SGM Journals
Bacteroidetes

Chryseobacterium piscicola sp. nov., isolated from diseased salmonid fish

Pedro Ilardi, Jorge Fernández, Ruben Avendaño-Herrera

  • Laboratorio de Veterquímica, Camino Melipilla 5641, Cerrillos, Santiago, Chile
  • Correspondence
    Ruben Avendaño-Herrera
    reavendano{at}yahoo.com

    • International Journal of Systematic and Evolutionary Microbiology 2009; 59(12):3001–3005 · https://doi.org/10.1099/ijs.0.007021-0

    View at publisher PubMed

    Abstract

    Eight bacterial strains isolated from diseased rainbow trout (n=5) and Atlantic salmon (n=3) were characterized by phenotypic and molecular taxonomic methods. The isolates were negative for the Gram-reaction, non-motile, rod-shaped and catalase- and oxidase-positive. Colonies on solid media were yellow, smooth, shiny and circular with regular edges. Growth occurred at 4–28 °C (optimum, 15 °C) and with 0–3 % NaCl (optimum, 0.5 %). Analysis of the 16S rRNA gene sequence allocated the micro-organisms to the genus Chryseobacterium, with Chryseobacterium soldanellicola PSD1-4T and Chryseobacterium soli JS6-6T as their closest relatives (96.9 and 97.1 % sequence similarity, respectively). The levels of DNA–DNA hybridization towards these nearest phylogenetic neighbours were below 17.1 %. The DNA G+C contents of strains VQ-6316sT and VQ-4836s were 32.5 and 32.3 mol%, respectively. The predominant menaquinone was MK-6 and the major fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 1ω9c, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω7t and/or iso-C15 : 0 2-OH). The eight isolates were classified as representatives of a novel species, Chryseobacterium piscicola sp. nov., with strain VQ-6316sT (=CECT 7357T=DSM 21068T) as the type strain.

    • The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains VQ-106r, VQ-4836s and VQ-6316sT are EU869188–EU869190, respectively.

    • An electron micrograph of cells of strain VQ-6316sT and a table giving API ZYM profiles are available with the online version of this paper.

    The family Flavobacteriaceae represents the main bacterial lineage in the phylum ‘Bacteroidetes’ (formerly the Cytophaga–Flavobacterium–Bacteroides group) (Bernardet et al., 1996, 2002). Within this family, the genera Bergeyella, Chryseobacterium and Riemerella form a separate branch on the basis of rRNA cistron similarity studies and phenotypic characterization (Vandamme et al., 1994). At the time of writing, the genus Chryseobacterium comprised 38 species, some of which are pathogenic to humans and animals (Bernardet et al., 2002, 2005, 2006).

    During the characterization of bacteria isolated from external lesions of diseased farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Osorno, Chile, eight yellow-pigmented strains were recovered on modified Anacker and Ordal's agar (AOA; 0.5 % tryptone, 0.05 % yeast extract, 0.02 % beef extract, 0.02 % sodium acetate, pH 7.2; Anacker & Ordal, 1955) incubated at 15 °C. Five strains were isolated from rainbow trout (VQ-106r, VQ-5916r, VQ-5946r, VQ-5926r, VQ-5966r) and the remaining three isolates (VQ-2206s, VQ-4836s, VQ-6316sT) were recovered from Atlantic salmon. Strains were routinely cultured aerobically on AOA at 15 °C for 3 days. Stock cultures were preserved at −80 °C in Criobilles tubes (AES Laboratory). The diseased fish showed the typical signs (i.e. skin and muscle ulcerative lesions on the flank and in the anus or peduncle area) observed in fish affected by Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease and rainbow trout fry syndrome (Ilardi & Avendaño-Herrera, 2008). However, a comparative analysis of 16S rRNA gene sequences indicated that all the isolates were members of the genus Chryseobacterium. Although they were only retrieved from superficial lesions, not from internal organs, their virulence for fish has been demonstrated (Ilardi & Avendaño-Herrera, 2008). In order to determine the exact taxonomic position of the new isolates, a taxonomic study was performed using a polyphasic approach.

