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Phylogenetic tree based on 16S rRNA gene sequence comparisons, obtained with the neighbour-joining algorithm, showing the relationships of Flavobacterium plurextorum sp. nov. with related species. Flexibacter flexilis ATCC 23079 T was used as an outgroup. Bootstrap values (expressed as a percentage of 1,000 replications) greater than 70% are given at the nodes. Solid circles indicate that the corresponding nodes (groupings) are also obtained on the maximum-likelihood tree. Open circles indicate that the corresponding nodes (groupings) are also obtained on the maximum-likelihood and parsimony trees. Sequence accession numbers are indicated in brackets. Bar, 1% sequence divergence. doi:10.1371/journal.pone.0067741.g001
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Five strains (1126-1H-08(T), 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a tempera...
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... 1126-1H-08 T and the type strains of the closest phylogenetically related species. DNA was extracted and purified by the method of Marmur [22]. Hybridization studies were carried out, using the membrane method of Johnson [23], described in detail by Arahal et al. [24]. The hybridization experiments were carried out under optimal conditions, at a temperature of 44 u C, which is within the limits of validity for the membrane method [25]. The percentages of hybridization were calculated as described by Johnson [26]. Three independent determinations were carried out for each experiment and the results reported as mean values. The type strains of species F. aquidurense CCUG 59847 T , F. araucananum CCUG 61031 T , F. hydatis DSM 2063 T , F. pectinovorum CCUG 58916 T , F. frigidimaris CCUG 59364 T , F. chungangense CCUG 58910T and F. oncorhynchi CECT 7678 T were included in this study. Respiratory quinones of strain 1126-1H-08 were extracted from 100 mg of freeze-dried cell material, using the two stage method described by Tindall [27,28], and further separated by thin layer chromatography on silica gel and analyzed, using HPLC, by the identification service of the DSMZ (Braunschweig, Germany). For cell fatty acid-fatty acid methyl ester (CFA-FAME) analyses, strain 1126-1H-08 T was grown on Columbia II agar base (BBL 4397596) with 5% horse blood, at 30 u C for 30–48 h, under aerobic conditions. The CFA-FAME profile was determined using gas chromatography (Hewlett Packard HP 5890) and a standard- ized protocol similar to that of the MIDI Sherlock MIS system [29], described previously [10]. CFAs were identified and the relative amounts were expressed as percentages of the total fatty acids of the respective strains. The minimal standards for the description of new taxa in the family Flavobacteriaceae [30] were followed for the phenotypic characterization of the strains. Gram-staining was performed as described by Smibert & Krieg [31]. Oxidase activity was determined by monitoring the oxidation of tetramethyl- p -phenyl- enediamine on filter paper and catalase activity was determined, using 3% H 2 O 2 solution [31]. Hydrolysis of L-tyrosine (0.5%, w/ v), lecithin (5%, w/v) [31], esculin (0.01% esculin and 0.05% ferric citrate, w/v), gelatin (4%; w/v), starch (0.2%, w/v), and casein [50% skimmed milk (Difco), v/v] were tested using nutrient agar as basal medium [30]. DNase test agar (Difco) was used for the DNase assay. Hydrolysis of urea (1%, w/v) was tested as described by Bowman et al. [32]. Growth in brain heart infusion broth was assessed at 15, 25, 30, 37 and 42 u C, with 3.0, 4.5 and 6.5% added NaCl, and under anaerobic (with 4–10% CO 2 ) and micro-aerobic (with 5–15% O 2 and 5–12% CO 2 ) conditions, using GasPak Plus and CampyPak Plus systems (BBL), respectively. Growth was tested on MacConkey (bioM ́rieux), nutrient (Difco) and trypticase-soy (bioM ́rieux) agar plates. The presence of gliding motility, using the hanging drop technique, and the production of flexirubin-type pigments and extracellular glycans were assessed, using the KOH and Congo red tests, respectively [1]. The strains were further biochemically characterized using the API 20NE and API ZYM systems (bioM ́rieux) according to the manufacturer’s instructions, except that incubation temperature was 25 u C. The type strains of species F. aquidurense CCUG 59847 T , F. araucananum CCUG 61031 T , F. hydatis DSM 2063 T , F. pectinovorum CCUG 58916 T , F. frigidimaris CCUG 59364 T , F. chungangense CCUG 58910 T and F. oncorhynchi CECT 7678 T were included in this study as references for the investigation of the phenotypic properties of the trout strains, using the same laboratory conditions. The five strains were characterized by pulsed-field gel electrophoresis (PFGE), after digestion of their genomic DNAs with the restriction enzymes Bsp120 I and Xho I, according to the specifica- tions of Chen et al. [33]. DNA fragments were resolved in a 1% agarose gel with a pulse-field gel electrophoresis apparatus, CHEF-DR III (Bio-Rad), at 6V/cm for 40 hours, with switching times ramped from 0.1 to 12 s at 14 u C, with an angle of 120 u . The gels were stained for 30 min with Syber-Safe and photographed under UV light (Gel-Doc, Bio-Rad). Strains differing in at least one band were considered different. 