso-C(15 : 1) G (18.5 %) and iso-C(17 : 0) 3-OH (8.1 %). The polar lipids consisted of phosphatidylethanolamine, one unknown aminophospholipid and nine unknown polar lipids. On the basis of the phenotypic, phylogenetic and genotypic data, strain CNURIC013(T) represents a novel species within the genus Tenacibaculum, for which the name Tenacibaculum jejuense sp. nov. is proposed. The type strain is CNURIC013(T) ( = KCTC 22618(T) = JCM 15975(T)).

A novel Gram-stain-negative rod-shaped gliding bacterial strain, designated 35/09(T), was isolated from diseased European sea bass (Dicentrarchus labrax L.) in Spain. Colonies were pale-yellow-pigmented with uneven edges and did not adhere to the agar. The DNA G+C content of the isolate was 31.3 mol%. 16S rRNA gene sequence analysis indicated affiliation to the genus Tenacibaculum (family Flavobacteriaceae, phylum 'Bacteroidetes'). Sequence similarities between the isolate and type strains of other members of the genus were 93.1-97.3 %. The major fatty acids (>5 % of the total fatty acids) were iso-C(15 : 0) (24.8 %), iso-C(15 : 0) 3-OH (18.0 %), anteiso-C(15 : 0) (8.1 %), C(15 : 1)ω6c (6.9 %) and iso-C(15 : 1) (6.2 %). Genotypic and phenotypic data indicate that strain 35/09(T) should be classified as a representative of a novel species in the genus Tenacibaculum, for which the name Tenacibaculum dicentrarchi sp. nov. is proposed; the type strain is 35/09(T) ( = CECT 7612(T) = NCIMB 14598(T)).

A Gram-negative, non-flagellated, non-spore-forming bacterium, designated YCS-6(T), that was motile by gliding, was isolated from seawater on the southern coast of Korea. Strain YCS-6(T) grew optimally at 30 °C and with 2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YCS-6(T) fell within the genus Tenacibaculum and was most closely associated with Tenacibaculum litopenaei B-I(T), with which the isolate exhibited 95.9% 16S rRNA gene sequence similarity. Sequence similarity between strain YCS-6(T) and other members of the genus Tenacibaculum was 93.8-95.7%. Strain YCS-6(T) contained menaquinone-6 (MK-6) as the predominant respiratory quinone and iso-C(15:0), summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1)ω7c), iso-C(15:0) 3-OH and iso-C(15:1) G as the major fatty acids. The DNA G+C content was 32.7 mol%. Differential phenotypic properties and phylogenetic distinctiveness distinguished strain YCS-6(T) from all other members of the genus Tenacibaculum. On the basis of our phenotypic, chemotaxonomic and phylogenetic data, strain YCS-6(T) is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum geojense sp. nov. is proposed. The type strain is YCS-6(T) (=KCTC 23423(T) =CCUG 60527(T)).

The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen., (2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)