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1 Institute of Bacteriology, Mycology and Hygiene, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria
2 Cientific Titular CSIC, GOI, IMEDEA (CSIC-UIB), C/Miquel Marqués 21, E-07190 Esporles, Mallorca, Spain
3 Institute of Microbiology and Genetics, University of Vienna, A-1030 Vienna, Austria
Correspondence
Hans-Jürgen Busse
hans-juergen.busse{at}vu-wien.ac.at
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The EMBL accession number for the 16S rDNA sequence of strain CW1T is AJ517414.
SDS-PAGE profiles of whole-cell lysates of the novel isolates and reference Staphylococcus species are available as supplementary material in IJSEM Online.
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; Lambert et al., 1998; Vernozy-Rozand et al., 2000). Several Staphylococcus species have been isolated from various mammals and birds. In the caprine host, Staphylococcus species have been recovered from skin and mucous membranes, as well as from goat products such as milk and cheese (Devriese et al., 1985; Valle et al., 1991; Ferrer et al., 1997; Klinger & Rosenthal, 1997; Mahanta et al., 1997; Bedidi-Madani et al., 1998; Vernozy-Rozand et al., 2000; Place et al., 2002). In the course of a study to determine the prevalence and significance of bacteria in the respiratory tract of goats with pneumonia from different areas of the Himalayan region, a small number of unidentified staphylococci were isolated. Preliminary routine investigations demonstrated that they were coagulase-negative and resistant towards novobiocin. The cluster of staphylococci that share these traits consists of Staphylococcus arlettae, Staphylococcus cohnii, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus kloosii, Staphylococcus saprophyticus, Staphylococcus succinus and Staphylococcus xylosus. Phylogenetically, these species are located on a separate subline within the genus Staphylococcus (Takahashi et al., 1999). Here, we report the characterization and classification of four isolates, for which we propose the name Staphylococcus nepalensis sp. nov. The four strains were recovered from three nasal swabs (PM34, RW78, CW1T) and one lung sample (MM3) collected from four goats with respiratory symptoms kept in different areas of Nepal (Pakhribas, Mangalbare, Rajarani, Chitwan). The organisms were isolated on blood agar base no. 2 (Oxoid) supplemented with 5 % (v/v) defibrinated sheep blood that was incubated aerobically at 37 °C for 24 h. Subcultivation was done on trypticase soy agar (BBL) or trypticase soy broth (BBL). Type strains of Staphylococcus species were supplied by the American Type Culture Collection, Manassas, VA, USA (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany (DSMZ), and the Czech Collection of Microorganisms, Masaryk University, Brno, Czech Republic (CCM).
16S rDNA sequence analyses
16S rDNA of strain CW1T was amplified and sequenced as described by Wieser et al. (1999) and PCR products were purified using a GenElute purification kit (Sigma). The derived sequence was aligned and compared with other bacterial 16S rRNA sequences available in the EMBL database using the Wisconsin package (GCG, 1995). The 16S rDNA sequence of strain CW1T consisted of 1470 nucleotides (positions 31–1492, Escherichia coli numbering; Brosius et al., 1978). Sequence comparisons using FASTA (Pearson & Lipman, 1988) demonstrated that strain CW1T is a member of the genus Staphylococcus. Highest sequence similarity values were obtained to S. cohnii subsp. urealyticus ATCC 49330T, S. saprophyticus subsp. saprophyticus ATCC 15305T, S. cohnii subsp. cohnii ATCC 29974T, S. arlettae ATCC 43957T, S. gallinarum ATCC 35539T, S. succinus ATCC 700337T and S. xylosus ATCC 29971T (99·0, 98·8, 98·8, 98·4, 98·2, 98·1 and 98·1 %, respectively).
Physiological characterization and antibiotic susceptibilities
Morphological and physiological characteristics as well as susceptibility to lysostaphin, lysozyme, furazolidone, novobiocin, bacitracin and the vibriostatic compound O/129 were examined as described previously (Hájek et al., 1992; Freney et al., 1999). Susceptibility to other antibiotics was determined on Mueller–Hinton agar (Oxoid) by a standard diffusion technique using antibiotic-impregnated disks (Oxoid). Any sign of growth inhibition, after 24 h incubation at 37 °C, was scored. Other traits were examined using API ID 32 Staph and API 50 CH strips (bioMérieux). Characteristics of the four strains PM34, MM3, RW78 and CW1T are summarized in the species description below and in Table 1.