16S rRNA gene sequences were determined for the five trout strains, displaying 100% 16S rRNA sequence similarity among them. Sequence searches showed that the 16S rRNA gene sequence of the strains were most similar to those of species of the genus Flavobacterium , exhibiting the highest levels of similarity with the sequence of the type strains of Flavobacterium oncorhynchi CECT 7678 T and Flavobacterium pectinovorum DSM 6368 T (98.5% and 97.9% sequence similarity, respectively). In addition, strains exhibited 16S rRNA gene sequence similarities greater than 97.0% with other seventeen other Flavobacterium species. It is clear from the phylogenetic analysis (Fig. 1) that the trout strains held a clear affiliation to the genus Flavobacterium and represented a distinct sub-lineage clustering with a cluster of four species that included F. pectinovorum, F. chilense, F. oncorhynchi and F. hercynium . However, their position within this sub-group was not supported by significant bootstrap values. The GenBank accession numbers for the 16S rRNA gene sequences of five strains sequenced in this study are shown in Fig. 1. Genomic DNA–DNA hybridizations between the trout strains yielded binding values of 87 to 100%. Flavobacterium species with 16S rRNA gene sequence similarities to the sequences of the trout strains lower than 98.0% correlated with levels of genomic DNA- DNA relatedness always lower than 70% [9–11,34–36]. For that reason, DNA-DNA hybridizations were carried out only between strain 1126-1H-08 T and the type strains of the phylogenetically closest related species; i.e. , those species with 16S rRNA gene sequence similarities greater than 97.5%. The levels of DNA-DNA relatedness for strain 1126-1H-08 T with respect to F. aquidurense CCUG 59847 T , F. araucananum CCUG 61031 T , F. hydatis DSM 2063 T , F. pectinovorum CCUG 58916 T , F. frigidimaris CCUG 59364 T , F. chungangense CCUG 58910 T and F. oncorhynchi CECT 7678 T ranged between 21 and 48%. These values were below the 70% cut-off point for species delineation [37,38] and clearly confirmed that the trout strains belong to a distinct genomic species of the genus Flavobacterium . The DNA G + C content of strain 1126-1H-08 T was 33.2 mol%, a value consistent with those of the genus Flavobacterium [1,30]. Chemotaxonomic characteristics of strain 1126-1H-08 T were in accordance with those of members of the genus Flavobacterium [5,6]: the major quinone was MK-6 (95%) with minor amounts of MK-5 (5%). The predominant cell fatty acids of strain 1126-1H- 08 T were iso-C 15:0 (19%) and C 15:0 (15%). Strain 1126-1H-08 T also contained moderate or small amounts of C 16:1 v 7c (10%), C 15:1 v 6c (9%), iso-C 15:0 3-OH, C 17:1 v 6c , isoG-C 15:1 (6%/each), iso-C 17:0 3-OH (5%), iso-C 17:1 v 9c, C 15:0 3-OH, C 16:0 3-OH (3%/ each), isoaldehyde-C 15:0 , C 16:0 , iso-C 16:0 3-OH, unknown fatty acids with an equivalent chain length of 11.5 (2%/each) and C 17:1 v 8c , iso-C16:0, C 12:1 , aldehyde-C 14:0 , anteiso-C 15:0 and unknown fatty acids with an equivalent chain lengths of 14.8 and 12.5 (1%/ each) (Table 1). The trout strains exhibited identical physiological and biochemical characteristics. Cells were Gram-negative rods, 0.7 m m wide and 3 m m long, non-endospore-forming, and non-gliding. Strains grew well under aerobic conditions and grew weakly under micro-aerobic conditions. Strains grew at 15–30 u C with optimal growth at approximately 25 u C, while no growth was observed at 37 u C or 42 u C. Growth occurred on trypticase-soy and nutrient agars but not on Marine agar after incubation at 25 u C for 72 hours. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25 u C. Colonies are non-hemolytic on Columbia agar after 72 hours incubation at 25 u C. Diffusible flexirubin-type pigments were produced and congo red was not absorbed by colonies. Growth did not occur in brain heart infusion broth containing 3, 4.5 and 6.5% NaCl. Catalase and oxidase were produced and nitrate and nitrite were reduced. Starch and tyrosine were degraded but DNA, gelatin, casein or agarose were not. A brown pigment was not produced on tyrosine agar. Aesculin was hydrolyzed but not urea, lecithin and arginine. Indole and H 2 S were not produced. Acid was not produced from D-glucose. Arabinose, D-glucose, mannose, N- acetyl-glucosamine, and maltose were used as sole carbon and energy sources but not citrate, mannitol, gluconate, caprate, adipate, and malate. Activities for alkaline phosphatase, leucine arylamidase, N-acetyl- b -glucosaminidase, a -glucosidase, acid phosphatase, and naphthol-AS-BI-phophohydrolase were detected. Esterase C4, valine arylamidase, b -galactosidase, ester lipase C8, lipase C14, cystine arylamidase, a -chymotrypsin, trypsin, a - galactosidase, b -glucuronidase, b -glucosidase, a -mannosidase and a -fucosidase were not detected. The phenotypic characteristics that differentiated the trout strains from phylogenetically related species are shown in Table 2. The new species also can be also differentiated from the clinically relevant fish pathogens F. columnare , F. psycrophilum and F. branchiophilum , by the inability of these three species to grow in trypticase-soy agar and to hydrolyze aesculin [4]. Other species isolated from diseased fish such as F. hydatis , F. jonshoniae and F. succinicans are motile (gliding), degrade DNA and produce acid from carbohydrates [4], while the new species exhibited opposite results for those tests. Moreover, the new species can be readily differentiated from F. chilense and F. araucananum because the latter species are motile ...
