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Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
Correspondence
Antonio Ventosa
ventosa{at}us.es
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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API ZYM profiles of strain VP48T and other Chryseobacterium and Elizabethkingia species are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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. It belongs to the family Flavobacteriaceae, phylum Bacteroidetes and currently includes 14 species (Table 1). Two previously recognized Chryseobacterium species, Chryseobacterium meningosepticum (King, 1959; Vandamme et al., 1994) and Chryseobacterium miricola (Li et al., 2003), have been transferred recently to the genus Elizabethkingia (Kim et al., 2005b). Chryseobacterium species are widely distributed in soil, water and clinical sources (Vandamme et al., 1994).
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). Plates were incubated at 28 °C for 7 days and colonies with different morphologies were subcultivated to obtain pure cultures. Among the organisms isolated in this study (about 600 pure cultures), strain VP48T was isolated on PCA during the sampling campaign of October 2003 and pure culture was obtained after subcultivation on R2A medium at 28 °C.
The shape and motility of bacterial cells were observed under a phase-contrast microscope (x1000 magnification) from a 24 h liquid culture in R2A medium. Growth at different temperatures (4–40 °C), pH values (pH 3–12) and NaCl concentrations (0–5 % NaCl) was tested on solid and liquid R2A medium. The isolate was also tested for its ability to grow on MacConkey agar (Difco) and TSA (Difco). H2S production was determined on Kligler iron agar (Difco). Oxidase activity was detected using a 1 % solution of tetramethyl-p-phenylenediamine (Difco) (Kovacs, 1956). Catalase activity was tested by picking up a young colony and smearing it in a drop of H2O2. The methyl red and Voges–Proskauer reactions were performed on Clark–Lubs medium (Scharlau). Indole production was determined with Kovacs' reagent on 1 % tryptone broth. Simmons' citrate test was tested on Simmons' citrate agar (Sigma). For determination of acid production from different carbohydrates, a medium containing 0.5 % peptone, 0.5 % NaCl and 0.001 % phenol red was used (Cowan & Steel, 1974). Nitrate reduction was tested on nitrate broth containing 0.2 % KNO3 (Skerman, 1967). Urease activity was studied on Christensen's medium (Christensen, 1946). Hydrolysis of gelatin, starch and DNA was tested on the corresponding agar media (Scharlau). Tween 80 hydrolysis was tested on R2A medium containing 1 % Tween 80 and 0.02 % CaCl2. Casein hydrolysis was tested on R2A medium supplemented with 2 % skim milk (Difco). Flexirubin-type pigments were detected as described by Bernardet et al. (2002). API 20NE and API ZYM strips (bioMérieux) were inoculated according to the manufacturer's instructions and incubated at 28 °C.
Phenotypic characteristics of strain VP48T are included in the species description; they clearly support its placement within the genus Chryseobacterium. Phenotypic features that differentiate strain VP48T from other species of Chryseobacterium and Elizabethkingia are shown in Table 1
. A table showing API ZYM profiles of strain VP48T and other Chryseobacterium and Elizabethkingia species is available as supplementary material in IJSEM Online.
The 16S rRNA gene was amplified using the universal primers 16F27 and 16R1488 as described previously by Mellado et al. (1995). The almost-complete nucleotide sequence was determined by NBT-Newbiotechnic (Sevilla, Spain) using an automated DNA sequencer model 3100 (Applied Biosystems). Alignment of 16S rRNA gene sequences was carried out using the ARB software program (Ludwig et al., 2004) and phylogenetic trees were inferred using the maximum-parsimony (Kluge & Farris, 1969), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood methods (Felsenstein, 1981).
The almost-complete 16S rRNA gene sequence of strain VP48T (1421 bp) was compared with sequences of related organisms retrieved from the GenBank/EMBL/DDBJ databases. Strain VP48T was most closely related to representatives of the family Flavobacteriaceae, especially to members of the genus Chryseobacterium. The highest sequence similarity was 96.0 % with Chryseobacterium indoltheticum and Chryseobacterium scophthalmum, whereas similarity values were 
96 % with the type strains of other species in the genus Chryseobacterium and 93.9–92.7 % with members of the genus Elizabethkingia. Moreover, phylogenetic analysis clearly showed that strain VP48T constitutes a branch that is separate from other Chryseobacterium species, although the branching patterns of the different trees were not completely stable (Fig. 1). Therefore, according to phylogenetic data, strain VP48T could be considered to be a representative of a novel species of the genus Chryseobacterium (Stackebrandt & Goebel, 1994).