    PCR amplification of the 16S rRNA genes of the eight isolates was conducted using universal primers 20F and 1500R (Weisburg et al., 1991) and PCR products were purified using QIAquick PCR purification kits (Qiagen). The 16S rRNA gene sequence was determined directly using the PCR-amplified DNA as a sequencing template on an ABI PRISM 310 sequencer (Applied Biosystems) according to the manufacturer's recommendations. The resulting sequences were compared with those available in GenBank, EMBL () and the Ribosomal Database Project using the program blast. Multiple alignments were performed using the clustal_x program (Thompson et al., 1997) and a phylogenetic tree was constructed using the neighbour-joining method with mega3 software (Kumar et al., 2004). All isolates formed a robust cluster within the genus Chryseobacterium. Data from sequence similarity analysis indicated that the closest relatives of the eight strains were Chryseobacterium soldanellicola PSD1-4T and Chryseobacterium soli JS6-6T, exhibiting 96.9 and 97.1 % 16S rRNA gene sequence similarities, respectively (Fig. 1⇓). Interestingly, when the sequence of strain VQ-6316sT was compared with those of Chryseobacterium strains isolated from fish published by Bernardet et al. (2005), it shared nearly 100 % sequence similarity with strains UOF CR4395 and UOF CR2995 (GenBank accession nos AY468455 and AY468454, respectively), isolated from Atlantic salmon in Finland.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between the fish isolates, other members of the genus Chryseobacterium and representatives of other related genera in the family Flavobacteriaceae. Weeksella virosa ATCC 43766T was used as an outgroup. Bootstrap values >50 % are shown at nodes. Bar, 0.01 substitutions per nucleotide position.

    DNA–DNA hybridization was performed as described by De Ley et al. (1970) with the modifications of Huß et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6×6 multicell changer and a temperature controller with in situ temperature probe (Varian). The levels of DNA–DNA relatedness between strain VQ-6316sT and strain VQ-5926r, C. soldanellicola PSD1-4T and C. soli JS6-6T were 99.2, 17.1 and 13.2 %, respectively, demonstrating that the isolates formed a tight group that was distinct from recognized species of the genus Chryseobacterium.

    Phenotypic characterization of the eight isolates was carried out as described by Bernardet et al. (2002) and according to standard methods (MacFaddin, 1980). The Gram reaction was tested by using the bioMérieux Gram-stain kit according to the manufacturer's instructions and the non-staining KOH method (Buck, 1982). Gliding motility was assessed by phase-contrast microscopy on a fresh Anacker and Ordal's broth culture by the hanging drop technique (Bernardet et al., 2002). The presence of flexirubin-type pigments and cell wall-associated galactosamine glycans was assessed by adding 20 % KOH or 0.01 % Congo red (Sigma), respectively, to 3-day-old colonies (Bernardet et al., 2002; Avendaño-Herrera et al., 2004). Cytochrome oxidase activity (using N,N,N′,N′-tetramethyl-p-phenylenediamine; Sigma) and catalase production (using 3 % H2O2) were determined on glass slides. The following tests were performed according to MacFaddin (1980): oxidation/fermentation reactions, Voges–Proskauer reaction and arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities.

    Hydrolysis of the following substrates was determined using AOA as the basal medium: gelatin (1 %), agar (3 %), starch (1 %), Tween 80 (1 %), casein (1 %), DNA (1 %), sheep blood (5 %) and tyrosine (0.1 %). Growth was tested at 0, 4, 15, 28, 37 and 42 °C on AOA. Growth in the presence of 0, 1, 1.5, 3 and 6 % (w/v) NaCl was also determined. Growth was tested on MacConkey, Simmons' citrate, blood, R2A, marine 2216, nutrient and tryptone soy agars. Enzymic activities were assessed using API ZYM (bioMérieux) strips according to the manufacturer's instructions, with the exception that the incubation temperature was 15 °C.

    The morphology of bacterial cells grown for 48 h on AOA was observed by light (1000× magnification) and transmission electron (100SX; JEOL) microscopy. Cells were prepared for electron microscopy as described by Spurr (1969) and Reynolds (1963).

    The results of the phenotypic analyses of the eight isolates are given in the species description and in Table 1⇓ and those from API ZYM tests are given in Supplementary Table S1 (available in IJSEM Online).

    Table 1.

    Phenotypic characteristics that differentiate Chryseobacterium piscicola sp. nov. from closely related Chryseobacterium species

    Taxa (the number of strains examined, when greater than 1, is given in parentheses): 1, C. piscicola sp. nov. (n=8); 2, C. soldanellicola PSD1-4T (Park et al., 2006); 3, C. scophthalmum (n=2); 4, C. piscium (n=4; de Beer et al., 2006); 5, C. balustinum LMG 8329T; 6, C. soli (n=2) (Weon et al., 2008); 7, C. indoltheticum LMG 4025T. Except where indicated otherwise, data for taxa 3, 5 and 6 are from Hugo et al. (2003). +, Positive; w, weakly positive; −, negative; v, variable; d, delayed; na, no data available.