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... of hybridization were calculated as described by Johnson [26]. Three independent determinations were carried out for each experiment and the results reported as mean values. The type strains of species F. aquidurense CCUG 59847 T , F. araucananum CCUG 61031 T , F. hydatis DSM 2063 T , F. pectinovorum CCUG 58916 T , F. frigidimaris CCUG 59364 T , F. chungangense CCUG 58910T and F. oncorhynchi CECT 7678 T were included in this study. Respiratory quinones of strain 1126-1H-08 were extracted from 100 mg of freeze-dried cell material, using the two stage method described by Tindall [27,28], and further separated by thin layer chromatography on silica gel and analyzed, using HPLC, by the identification service of the DSMZ (Braunschweig, Germany). For cell fatty acid-fatty acid methyl ester (CFA-FAME) analyses, strain 1126-1H-08 T was grown on Columbia II agar base (BBL 4397596) with 5% horse blood, at 30 u C for 30–48 h, under aerobic conditions. The CFA-FAME profile was determined using gas chromatography (Hewlett Packard HP 5890) and a standard- ized protocol similar to that of the MIDI Sherlock MIS system [29], described previously [10]. CFAs were identified and the relative amounts were expressed as percentages of the total fatty acids of the respective strains. The minimal standards for the description of new taxa in the family Flavobacteriaceae [30] were followed for the phenotypic characterization of the strains. Gram-staining was performed as described by Smibert & Krieg [31]. Oxidase activity was determined by monitoring the oxidation of tetramethyl- p -phenyl- enediamine on filter paper and catalase activity was determined, using 3% H 2 O 2 solution [31]. Hydrolysis of L-tyrosine (0.5%, w/ v), lecithin (5%, w/v) [31], esculin (0.01% esculin and 0.05% ferric citrate, w/v), gelatin (4%; w/v), starch (0.2%, w/v), and casein [50% skimmed milk (Difco), v/v] were tested using nutrient agar as basal medium [30]. DNase test agar (Difco) was used for the DNase assay. Hydrolysis of urea (1%, w/v) was tested as described by Bowman et al. [32]. Growth in brain heart infusion broth was assessed at 15, 25, 30, 37 and 42 u C, with 3.0, 4.5 and 6.5% added NaCl, and under anaerobic (with 4–10% CO 2 ) and micro-aerobic (with 5–15% O 2 and 5–12% CO 2 ) conditions, using GasPak Plus and CampyPak Plus systems (BBL), respectively. Growth was tested on MacConkey (bioM ́rieux), nutrient (Difco) and trypticase-soy (bioM ́rieux) agar plates. The presence of gliding motility, using the hanging drop technique, and the production of flexirubin-type pigments and extracellular glycans were assessed, using the KOH and Congo red tests, respectively [1]. The strains were further biochemically characterized using the API 20NE and API ZYM systems (bioM ́rieux) according to the manufacturer’s instructions, except that incubation temperature was 25 u C. The type strains of species F. aquidurense CCUG 59847 T , F. araucananum CCUG 61031 T , F. hydatis DSM 2063 T , F. pectinovorum CCUG 58916 T , F. frigidimaris CCUG 59364 T , F. chungangense CCUG 58910 T and F. oncorhynchi CECT 7678 T were included in this study as references for the investigation of the phenotypic properties of the trout strains, using the same laboratory conditions. The five strains were characterized by pulsed-field gel electrophoresis (PFGE), after digestion of their genomic DNAs with the restriction enzymes Bsp120 I and Xho I, according to the specifica- tions of Chen et al. [33]. DNA fragments were resolved in a 1% agarose gel with a pulse-field gel electrophoresis apparatus, CHEF-DR III (Bio-Rad), at 6V/cm for 40 hours, with switching times ramped from 0.1 to 12 s at 14 u C, with an angle of 120 u . The gels were stained for 30 min with Syber-Safe and photographed under UV light (Gel-Doc, Bio-Rad). Strains differing in at least one band were considered different. 16S rRNA gene sequences were determined for the five trout strains, displaying 100% 16S rRNA sequence similarity among them. Sequence searches showed that the 16S rRNA gene sequence of the strains were most similar to those of species of the genus Flavobacterium , exhibiting the highest levels of similarity with the sequence of the type strains of Flavobacterium oncorhynchi CECT 7678 T and Flavobacterium pectinovorum DSM 6368 T (98.5% and 97.9% sequence similarity, respectively). In addition, strains exhibited 16S rRNA gene sequence similarities greater than 97.0% with other seventeen other Flavobacterium species. It is clear from the phylogenetic analysis (Fig. 1) that the trout strains held a clear affiliation to the genus Flavobacterium and represented a distinct sub-lineage clustering with a cluster of four species that included F. pectinovorum, F. chilense, F. oncorhynchi and F. hercynium . However, their position within this sub-group was not supported by significant bootstrap values. The GenBank accession numbers for the 16S rRNA gene sequences of five strains sequenced in this study are shown in Fig. 1. Genomic DNA–DNA hybridizations between the trout strains yielded binding values of 87 to 100%. Flavobacterium species with 16S rRNA gene sequence similarities to the sequences of the trout strains lower than 98.0% correlated with levels of genomic DNA- DNA relatedness always lower than 70% [9–11,34–36]. For that