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; Altenburger et al., 1996). Quinone systems consisted of the major menaquinone MK-7 (71–74 %) and minor amounts of MK-6 (10–16 %) and MK-8 (10–18 %). A similar quinone system with the predominant compound MK-7 has been reported for numerous Staphylococcus species, including strains of S. cohnii, S. saprophyticus and S. xylosus (Nahaie et al., 1984). Polar lipids were extracted and analysed by two-dimensional TLC according to Ventosa et al. (1993). The polar lipid profiles of the three analysed strains were homogeneous. They consisted of the major lipids diphosphatidylglycerol, phosphatidylglycerol and a glycolipid. The chromatographic behaviour of this glycolipid corresponded to a glycolipid G1, which was assumed to be 
-gentiobiosyl diacylglycerol (Nahaie et al., 1984). Additionally, two unknown aminophospholipids, three unknown glycolipids and one unknown polar lipid were detected. Concerning the major components, this polar lipid profile was in good agreement with the profiles of S. cohnii NCTC 11041T, S. saprophyticus SCH94T and S. xylosus NCTC 11043T (Nahaie et al., 1984). Diagnostic cell-wall diamino acids were extracted from cell walls as described by Schleifer (1985) and identified by comparison with the chromatographic behaviour of references. The characteristic cell-wall diamino acid of strains CW1T, PM34 and MM3 was L-lysine, which was reported to be characteristic of staphylococci (Kloos et al., 1992).
Fatty acids were extracted from biomass grown at 28 °C for 24 h on trypticase soy agar according to Osterhout et al. (1991) and analysed using GLC (Wieser et al., 1999). In the fatty acid profiles, the predominant fatty acids were ai-C15 : 0, i-C15 : 0 and ai-C17 : 0, whilst i-C17 : 0, C18 : 0, C16 : 0 and C20 : 0 were present in moderate amounts (Table 2). Similar fatty acid profiles were reported for several Staphylococcus species by Kotilainen et al. (1991). The combination of these chemotaxonomic characteristics supports the assignment of the isolates to the genus Staphylococcus.
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; Rosengarten et al., 1994). Each of the 11 species and subspecies displayed a unique band profile, allowing their differentiation from each other (see supplementary material available in IJSEM Online). Partial similarities were observed between S. succinus subsp. succinus ATCC 700337T and S. kloosii CCM 3834T, S. arlettae CCM 3830T, S. cohnii subsp. cohnii CCM 2736T and S. cohnii subsp. urealyticus CCM 4294T, and S. saprophyticus subsp. saprophyticus CCM 883T and CW1T, but almost identical protein profiles were only detected for isolates PM34, MM3, RW78 and CW1T. These results support assignment of the four isolates to a single species that has not yet been recognized.
Genomic analysis
The DNA extraction protocol was based on a previously described procedure (Lambert et al., 1998). Determination of DNA G+C content was performed as described by Wieser et al. (1999). The G+C content of the genomic DNA of CW1T was 33 mol%, which is in the range reported for other Staphylococcus species (Kloos & Schleifer, 1986).
Enterobacterial repetitive intergenic consensus (ERIC)-PCR has been shown to be a useful approach for identification of Staphylococcus epidermidis and its differentiation from related species. In order to examine this approach for identification of our four isolates, their ERIC-fingerprints, generated as described by Wieser & Busse (2000) using a Gene Amp PCR System 2400 thermal cycler (Perkin Elmer Applied Biosystems), were analysed. Each isolate displayed a single band consisting of approximately 640 bp (Fig. 1). Other Staphylococcus species that were considered to be the nearest relatives of CW1T based on 16S rDNA sequence similarity values exhibited clearly distinguishable banding patterns. These observations indicated that the four isolates are members of a single novel species.
