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1 School for Agriculture and Environmental Science, Central University of Technology: Free State, P/bag x20539, 1 Pres. Brandt Street, Bloemfontein 9300, South Africa
2 Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa
3 Department of Biotechnology and Food Technology, Tshwane University of Technology, Pretoria, South Africa
4 Laboratorium of Microbiology, Ghent University, K.L. Ledeganckstraat 35, Ghent B-9000, Belgium
5 BCCM/LMG Bacteria Collection, Ghent University, K.L. Ledeganckstraat 35, Ghent B-9000, Belgium
Correspondence
Hanli de Beer
hdebeer{at}cut.ac.za
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Chryseobacterium vrystaatense LMG 22846T and LMG 22954 are AJ871397 and AJ871398, respectively.
A dendrogram derived from the unweighted pair group mean linkage of correlation coefficients between the whole-cell protein profiles of C. vrystaatense and reference strains of related taxa and a table detailing the strains investigated and their origin are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). In the literature on meat spoilage, ‘flavobacteria’ is used as a generic name for yellow-pigmented rods (Hendrie et al., 1969). Usually, pathogens such as Salmonella and Campylobacter are associated with poultry, but large numbers of other bacteria often associated with spoilage are found on poultry carcasses. These include many so-called flavobacteria that may originate from the poultry itself or from the abattoir environment and which are responsible for spoilage (Hang'ombe et al., 1999). Mai & Conner (2001) found that the incidence of members of the genus Pseudomonas and flavobacteria on chicken carcasses was 17 and 16 %, respectively. The incidence of flavobacteria on poultry is much higher than on other fresh meat (Nychas & Drosinos, 1999).
Over the past decade, many changes have taken place in the taxonomy of the family Flavobacteriaceae, and the revised genus Flavobacterium comprises mainly aquatic bacteria that are not known in food microbiology (Bernardet et al., 1996). Several former Flavobacterium species were transferred to the novel genus Chryseobacterium (Vandamme et al., 1994) and novel species have been described, including Chryseobacterium joostei (Hugo et al., 2003), Chryseobacterium defluvii (Kämpfer et al., 2003), Chryseobacterium miricola (Li et al., 2003) and, most recently, Chryseobacterium formosense (Young et al., 2005), Chryseobacterium daecheongense (Kim et al., 2005a) and Chryseobacterium taichungense (Shen et al., 2005). ‘Chryseobacterium proteolyticum’ was described by Yamaguchi & Yokoe (2000), but its name has not been validly published. In addition, two Chryseobacterium species, Chryseobacterium meningosepticum and Chryseobacterium miricola, have been reclassified into the novel genus Elizabethkingia (Kim et al., 2005b). In general, these Chryseobacterium species are widely distributed in water, soil and the clinical environment, but they are also found in food specimens, such as milk, meat, poultry and fish (Jooste & Hugo, 1999).
During a study investigating the incidence of yellow-pigmented strains isolated from meat, 36 yellow-pigmented isolates were obtained from raw chicken, at different stages of processing, from a local chicken-processing plant in the Vrystaat province (Free State, South Africa). Informtion regarding the investigated isolates, their origin and number is given in the Supplementary Table (available in IJSEM Online). The spoilage ability of this group of strains has not yet been determined. Chryseobacterium reference strains were available from the BCCM/LMG Bacteria Collection. After growth for 24 h on trypticase soy agar (BBL), cells were harvested and whole-cell protein extracts were prepared and subjected to whole-cell protein electrophoresis as described by Pot et al. (1994). A densitometric analysis, normalization and interpolation of the protein profiles, and a numerical analysis were performed by using the GelCompar software package (Applied Maths). The 36 isolates formed three subgroups comprising 19, four and 13 isolates, respectively. Isolates belonging to the three subgroups differed mainly in the density of some protein bands (see Supplementary Figure in IJSEM Online).