reason, DNA-DNA hybridizations were carried out only between strain 1126-1H-08 T and the type strains of the phylogenetically closest related species; i.e. , those species with 16S rRNA gene sequence similarities greater than 97.5%. The levels of DNA-DNA relatedness for strain 1126-1H-08 T with respect to F. aquidurense CCUG 59847 T , F. araucananum CCUG 61031 T , F. hydatis DSM 2063 T , F. pectinovorum CCUG 58916 T , F. frigidimaris CCUG 59364 T , F. chungangense CCUG 58910 T and F. oncorhynchi CECT 7678 T ranged between 21 and 48%. These values were below the 70% cut-off point for species delineation [37,38] and clearly confirmed that the trout strains belong to a distinct genomic species of the genus Flavobacterium . The DNA G + C content of strain 1126-1H-08 T was 33.2 mol%, a value consistent with those of the genus Flavobacterium [1,30]. Chemotaxonomic characteristics of strain 1126-1H-08 T were in accordance with those of members of the genus Flavobacterium [5,6]: the major quinone was MK-6 (95%) with minor amounts of MK-5 (5%). The predominant cell fatty acids of strain 1126-1H- 08 T were iso-C 15:0 (19%) and C 15:0 (15%). Strain 1126-1H-08 T also contained moderate or small amounts of C 16:1 v 7c (10%), C 15:1 v 6c (9%), iso-C 15:0 3-OH, C 17:1 v 6c , isoG-C 15:1 (6%/each), iso-C 17:0 3-OH (5%), iso-C 17:1 v 9c, C 15:0 3-OH, C 16:0 3-OH (3%/ each), isoaldehyde-C 15:0 , C 16:0 , iso-C 16:0 3-OH, unknown fatty acids with an equivalent chain length of 11.5 (2%/each) and C 17:1 v 8c , iso-C16:0, C 12:1 , aldehyde-C 14:0 , anteiso-C 15:0 and unknown fatty acids with an equivalent chain lengths of 14.8 and 12.5 (1%/ each) (Table 1). The trout strains exhibited identical physiological and biochemical characteristics. Cells were Gram-negative rods, 0.7 m m wide and 3 m m long, non-endospore-forming, and non-gliding. Strains grew well under aerobic conditions and grew weakly under micro-aerobic conditions. Strains grew at 15–30 u C with optimal growth at approximately 25 u C, while no growth was observed at 37 u C or 42 u C. Growth occurred on trypticase-soy and nutrient agars but not on Marine agar after incubation at 25 u C for 72 hours. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25 u C. Colonies are non-hemolytic on Columbia agar after 72 hours incubation at 25 u C. Diffusible flexirubin-type pigments were produced and congo red was not absorbed by colonies. Growth did not occur in brain heart infusion broth containing 3, 4.5 and 6.5% NaCl. Catalase and oxidase were produced and nitrate and nitrite were reduced. Starch and tyrosine were degraded but DNA, gelatin, casein or agarose were not. A brown pigment was not produced on tyrosine agar. Aesculin was hydrolyzed but not urea, lecithin and arginine. Indole and H 2 S were not produced. Acid was not produced from D-glucose. Arabinose, D-glucose, mannose, N- acetyl-glucosamine, and maltose were used as sole carbon and energy sources but not citrate, mannitol, gluconate, caprate, adipate, and malate. Activities for alkaline phosphatase, leucine arylamidase, N-acetyl- b -glucosaminidase, a -glucosidase, acid phosphatase, and naphthol-AS-BI-phophohydrolase were detected. Esterase C4, valine arylamidase, b -galactosidase, ester lipase C8, lipase C14, cystine arylamidase, a -chymotrypsin, trypsin, a - galactosidase, b -glucuronidase, b -glucosidase, a -mannosidase and a -fucosidase were not detected. The phenotypic characteristics that differentiated the trout strains from phylogenetically related species are shown in Table 2. The new species also can be also differentiated from the clinically relevant fish pathogens F. columnare , F. psycrophilum and F. branchiophilum , by the inability of these three species to grow in trypticase-soy agar and to hydrolyze aesculin [4]. Other species isolated from diseased fish such as F. hydatis , F. jonshoniae and F. succinicans are motile (gliding), degrade DNA and produce acid from carbohydrates [4], while the new species exhibited opposite results for those tests. Moreover, the new species can be readily differentiated from F. chilense and F. araucananum because the latter species are motile (gliding), grow in 3% NaCl and assimilate mannitol [9] and from F. oncorhynchi which produces b -galactosidase while the new species give opposite results for this test [10]. After PFGE typing, the trout strains were characterized by 3 different restriction profiles with the enzymes Bsp120 I (Fig. 2) and Xho I (not shown). Strains 986-08 and 1084B-08 exhibited indistinguishable restriction profiles with both enzymes and strain 51B-09 could not be ...
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... sequence of the type strains of Flavobacterium oncorhynchi CECT 7678 T and Flavobacterium pectinovorum DSM 6368 T (98.5% and 97.9% sequence similarity, respectively). In addition, strains exhibited 16S rRNA gene sequence similarities greater than 97.0% with other seventeen other Flavobacterium species. It is clear from the phylogenetic analysis (Fig. 1) that the trout strains held a clear affiliation to the genus Flavobacterium and represented a distinct sub-lineage clustering with a cluster of four species that included F. pectinovorum, F. chilense, F. oncorhynchi and F. hercynium. However, their position within this sub-group was not supported by significant bootstrap values. The ...