    The respiratory quinones of strains VQ-6316sT and VQ-4836s were analysed using reverse-phase HPLC by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany). The major peak identified was that of menaquinone MK-6, which is consistent with that present in all other members of the family Flavobacteriaceae.

    The fatty acids of strains VQ-6316sT and VQ-4836s were extracted from cells grown on AOA at 15 °C for 48 h, methylated and analysed using the standard protocol of the Microbial Identification System (MIDI, Microbial ID) at the DSMZ as described by Sasser (1990). The predominant fatty acids (mean values) were: iso-C15 : 0, 36.2 %; anteiso-C15 : 0, 13.9 %; iso-C17 : 1ω9c, 11.1 %; iso-C17 : 0 3-OH, 10.5 %; and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω7t and/or iso-C15 : 0 2-OH), 11.2 %. Detailed fatty acid profiles of the novel isolates and their closest relatives are presented in Table 2⇓.

    Table 2.

    Cellular fatty acid content (%) of Chryseobacterium piscicola sp. nov. and closely related species of the genus Chryseobacterium

    Taxa: 1, C. piscicola sp. nov. (n=2); 2, C. soldanellicola PSD1-4T (Park et al., 2006); 3, C. scophthalmum (n=2); 4, C. piscium (n=4; de Beer et al., 2006); 5, C. balustinum LMG 8329T; 6, C. soli JS6-6T (Weon et al., 2008); 7, C. indoltheticum LMG 4025T. Data for taxa 3, 5 and 6 are taken from Hugo et al. (2003). Values derived from more than one strain are mean±sd. nd, Not detected; nr, not reported; tr, trace (less than 1.0 %). Fatty acids amounting to <1 % of the total fatty acids in all strains studied are omitted. Different culture conditions were used.

    Genomic DNA was purified following the method of Cashion et al. (1977) and the DNA G+C content was determined using the HPLC method according to Mesbah et al. (1989) by the DSMZ. The DNA G+C contents of strains VQ-6316sT and VQ-4836s were 32.5 and 32.3 mol%, respectively. These values fall within the range (29–39 mol%) reported for members of the genus Chryseobacterium by Bernardet et al. (2006).

    On the basis of the results obtained, the new isolates are sufficiently distinct from all recognized species of the genus Chryseobacterium to be assigned as representatives of a novel species of this genus, for which the name Chryseobacterium piscicola sp. nov. is proposed.

    Description of Chryseobacterium piscicola sp. nov.

    Chryseobacterium piscicola [pis.ci′cola. L. n. piscis fish; L. suff. -cola (from L. n. incola) inhabitant; N.L. n. piscicola an inhabitant of fish].

    Cells are Gram-reaction-negative, non-motile rods approximately 1.6–3.5 μm in length and 0.8–1.2 μm in diameter (see Supplementary Fig. S1 available in IJSEM Online). Catalase- and oxidase-positive and non-fermentative. Colonies are smooth, shiny, circular with regular edges and yellow in colour. Flexirubin pigments are produced, but Congo red is not absorbed. Grows at 4–28 °C (optimum, 15 °C) and with 0–3 % NaCl (optimum, 0.5 %). Good growth occurs on blood (α-haemolytic), R2A, nutrient and tryptone soy agars, but no growth occurs on MacConkey, marine 2216 or Simmons' citrate agars. Gelatin and aesculin are hydrolysed, but Tween 80, tyrosine, agar, starch and casein are not. All strains are negative for arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities. DNA reaction is weakly positive. Nitrate and nitrite are not reduced. The methyl red and Voges–Proskauer tests are negative. In the API ZYM system, alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-glucuronidase, α-glucosidase, β-glucosidase and N-acetyl-β-glucosaminidase activities are present, but trypsin, α-galactosidase, β-galactosidase, α-mannosidase and α-fucosidase activities are absent. The predominant menaquinone is MK-6 and the major (>10 %) fatty acids are iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 1ω9c, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω7t and/or iso-C15 : 0 2-OH).

    The type strain, VQ-6316sT (=CECT 7357T=DSM 21068T), was isolated from diseased Atlantic salmon (Salmo salar) farmed in Osorno, Chile. The DNA G+C content of the type strain is 32.5 mol%.

    Acknowledgments

    This work was supported by grant IPC 019 from the Program Bicentenario Ciencia y Tecnología, CONICYT, Chile. R. A.-H was also supported in part by this Grant. Thanks are expressed to members of the technical team of the laboratories of Veterquímica. The authors wish to express their gratitude to Dr J.-F. Bernardet, Institut National de la Recherche Agronomique, Unité de Virologie et Immunologie Moléculaires, for reviewing and editing the manuscript.

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