High-molecular-mass DNA was prepared according to the methods of Pitcher et al. (1989) from a representative isolate of each of these three subgroups, i.e. isolates LMG 22846T, LMG 22848 and LMG 22847, and from Chryseobacterium reference strains (see below). DNA–DNA hybridizations were performed with photobiotin-labelled probes in microplate wells as described by Ezaki et al. (1989), using an HTS7000 Bio Assay Reader (Perkin Elmer) for the fluorescence measurements. The hybridization temperature was 33 °C. Hybridization values obtained among the three isolates were >92 %, demonstrating that the three subgroups formed a homogeneous genospecies.
In order to determine the phylogenetic position of this taxon, the 16S rRNA gene sequences of strains LMG 22846T and LMG 22954 were determined as described by Willems et al. (2003). The 16S rRNA gene sequences of strains LMG 22846T and LMG 22954 showed 99·9 % similarity to each other and 96·9, 97·1 and 97·1 % similarity to those of C. joostei, Chryseobacterium indologenes and Chryseobacterium gleum, respectively, demonstrating that the taxon represented by these two strains belonged to the genus Chryseobacterium. Values towards the other type strains investigated were <96 %. Fig. 1 illustrates the phylogenetic position of this taxon within the genus Chryseobacterium. Subsequent DNA–DNA hybridizations between the type strains of the latter three species yielded a value of 46 % between strain LMG 22846T and C. joostei LMG 18212T; the other values were between 27 and 32 %.
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. The nucleoside mixture obtained was then separated by HPLC using a Waters SymmetryShield C8 column regulated at 37 °C. The solvent was 0·02 M NH4H2PO4, pH 4·0, with 1·5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference. The detector used was a Waters model 484 UV-VIS absorbance detector set at 270 nm. The DNA G+C contents of strains LMG 22846T, LMG 22847 and LMG 22848 were 37·1, 37·0 and 37·6 mol%, respectively, within the range of 34–38 mol% reported for the genus Chyseobacterium (Vandamme et al., 1994).
The whole-cell fatty acid components of seven strains representing the three subgroups and of Chryseobacterium reference strains were prepared from a loopful of well-grown cells incubated for 24 h at 28 °C. Separation and identification of esters were performed with the Sherlock Microbial Identification system (MIDI version 3.0) as described by Vandamme et al. (1992). Mean percentages and SD were calculated and are shown in Table 1. As for other Chryseobacterium species, the predominant fatty acids were iso-C15 : 0 (40–43 %), iso-C17 : 1
9c (16–21 %) and iso-C17 : 0 3-OH (15–18 %).
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, MacFaddin (1980), Gerhardt et al. (1981) and Hugo et al. (2003). Twenty-two isolates were subjected to Biolog GN2 MicroPlate (Biolog Inc.) tests according to the manufacturer's protocol. Test results are given below in the species description and in Table 2, which includes the biochemical tests useful for the differentiation of the novel species from recognized Chryseobacterium species.
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Description of Chryseobacterium vrystaatense sp. nov.
Chryseobacterium vrystaatense (vry.staa.ten'se. N.L. neut. adj. vrystaatense named after Vrystaat (Free State), the South African province where these bacteria were isolated).