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... sub-lineage clustering with a cluster of four species that included F. pectinovorum, F. chilense, F. oncorhynchi and F. hercynium. However, their position within this sub-group was not supported by significant bootstrap values. The GenBank accession numbers for the 16S rRNA gene sequences of five strains sequenced in this study are shown in Fig. ...
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Citations
... Flavobacterial disease impacts farmed and wild fish alike, causing severe economic losses in the fishing industry (Ostland et al., 1994;Starliper, 2011;Thomas-Jinu & Goodwin, 2004). In addition, Flavobacterium plurextorum, Flavobacterium araucananum and Flavobacterium piscis have been isolated from diseased fish (Kämpfer et al., 2012;Zamora et al., 2013;Zamora et al., 2014), which have not been studied much yet, but are thought to have potential pathogenicity (Kumru et al., 2020;Loch & Faisal, 2015). Among them, F. plurextorum was first isolated from diseased rainbow trout (Oncorhynchus mykiss) in 2008 (Zamora et al., 2013). ...
... In addition, Flavobacterium plurextorum, Flavobacterium araucananum and Flavobacterium piscis have been isolated from diseased fish (Kämpfer et al., 2012;Zamora et al., 2013;Zamora et al., 2014), which have not been studied much yet, but are thought to have potential pathogenicity (Kumru et al., 2020;Loch & Faisal, 2015). Among them, F. plurextorum was first isolated from diseased rainbow trout (Oncorhynchus mykiss) in 2008 (Zamora et al., 2013). Largescale comparative genomic analysis has revealed that F. plurextorum has partial type IV secretion system (T4SS) and type VI secretion system subtype 3 (T6SS iii ) which can contribute to pathogenicity (Kumru et al., 2020). ...
... The 16S rRNA gene sequence of RSG-18 was completely identical to F. plurextorum CCUG 60112 T , isolated from Trout eggs (O. mykiss) in Spain in 2008 (Table S3) (Zamora et al., 2013). RSG-18 was isolated from the gut of S. schlegelii in 2013, showing the mutational robustness of the 16S rRNA gene despite differences in the time of isolation, fish species, etc. ...
Flavobacterium plurextorum is a potential fish pathogen of interest, previously isolated from diseased rainbow trout (Oncorhynchus mykiss) and oomycete‐infected chum salmon (Oncorhynchus keta) eggs. We report here the first complete genome sequence of F. plurextorum RSG‐18 isolated from the gut of Schlegel's black rockfish (Sebastes schlegelii). The genome of RSG‐18 consists of a circular chromosome of 5,610,911 bp with a 33.57% GC content, containing 4858 protein‐coding genes, 18 rRNAs, 63 tRNAs and 1 tmRNA. A comparative analysis was conducted on 11 Flavobacterium species previously reported as pathogens or isolated from diseased fish to confirm the potential pathogenicity of RSG‐18. In the SEED classification, RSG‐18 was found to have 36 genes categorized in ‘Virulence, Disease and Defense’. Across all Flavobacterium species, a total of 16 antibiotic resistance genes and 61 putative virulence factors were identified. All species had at least one phage region and type I, III and IX secretion systems. In pan‐genomic analysis, core genes consist of genes linked to phages, integrases and matrix‐tolerated elements associated with pathology. The complete genome sequence of F. plurextorum RSG‐18 will serve as a foundation for future research, enhancing our understanding of Flavobacterium pathogenicity in fish and contributing to the development of effective prevention strategies.
... . plurextorum caused a clinical episode of septicemia in a rainbow trout farm located in the central region of Spain(Zamora, Fernandez-Garayzabal, et al., 2013), and F. bernardetii was isolated from diseased rainbow trout farmed in Rize city (Turkey), which show neurological symptoms (Data SS1). Additionally, novel Flavobacterium species were identified from seemingly healthy fish, including F. erciyesense and F. oncorhynchi, isolated from the skin mucus of rainbow trout, and F. muglaense, isolated from the internal organs of an apparently healthy rainbow trout, both in TurkeySaticioglu, 2021;.Moreover, F. collinsii, F. branchiarum, and F. branchiicola were recovered from the liver and gills of twelve different trout in the same fish farm in two different years(2008 and 2009) ...
Outbreaks of bacterial infections in aquaculture have emerged as significant threats to the sustainable production of rainbow trout ( Oncorhynchus mykiss ) worldwide. Understanding the dynamics of these outbreaks and the bacteria involved is crucial for implementing effective management strategies. This comprehensive review presents an update on outbreaks of bacteria isolated from rainbow trout reported between 2010 and 2022. A systematic literature survey was conducted to identify relevant studies reporting bacterial outbreaks in rainbow trout during the specified time frame. More than 150 published studies in PubMed, Web of Science, Scopus, Google Scholar and relevant databases met the inclusion criteria, encompassing diverse geographical regions and aquaculture systems. The main bacterial pathogens implicated in the outbreaks belong to both gram‐negative, namely Chryseobacterium, Citrobacter, Deefgea Flavobacterium, Janthinobacterium, Plesiomonas, Pseudomonas, Shewanella, and gram‐positive genera, including Lactococcus and Weissella, and comprise 36 new emerging species that are presented by means of pathogenicity and disturbance worldwide. We highlight the main characteristics of species to shed light on potential challenges in treatment strategies. Moreover, we investigate the role of various risk factors in the outbreaks, such as environmental conditions, fish density, water quality, and stressors that potentially cause outbreaks of these species. Insights into the temporal and spatial patterns of bacterial outbreaks in rainbow trout aquaculture are provided. Furthermore, the implications of these findings for developing sustainable and targeted disease prevention and control measures are discussed. The presented study serves as a comprehensive update on the state of bacterial outbreaks in rainbow trout aquaculture, emphasizing the importance of continued surveillance and research to sustain the health and productivity of this economically valuable species.
