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1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 Thailand Institute of Scientific and Technological Research, Pathumthani 12120, Thailand
3 Korean Collection for Type Cultures (KCTC), Biological Resource Center (BRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejeon 305-806, Korea
4 Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Kisarazu, Chiba 292-0818, Japan
Correspondence
Somboon Tanasupawat
Somboon.T{at}chula.ac.th
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subunit (rpoA) gene sequence analysis, strain FP15-1T was closely related to Enterococcus italicus KCTC 5373T, with 99.2 and 93.8 % similarity, respectively. The strain could be clearly distinguished from E. italicus ATCC 5373T by low DNA–DNA relatedness (
33.8 %) and phenotypic characteristics. Therefore, this strain represent a novel species of the genus Enterococcus, for which the name Enterococcus camelliae sp. nov. is proposed. The type strain is FP15-1T (=KCTC 13133T =NBRC 101868T =NRIC 0105T =TISTR 932T =PCU 277T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rpoA gene sequences of strain FP15-1T are respectively EF154454 and EF197993.
16S rRNA gene sequence-based maximum-likelihood and maximum-parsimony trees are available as supplementary material with the online version of this paper.
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; Merquior et al., 1994; Naser et al., 2005). At the time of writing, 33 Enterococcus species names have been validly published (Euzéby, 1997; last full update 1 February 2007). Four species groups, the Enterococcus faecium, E. avium, E. gallinarum and E. cecorum groups, and other ungrouped species, including Enterococcus faecalis, E. saccharolyticus, E. sulfureus and E. dispar, were revealed by comparative 16S rRNA gene sequence analysis (Hardie & Whiley, 1997). Recently, Enterococcus saccharominimus, represented by isolates from contaminated pasteurized cow's milk (Vancanneyt et al., 2004), was reclassified as a later synonym of Enterococcus italicus, isolated from artisanal Italian cheeses (Fortina et al., 2004), by comparison of partial sequences for three housekeeping genes, phenylalanyl-tRNA synthase subunit (pheS), RNA polymerase subunit (rpoA) and the subunit of ATP synthase (atpA), and as confirmed by DNA–DNA hybridization (Naser et al., 2006). In the present paper, we describe a novel Enterococcus strain from fermented tea leaves (‘miang’) based on phenotypic and chemotaxonomic characteristics, DNA–DNA relatedness and 16S rRNA and rpoA gene sequence analysis.
Samples of fermented tea leaves were collected from Chiangmai province, in the northern part of Thailand. Cocci in chains were isolated from the samples using GYP-CaCO3 agar (Tanasupawat et al., 1992). GYP-sodium acetate-mineral salt broth (Tanasupawat et al., 1992) (pH 7.2) was used for working cultures. All tests were performed by incubating the cultures at 30 °C. Cell shape, size and arrangement and colony appearance were examined using cells grown on GYP agar for 3 days. Gram staining was done as described by Hucker & Conn (1923). Spore formation was examined in the Gram-stained specimen. Results of the oxidation/fermentation test and motility were examined in soft agar (Whittenbury, 1963). Catalase activity, hydrolysis of gelatin, aesculin, arginine and starch, nitrate reduction, production of gas from glucose, gluconate and citrate and acid formation from carbohydrates were tested as reported by Tanasupawat et al. (1992). Additional biochemical characteristics were recorded after 2 days incubation in API 50 CH galleries. Hydrolysis of horse blood was assessed as described by Barrow & Feltham (1993). Growth on Slanetz–Bartley agar (Oxoid) and on kanamycin aesculin azide agar (Merck) was tested. The reaction in litmus milk (Difco) was investigated after incubating cultures for 3, 7 and 14 days. Effects of temperature (10–45 °C), initial pH (pH 4.0, 4.5, 5.0, 7.5, 9.0 and 9.6) and NaCl concentration (2, 4, 6, 6.5, 8 and 10 %, w/v) were observed by examining growth in GYP-sodium acetate-mineral salt broth. Vitamin requirements were examined by the method of Kihara & Snell (1960) with modifications. The isomer of lactic acid was analysed enzymically (Okada et al., 1978). Methyl esters of fatty acids were prepared as described by Ikemoto et al. (1978) and cellular fatty acid compositions were analysed by GLC [model GC-14A (Shimadzu), equipped with a CBP1 (OV-1) type capillary column, 25 m by 0.25 mm inside diameter, at 180–220 °C, and a flame ionization detector]. Gas-liquid chromatograms were calculated by the Chromatopac C-R 4A data-processor (Shimadzu). Quinones were extracted from freeze-dried cells and purified as described by Collins et al. (1977) and Collins & Jones (1979). Purified quinones were analysed by HPLC (Tamaoka et al., 1983).
DNA was isolated and purified by the method of Saito & Miura (1963). The DNA base composition was determined by reversed-phase HPLC (Tamaoka & Komagata, 1984). PCR amplification and sequencing of the 16S rRNA and rpoA genes were performed as described by Lane (1991) and Naser et al. (2005), respectively. The sequences were assembled and compared with deposited type strain sequences available in GenBank/EMBL/DDBJ by using BioEdit version 7.0.1 (Hall, 1999) and CLUSTAL_X version 1.83 (Thompson et al., 1997). Phylogenetic trees were constructed based on the neighbour-joining method (Saitou & Nei, 1987) by the NJPlot program (Perrière & Gouy, 1996) and on the maximum-likelihood and the maximum-parsimony methods using MEGA version 3.1 (Kumar et al., 2004). Confidence values of branches of the phylogenetic tree were determined using bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings. DNA–DNA hybridization experiments were performed at 40 °C for 16 h, as reported by Ezaki et al. (1989).