Colonies are shiny, orange–yellow and translucent with entire edges. Cells are Gram-negative, catalase- and oxidase-positive and produce flexirubin-type pigments; they are non-motile and neither oxidative nor fermentative. Colonies are seen after 24 h growth at 4, 15, 25 and 32 °C on nutrient agar, but no growth occurs at 37 °C or above. Growth on MacConkey agar no. 3 (Oxoid CM115) is observed after 48 h at 15, 25 and 32 °C for all but one strain that grows only on MacConkey agar after 48 h at 32 °C. All strains grow in nutrient broth containing 1 and 2 % NaCl (w/v); growth in 3 % NaCl broth is strain-dependent (14 strains, excluding the type strain, test positive for growth). Although several strains initially show growth at different salt concentrations, only five show growth in 4 % NaCl broth after 5 days incubation (R-23493, R-23569, R-23575, R-23576 and R-23595). Strains show strong DNase activity and also urease and lecithinase activity, but no starch or tyrosine hydrolysis. Reactions on triple-sugar agar (Oxoid CM277) and 10 % lactose are alkaline. Ability to produce H2S varies depending on incubation time on triple-sugar agar and SIM medium (Oxoid CM435). Both media contain sodium thiosulfate, but SIM medium also contains ferrous ammonium sulfate and >50 % of strains give a positive reaction. All strains tested (n=22) show positive reactions in the Biolog system for Tweens 40 and 80, gentobiose, 
-D-glucose, D-mannose, D-trehalose, succinic acid monomethyl ester, acetic acid and L-asparagine. At least 19 of the strains (85 %) react for glycyl L-glutamic acid, glycerol, L-glutamic acid, L-serine and L-aspartic acid. Reactions in which more than 50 % of the strains give a positive result are for dextrin (n=16), D-mannitol (n=18), -ketovaleric acid (n=15), L-alanylglycine (n=12), L-threonine (n=16), inosine (n=13), uridine (n=15) and thymidine (n=12).
The type strain, LMG 22846T (=CCUG 50970T), was isolated from a raw chicken-portion sample obtained from a chicken-processing plant. The other (35) isolates were obtained from raw-chicken samples, collected at different processing stages (portion, whole bird, automatic line, manual line and the spiral freezing process) on different occasions. Three additional isolates have been deposited in the BCCM/LMG Bacteria Collection as LMG 22847, LMG 22848 and LMG 22954.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Cowan, S. T. (1974). Cowan and Steel's Manual for the Identification of Medical Bacteria, 2nd edn. Cambridge: Cambridge University Press.
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Kim, K. K., Bae, H.-S., Schumann, P. & Lee, S.-T. (2005a). Chryseobacterium daecheongense sp. nov., isolated from freshwater lake sediment. Int J Syst Evol Microbiol 55, 133–138.
Kim, K. K., Kim, M.-K., Lim, J. H., Park, H. Y. & Lee, S.-T. (2005b). Transfer of Chryseobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen. nov. as Elizabethkingia meningoseptica comb. nov. and Elizabethkingia miricola comb. nov. Int J Syst Evol Microbiol 55, 1287–1293.
Li, Y., Kawamura, Y., Fujiwara, N., Naka, T., Liu, H., Huang, X., Kobayashi, K. & Ezaki, T. (2003). Chryseobacterium miricola sp. nov., a novel species isolated from condensation water of space station Mir. Syst Appl Microbiol 26, 523–528.[CrossRef][Medline]
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Shen, F.-T., Kämpfer, P., Young, C.-C., Lai, W.-A. & Arun, A. B. (2005). Chryseobacterium taichungense sp. nov., isolated from contaminated soil. Int J Syst Evol Microbiol 55, 1301–1304.
Vandamme, P., Vancanneyt, M., Pot, B. & 10 other authors (1992). Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens. Int J Syst Bacteriol 42, 344–356.
Vandamme, P., Bernardet, J.-F., Segers, P., Kersters, K. & Holmes, B. (1994). New perspectives in the classification of the flavobacteria: description of Chryseobacterium gen. nov., Bergeyella gen. nov., and Empedobacter nom. rev. Int J Syst Bacteriol 44, 827–831.
Willems, A., Fernández-López, M., Muñoz-Adelantado, E., Goris, J., De Vos, P., Martínez-Romero, E., Toro, N. & Gillis, M. (2003). Description of new Ensifer strains from nodules and proposal to transfer Ensifer adhaerens Casida 1982 to Sinorhizobium as Sinorhizobium adhaerens comb. nov. Request for an Opinion. Int J Syst Evol Microbiol 53, 1207–1217.
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