... Flavobacterium species have been previously isolated from the liver of naturally infected salmonids and non-salmonids (Loch & Faisal, 2015). In most cases, fish suffered from septicaemia and exhibited multifocal hepatic necrosis (Zamora et al., 2013(Zamora et al., , 2014. In this way, Timur and colleagues showed multifocal necrotic areas and haemorrhages in the liver of Russian sturgeons coinfected with Aeromonas hydrophila and Flavobacterium hydatis (Timur et al., 2010). ...
Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. Here, F. psychrophilum was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.
... Clade D contains F. succinicans, previously implicated in fish disease (Anderson and Ordal, 1961;Good et al., 2015;Adamek et al., 2018;Loch and Faisal, 2018;Einarsdottir et al., 2020), but isolates ranged in pairwise 16S rRNA gene similarity from 98.2-99.9%. The type-strain in Clade H, F. tructae, is one of the few atypical Flavobacterium with demonstrated pathogenicity in fish (Zamora et al., 2013a;Loch and Faisal, 2016b). Flavobacterium tructae has only been reported in salmonids, but Clade H also included an isolate from Gambusia with high 16S rRNA gene similarity (99.6% PI). ...
... oncorhynchi and F. plurextorum. Both species have been reported in association with salmonids and other species, in diseased or healthy farmed and wild fish (Zamora et al., 2012a;Loch et al., 2013;Zamora et al., 2013a;Loch and Faisal, 2014a;Loch and Faisal, 2018). Flavobacterium oncorhynchi has also been implicated in a fatal human fetal infection (Ferreira et al., 2022). ...
Flavobacterial diseases, caused by bacteria in the order Flavobacteriales, are responsible for devastating losses in farmed and wild fish populations worldwide. The genera Flavobacterium (Family Flavobacteriaceae) and Chryseobacterium (Weeksellaceae) encompass the most well-known agents of fish disease in the order, but the full extent of piscine-pathogenic species within these diverse groups is unresolved, and likely underappreciated. To identify emerging agents of flavobacterial disease in US aquaculture, 183 presumptive Flavobacterium and Chryseobacterium isolates were collected from clinically affected fish representing 19 host types, from across six western states. Isolates were characterized by 16S rRNA gene sequencing and phylogenetic analysis using the gyrB gene. Antimicrobial susceptibility profiles were compared between representatives from each major phylogenetic clade. Of the isolates, 52 were identified as Chryseobacterium species and 131 as Flavobacterium. The majority of Chryseobacterium isolates fell into six clades (A-F) consisting of ≥ 5 fish isolates with ≥ 70% bootstrap support, and Flavobacterium into nine (A-I). Phylogenetic clades showed distinct patterns in antimicrobial susceptibility. Two Chryseobacterium clades (F & G), and four Flavobacterium clades (B, G-I) had comparably high minimal inhibitory concentrations (MICs) for 11/18 antimicrobials tested. Multiple clades in both genera exhibited MICs surpassing the established F. psychrophilum breakpoints for oxytetracycline and florfenicol, indicating potential resistance to two of the three antimicrobials approved for use in finfish aquaculture. Further work to investigate the virulence and antigenic diversity of these genetic groups will improve our understanding of flavobacterial disease, with applications for treatment and vaccination strategies.
... Another species, F. branchiophilum, is also known as a fish pathogen, but within more restricted geographical areas. In addition, the following species, often represented by a very small number of isolates, were recovered from diseased fish tissues and have been suspected to be pathogenic: F. araucananum from kidney and external lesions of Atlantic salmon (Salmo salar) (5); F. bernardetii from kidney and liver of rainbow trout (Oncorhynchus mykiss) (6); F. turcicum and F. kayseriense from rainbow trout kidney and spleen, respectively (7); F. branchiarum and F. branchiicola from rainbow trout gills (8); F. chilense from external lesions of rainbow trout (5); F. collinsii from the liver of rainbow trout (8); F. hydatis from the gills of diseased salmon (9, 10); F. inkyongense from diseased chocolate cichlids (Hypselecara coryphaenoides) (11); F. johnsoniae-like isolates from various diseased fish species (12); F. oncorhynchi from liver and gills of rainbow trout (13); F. piscis from liver, gills, and kidney of rainbow trout (14); F. plurextorum from liver and eggs of rainbow trout (15); and F. succinicans from gills of rainbow trout suffering bacterial gill disease (16). At least one species, F. tructae, which was isolated from liver, gills, and kidney of rainbow trout (14) and concurrently from kidney of feral spawning adult Chinook salmon (Oncorhynchus tshawytscha) under the alternative name of F. spartansii (17), may be considered a salmonid pathogen, as two isolates were able to induce pathological changes and mortality in experimentally infected Chinook salmon, though only using very high infectious doses (18). ...
Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare cause considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a Flavobacterium collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii stands within a cluster of species associated with diseased fish, the closest one being F. tructae, which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of Flavobacterium bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs or mortalities were observed with F. bernardetii. F. collinsii showed very low virulence but was isolated from the internal organs of survivors, indicating that the bacterium is able to survive inside the host and may provoke disease in fish under compromised conditions such as stress and/or wounds. Our results suggest that members of a phylogenetic cluster of fish-associated Flavobacterium species may be opportunistic fish pathogens causing disease under specific circumstances. IMPORTANCE Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development, and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii, which belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium survived inside the host, likely escaping clearance by the immune system but without provoking massive mortality, suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.