Cells of strain FP15-1T were Gram-positive, facultatively anaerobic, non-motile, non-spore-forming, spherical or ovoid and arranged in pairs or in chains. The detailed morphological, cultural, physiological and biochemical properties, including chemotaxonomic characteristics, are listed in the species description. Demethylmenaquinones with seven to nine isoprene units [DMK-7, 94.7 %; DMK-8, 4.2 %; DMK-9, 1.1 %] were found. The strain contained the straight-chain unsaturated fatty acids C16 : 1 (30.5 %) and C18 : 1 (20.9 %) as the dominant components. The remainder of the fatty acid profile consisted of C14 : 1 (4.6 %), C14 : 0 (5.4 %), C16 : 0 (5.0 %), C18 : 0 (3.6 %), C20 : 1 (6.7 %), cyclopropane acids of C17 (3.4 %) and C19 (9.1 %), a trace of C12 : 0 and unidentified components (7.2 %). Strain FP15-1T contained roughly the same fatty acid pattern as its closest relatives, E. italicus KCTC 5373T and E. saccharolyticus ATCC 43076T. The amount of C16 : 1 was a significant marker to differentiate strain FP15-1T from E. italicus KCTC 5373T and E. saccharolyticus ATCC 43076T, in which it has been reported to be present in small amounts. The cyclopropane acid of C17 was absent in both E. italicus KCTC 5373T and E. saccharolyticus ATCC 43076T (Fortina et al., 2004). The DNA G+C content of the strain was 37.8 mol%. The almost-complete 16S rRNA gene sequence (1325 bp) of strain FP15-1T indicated that the strain was closely related to E. italicus KCTC 5373T (99.2 %), E. saccharolyticus ATCC 43076T (98.3 %), E. sulfureus ATCC 49903T (98.1 %) and Enterococcus casseliflavus NCIMB 11449T (97.0 %) (Fortina et al., 2004). Lower sequence similarities (<97 %) were found to other described species of the genus Enterococcus (Fig. 1). Trees constructed by the maximum-likelihood and maximum-parsimony methods are available as Supplementary Figs S1 and S2 in IJSEM Online. The rpoA gene sequence (615 bp) of strain FP15-1T showed 93.8, 88.3 and 88.1 % similarity, respectively, to those of E. italicus KCTC 5373T, E. sulfureus ATCC 49903T and E. saccharolyticus ATCC 43076T (Fig. 2). The application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on the rpoA gene (Naser et al., 2005, 2006) confirmed the separation of strain FP15-1T from related species. Strain FP15-1T showed low DNA–DNA relatedness to E. italicus KCTC 5373T (21.6 %), with 33.8 % relatedness in the reciprocal reaction. In addition, strain FP15-1T could be differentiated from closely related Enterococcus species by growth in 6.5 % NaCl and at 10 °C, acid production and DNA G+C content (Table 1). Therefore strain FP15-1T should be classified in the genus Enterococcus as the type strain of a novel species, Enterococcus camelliae sp. nov.
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Cells are Gram-positive, facultatively anaerobic, non-motile, non-spore-forming, spherical or ovoid, 0.5–1 µm in diameter and arranged in pairs or in chains. Colonies on GYP agar plates are circular, raised or low-convex with entire margins, and non-pigmented. Small, red colonies appear on Slanetz–Bartley agar. No growth is observed on kanamycin aesculin azide agar. Positive for hydrolysis of aesculin but weak reactions for starch hydrolysis and blood haemolysis. Negative for catalase, hydrolysis of arginine and gelatin, reduction of nitrate and production of gas from glucose, gluconate and citrate. Utilizes glucose fermentatively. No acidification, coagulation, reduction or liquefaction in litmus milk. Grows at pH 5.0–9.6, at 15–45 °C and in 2–6 % NaCl. Acid is produced from D-glucose, D-fructose, D-cellobiose, aesculin, D-mannose, maltose, mannitol, N-acetylglucosamine, trehalose, sucrose and salicin, but not from glycerol, erythritol, D- or L-arabinose, D-ribose, D- or L-xylose, adonitol, methyl 
-xyloside, D-galactose, D-sorbose, rhamnose, dulcitol, inositol, methyl -D-mannoside, methyl -D-glucoside, D-amygdalin, arbutin, lactose, inulin, D-melibiose, D-melezitose, D-sorbitol, raffinose, starch, glycogen, xylitol, -gentiobiose, D-turanose, D-lyxose, D-tagatose, D- or L-fucose, D- or L-arabitol, gluconate or 2- or 5-ketogluconate. Riboflavin, niacin and calcium pantothenate are required for growth. DMK-7 is the major menaquinone. C16 : 1 is the dominant component of the fatty acid profile. The DNA G+C content of the type strain is 37.8 mol%.
The type strain, FP15-1T (=KCTC 13133T =NBRC 101868T =NRIC 0105T =TISTR 932T =PCU 277T), was isolated from fermented tea leaves (‘miang’) produced in Thailand.
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