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under optimal conditions at 58 °C (30° below the melting temperature of the homoduplex DNA; Tm=88 °C). The results (Table 3) demonstrated that strain CW1T represents a novel species of the genus Staphylococcus.
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Based on the results presented here, it is demonstrated that strains PM34, MM3, RW78 and CW1T are members of a novel species of the genus Staphylococcus, for which we propose the name Staphylococcus nepalensis sp. nov.
Description of Staphylococcus nepalensis sp. nov.
Staphylococcus nepalensis (ne.pa.len'sis. N.L. masc. adj. nepalensis pertaining to the kingdom of Nepal, where clinical samples for bacteriological examination were collected from local goats).
Cells are Gram-positive cocci, 1·1–1·6 µm in diameter, that occur singly, in pairs and in irregular clusters. Motility is not observed. Colonies after 2 days on P agar are circular, low-convex, smooth, glossy, opaque white and 2–6 mm in diameter. Growth occurs aerobically and anaerobically in the presence of 0–7·5 % NaCl (w/v). The type strain grows well in the presence of 10 % NaCl (w/v), but growth is variable for other strains. No growth is observed in the presence of 15 % NaCl. Growth occurs between 20 and 40 °C, best growth at 30 °C. No growth at 15 or 45 °C. Oxidase-negative. Catalase-positive. Produces urease, alkaline phosphatase, pyrrolidonyl arylamidase, -galactosidase and -glucuronidase. Hydrolyses aesculin and Tween 80. Reduces nitrate to nitrite. Positive for aerobic production of acid from D-glucose, D-fructose, D-mannose, maltose, lactose, trehalose, mannitol, sucrose, L-arabinose, N-acetylglucosamine, galactose, glycerol, erythritol, D-xylose, arbutin and salicin. Negative for oxidase activity, clumping factor, coagulase, hyaluronidase, arginine dihydrolase, ornithine decarboxylase, acetoin, arginine arylamidase, 
- and -haemolysins, heat-stable and heat-labile nucleases, indole, hydrogen sulphide and lecithinase. Acid is not formed aerobically from D-raffinose, ribose, D-cellobiose, D-arabinose, L-xylose, adonitol, methyl -D-xyloside, rhamnose, L-sorbose, dulcitol, starch, inositol, methyl -D-mannoside, methyl -D-glucoside, amygdalin, melibiose, melezitose, gentiobiose, glycogen, inulin, D-tagatose, D-lyxose, cellobiose, D-fucose, L-fucose, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluconate. Acid production from D-arabitol, sorbitol, turanose and xylitol is variable; the type strain is positive. Resistant to novobiocin, bacitracin, vibriostatic agent O/129, lysozyme, metronidazole and optochin. Susceptible to lysostaphin, furazolidone, ampicillin, amoxicillin, amoxicillin/clavulanic acid, ceftiofur, cephalexin, cephalothin, chloramphenicol, clindamycin, colistin sulphate, enrofloxacin, erythromycin, florfenicol, fosfomycin, fusidic acid, gentamicin, kanamycin, lincomycin, neomycin, nitrofurantoin, oxacillin, penicillin G, polymyxin B, sulfamethoxazole/trimethoprim, tetracycline and vancomycin. The quinone system consists of the major menaquinone MK-7 and minor amounts of MK-6 and MK-8. Predominant fatty acids are ai-C15 : 0, i-C15 : 0 and ai-C17 : 0, whilst i-C17 : 0, C18 : 0, C16 : 0, C20 : 0 and i-C19 : 0 are present in moderate amounts. Polar lipid profile consists of the major lipids diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid (assumed to be -gentiobiosyl diacylglycerol). Additionally, minor amounts of two unknown aminophospholipids and three unknown glycolipids are detected. The G+C content of the type strain is 33 mol% (HPLC).
The type strain is CW1T (=DSM 15150T=CCM 7045T) and the most dissimilar strain is PM34 (=DSM 15151=CCM 7046).
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ACKNOWLEDGEMENTS |
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90 % of strains positive; d, 11–89 % of strains positive; -, 0–10 % of strains positive.