... Another species, F. branchiophilum, is also known as a fish pathogen, but within more restricted geographical areas. In addition, the following species, often represented by a very small number of isolates, were recovered from diseased fish tissues and have been suspected to be pathogenic: F. araucananum from kidney and external lesions of Atlantic salmon (Salmo salar) (5); F. bernardetii from kidney and liver of rainbow trout (Oncorhynchus mykiss) (6); F. turcicum and F. kayseriense from rainbow trout kidney and spleen, respectively (7); F. branchiarum and F. branchiicola from rainbow trout gills (8); F. chilense from external lesions of rainbow trout (5); F. collinsii from the liver of rainbow trout (8); F. hydatis from the gills of diseased salmon (9, 10); F. inkyongense from diseased chocolate cichlids (Hypselecara coryphaenoides) (11); F. johnsoniae-like isolates from various diseased fish species (12); F. oncorhynchi from liver and gills of rainbow trout (13); F. piscis from liver, gills, and kidney of rainbow trout (14); F. plurextorum from liver and eggs of rainbow trout (15); and F. succinicans from gills of rainbow trout suffering bacterial gill disease (16). At least one species, F. tructae, which was isolated from liver, gills, and kidney of rainbow trout (14) and concurrently from kidney of feral spawning adult Chinook salmon (Oncorhynchus tshawytscha) under the alternative name of F. spartansii (17), may be considered a salmonid pathogen, as two isolates were able to induce pathological changes and mortality in experimentally infected Chinook salmon, though only using very high infectious doses (18). ...
Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare are causing considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a F. collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii is standing within a cluster of species associated to diseased fish, the closest one being F. tructae which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of F. bernardetii F-372 T , another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs nor mortalities were observed. However, F. collinsii was isolated from the internal organs of wounded fish, suggesting that the bacterium could invade fish under compromised conditions such as stress and/or wounds. Our results suggest that some fish-associated Flavobacterium species should be considered as opportunistic fish pathogens causing disease under specific circumstances.
IMPORTANCE
Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii that belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium colonized only oppressed fish facing stressful conditions suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.
... Among these pathogens, the Flavobacterium and Legionella genera were the most prevalent in both layers. Although most Flavobacteria are harmless, some are opportunistic or true pathogens that cause diseases in animals, plants, and humans [86][87][88]. The majority of Legionella species are considered pathogenic and have been reported as one of the leading bacterial etiological pathogens of waterborne outbreaks in the United States between 2007 and 2009 [89]. ...
Here, we describe the bacterial diversity and physicochemical properties in freshwater samples from the surface and bottom layers of the Billings Reservoir, the largest open-air storage ecosystem in the São Paulo (Brazil) metropolitan area. Forty-four samples (22 from the surface and 22 from the bottom layers) were characterized based on 16S rRNA gene analysis using Illumina MiSeq. Taxonomical composition revealed an abundance of the Cyanobacteria phylum, followed by Proteobacteria, which were grouped into 1903 and 2689 different genera in the surface and the deep-water layers, respectively. Chroobacteria, Actinobacteria, Betaproteobacteria, and Alphaproteobacteria were the most dominant classes. The Shannon diversity index was in the range of 2.3–5.39 and 4.04–6.86 in the surface and bottom layers, respectively. Flavobacterium was the most predominant pathogenic genus. Temperature and phosphorus concentrations were among the most influential factors in shaping the microbial communities of both layers. Predictive functional analysis suggests that the reservoir is enriched in motility genes involved in flagellar assembly. The overall results provide new information on the diversity composition, ecological function, and health risks of the bacterial community detected in the Billings freshwater reservoir. The broad bacterial diversity indicates that the bacterioplankton communities in the reservoir were involved in multiple essential environmental processes.
... Numerous Flavobacterium spp. have been found in association with fish and implicated and/or confirmed as disease causing agents, including F. johnsoniae [64], F. hydatis [66], F. succinicans [24], F. spartansi [45], F. inkyongense [16], F. chilense [28], F. araucananum [28], F. oncorhynchi [79], F. plurextorum [78], F. tructae [81], F. piscis [81], F. collinsii [80], F. branchiarum [80], and F. branchiicola [80]. However, the three species that are most recognized as freshwater fish pathogens and thus have been most extensively studied are F. branchiophilum (bacterial gill disease), F. psychrophilum (bacterial cold water disease, rainbow trout fry syndrome), and F. columnare (columnaris disease) [46]. ...
Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT shared less than <98.8 % sequence identity to F. columnare ATCC 23463T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36T), F. davisii sp. nov. (90-106T), and F. oreochromis sp. nov. (Costa Rica 04-02-TNT) are proposed to represent genetic groups 2, 3, and 4, respectively.
... As shown in Table 1, the amino acid sequence identity of Tet(X) protein produced by Epilithonimonas strains exhibited a 100% identity with those synthesized by the Chryseobacterium strains (SNU WT5 and SNU WT7) from South Korea [22,23]. Tet(X) proteins from Chilean strains showed an approximately 84% identity with the Tet(X) of Chryseobacterium from the UK (MOF25P and BGARF1) [16], Turkey (C2), and Spain (701B-08) [24,25], Flvobacterium kayseriense from Turkey (F-47 and F-380) [26], and Flavobacterium plurextorum from Spain (CCUG 60112) [27,28]. All of these strains were isolated from fish, and their whole genomes are currently included in the GenBank database ( Table 2). ...