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. The G+C content of genomic DNA was determined by the method of Marmur & Doty (1962) using the equation of Owen & Hill (1979). The DNA G+C content of strain VP48T was 34.3 mol%, a value that falls within the range described for the genus Chryseobacterium (Vandamme et al., 1994).
The whole-cell fatty acid composition was determined using the Microbial Identification System (MIDI, Inc.). Cells were cultured for 24 h at 28 °C on TSA medium at pH 7.0 and cellular fatty acids were analysed using GC at the Belgian Co-ordinated Collections of Microorganisms (Laboratory of Microbiology, University of Gent, Gent, Belgium). The predominant fatty acids of strain VP48T were summed feature 3 (15 : 0 iso 2-OH and/or 16 : 1
7c, 26.7 %), 15 : 0 iso (26.1 %) and 17 : 0 iso 3-OH (17.6 %). Significant amounts of these fatty acids are characteristic of members of the genus Chryseobacterium (Vandamme et al., 1994). However, the fatty acid profile of strain VP48T differs from those of other Chryseobacterium species in its larger amount of summed feature 3 and smaller amounts of 15 : 0 iso and 17 : 1 iso 9c and in the presence of 16 : 15c. A detailed fatty acid composition of strain VP48T is given in the species description.
Phylogenetic data and several phenotypic features enabled strain VP48T to be differentiated from other members of the genus Chryseobacterium. A novel species, Chryseobacterium hispanicum sp. nov., is therefore proposed to accommodate this strain.
Description of Chryseobacterium hispanicum sp. nov.
Chryseobacterium hispanicum (his.pa'ni.cum. L. neut. adj. hispanicum from Spain).
Gram-negative rods, 0.9x1.5–4.0 µm, occurring singly, in pairs or in short chains. Cells are non-motile and non-spore-forming. Colonies on TSA plates are white–yellow, opaque, round, convex and 2–3 mm in diameter after 48 h at 28 °C. Strictly aerobic. Flexirubin reaction is weakly positive. Growth occurs between 5 and 30 °C (optimal growth at 25–28 °C) and from pH 5 to 10 (optimal growth at pH 7.0). Growth occurs with 0–2 % NaCl (optimal growth without NaCl). Good growth occurs on R2A, PCA and TSA, but not on MacConkey agar. Positive for catalase and weakly positive for oxidase; urease activity is absent. Indole is weakly positive. Methyl red and Voges–Proskauer reactions are negative. Does not produce H2S. Casein, starch, aesculin and gelatin are hydrolysed, but Tween 80 and DNA are not. Simmons' citrate test is negative. Nitrate is reduced to nitrite. Acid is produced oxidatively from D-arabinose, D-fructose, D-glucose, glycerol, D-maltose, D-mannose and D-xylose, but not from D-galactose, lactose, D-mannitol or D-trehalose. Glucose fermentation is negative. D-Glucose, L-arabinose, D-mannose and D-maltose are used as sole carbon sources. N-Acetylglucosamine, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid are not used as sole carbon sources. Alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, 
-chymotrypsin, naphthol-AS-BI-phosphohydrolase, -glucosidase and 
-glucosidase activities are present. Arginine dihydrolase, lipase (C14), -galactosidase, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase activities are absent. Cellular fatty acids amounting to more than 1 % are: 13 : 0 iso, 2.5 %; unknown 13.565, 1.0 %; 15 : 0 iso, 26.1 %; 15 : 0 anteiso, 3.6 %; summed feature 3 (16 : 17c and/or 15 : 0 iso 2-OH), 26.7 %; 16 : 15c, 5.3 %; 16 : 0, 2.4 %; 15 : 0 iso 3-OH, 4.0 %; 17 : 1 iso 9c, 1.3 %; unknown 16.582, 1.9 %; 16 : 0 3-OH, 4.4 %; and 17 : 0 iso 3-OH, 17.6 %. The DNA G+C content of the type strain is 34.3 mol% (Tm method).
The type strain is VP48T (=CECT 7129T=CCM 7359T=JCM 13554T), isolated from drinking water in Sevilla, Spain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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