The main objective of this study was to characterize the tet(X) genes, which encode a
monooxygenase that catalyzes the degradation of tetracycline antibiotics, carried by the resistant
strains FP105 and FP233-J200, using whole-genome sequencing analysis. The isolates were recovered
from fin lesion and kidney samples of diseased rainbow trout Oncorhynchus mykiss, during two
Flavobacteriosis outbreaks occurring in freshwater farms located in Southern Chile. The strains were
identified as Epilithonimonas spp. by using biochemical tests and by genome comparison analysis
using the PATRIC bioinformatics platform and exhibited a minimum inhibitory concentration (MIC)
of oxytetracycline of 128 �g/mL. The tet(X) genes were located on small contigs of the FP105 and
FP233-J200 genomes. The sequences obtained for the tet(X) genes and their genetic environment were
compared with the genomes available in the GenBank database of strains of the Chryseobacterium
clade belonging to the Flavobacterium family, isolated from fish and carrying the tet(X) gene. The Tet(X)
proteins synthesized by the Chilean Epilithonimonas strains showed a high amino acid similarity
(range from 84% to 100%), with the available sequences found in strains belonging to the genus
Chryseobacterium and Flavobacterium isolated from fish. An identical neighborhood of tet(X) genes
from both Chilean strains was observed. The genetic environment of tet(X) observed in the two
strains of Epilithonimonas studied was characterized by the upstream location of a sequence encoding
a hypothetical protein and a downstream located alpha/beta hydrolase-encoding gene, similar to the
observed in some of the tet(X) genes carried by Chryseobacterium and Flavobacterium strains isolated
from fish, but the produced proteins exhibited a low amino acid identity (25–27%) when compared to
these synthesized by the Chilean strains. This study reports for the first time the carriage of the tet(X)
gene by the Epilithonimonas genus and their detection in fish pathogenic bacteria isolated from farmed
salmonids in Chile, thus limiting the use of therapies based on oxytetracycline, the antimicrobial
most widely used in Chilean freshwater salmonid farming. This results suggest that pathogenic
strains of the Chryseobacterium clade occurring in Chilean salmonid farms may serve as important
reservoirs of tet(X) genes.
... For example, F. branchiophilum, F. columnaris, and F. psychrophilum cause bacterial gill disease, columnaris infection, and BCWD, respectively (Noga, 2010). In addition to these three species, at least 10 species have been isolated from diseased salmon fish (Anderson and Ordal, 1961;Bernardet et al., 1996;Kämpfer et al., 2012;Suebsing and Kim, 2012;Zamora et al., 2012Zamora et al., , 2013Zamora et al., , 2014Loch and Faisal, 2014). F. plurextorum has been isolated from the liver, gills, and eggs of sick rainbow trout fishes, although there have been no reports of this species being isolated from chum salmon until now (Zamora et al., 2013). ...
... In addition to these three species, at least 10 species have been isolated from diseased salmon fish (Anderson and Ordal, 1961;Bernardet et al., 1996;Kämpfer et al., 2012;Suebsing and Kim, 2012;Zamora et al., 2012Zamora et al., , 2013Zamora et al., , 2014Loch and Faisal, 2014). F. plurextorum has been isolated from the liver, gills, and eggs of sick rainbow trout fishes, although there have been no reports of this species being isolated from chum salmon until now (Zamora et al., 2013). The F. plurextorum detected in this study potentially infects chum salmon eggs. ...
To elucidate the localization of oomycetes (water molds) and bacteria in a single egg of chum salmon, Oncorhynchus keta, eggs infected with oomycetes were examined by bright-field microscopy, scanning electron microscopy (SEM), and fluorescent in situ hybridization (FISH) using oomycete universal and bacterial species-specific probes. Furthermore, DNA barcoding for oomycetes and bacteria was performed on the chorion and cytoplasm of the eggs using genomic DNA extracted from the sections of eggs used for FISH analyses. Using bright-field microscopy and SEM, it was shown that oomycete hyphae penetrated the chorion and invaded the cytoplasm of the eggs. Based on DNA barcoding, two oomycetes (Pythium monospermum and Pythium sp. SE-OP1), and three bacterial species (Flavobacterium plurextorum, Flavobacterium sp. SE-BF1 and Undibacterium pigrum) were predominantly detected in the infected eggs. FISH analyses indicated that localization of the oomycetes was not noticeable different between the chorion and cytoplasm of the eggs. In contrast, FISH analyses using bacterial species-specific probes demonstrated clear differences in their localization. Flavobacterium sp. SE-BF1 and U. pigrum were predominantly found inside the chorion and around the oomycete hyphae invading the cytoplasm, respectively. These results are the first to indicate the possibility that U. pigrum has a specific relationship with oomycetes and coordinately invades eggs with oomycetes. However, it seems that Flavobacterium sp. SE-BF1 invades by a mechanism different from that of U. pigrum. Although this study is preliminary and the interaction between oomycetes and U. pigrum associated with chum salmon egg infection needs to be elucidated in detail, this finding may provide new insights into the infection mechanism of bacteria in salmon eggs, which is a longstanding problem in salmon hatcheries.
