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1 Max von Pettenkofer Institute for Hygiene and Medical Microbiology, University of Munich, Munich, Germany
2 Institute of Medical Microbiology, University of Münster, Hospital and Clinics, Münster, Germany
3 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
4 Institute of Hygiene, University of Münster, Hospital and Clinics, Münster, Germany
Correspondence
Karsten Becker
kbecker{at}uni-muenster.de
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type L-lys–gly2–4–L-Ser–Gly), menaquinone pattern (MK-7, MK-6 and MK-8) and major cellular fatty acids (ai-C15 : 0, ai-C17 : 0 and i-C15 : 0) that corresponded to those of staphylococci. Phenotypically, the isolates most closely resembled Staphylococcus capitis subsp. capitis and Staphylococcus auricularis, but they could be distinguished from these species by physiological tests and chemotaxonomic investigations. The results of DNA–DNA hybridization, chemotaxonomic investigations and 16S rRNA gene and RNA polymerase B gene (rpoB) sequence analysis enabled strains B3117T, K6999, 229 and 230 to be differentiated genotypically and phenotypically from known Staphylococcus species, indicating that these isolates are representatives of a novel species. The name Staphylococcus pettenkoferi sp. nov. is proposed for this novel species, with strain B3117T (=CIP 107711T=CCUG 51270T) as the type strain. Due to differences in the results of physiological and chemotaxonomic investigations and DNA–DNA hybridization data, strain A6664 was not included in the description of the novel species.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains B3117T, K6999, 229, 230 and A6664 are AF322002, AM265622, DQ538517, DQ538518 and DQ538520, respectively.
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; von Eiff et al., 2005, 2006). Furthermore, CoNS infect immunocompromised patients such as premature infants and patients undergoing chemotherapy, as well as patients with malignant disease or organ transplants (Pagano et al., 1997; Pessoa-Silva et al., 2001). Two strains of CoNS of human origin have been isolated and described as ‘Staphylococcus pettenkoferi’ (Trülzsch et al., 2002) but the name was not validly published. These two strains and three additional strains from different human sources were subjected to a polyphasic study, including 16S rRNA gene and RNA polymerase B gene (rpoB) sequence comparisons, DNA–DNA hybridization and biochemical and chemotaxonomic analyses.
Five bacterial strains were cultured from human clinical specimens in Belgium and Germany. Strains B3117T and A6664 were isolated from different patients (from a blood culture and wound infection, respectively) at the University of Munich Medical Center, Munich, Germany, in August and November 2000 (designated ‘S. pettenkoferi’; Trülzsch et al., 2002). Strain K6999 was recovered from a blood culture at the Institute of Medical Microbiology, University of Münster, Münster, Germany. Isolates 229 and 230 were grown from blood cultures of different patients in Gosselies Hospital, Gosselies, Belgium. Blood cultures (Bactec 9240 System; Becton Dickinson) were subcultured aerobically for 24 h on Columbia agar supplemented with 5 % sheep blood (Becton Dickinson) and anaerobically using GENbox anaer (bioMérieux) on Schaedler agar supplemented with 5 % sheep blood (Difco) for 48 h.
Nutrient broth (Oxoid) was used as liquid or semi-solid medium. Colony morphology was observed after 2 days growth at 35 °C. Pigment production was tested after incubation for 3–5 days at room temperature. All physiological tests were performed at 37 °C (Cowan & Steel, 1974; Kloos et al., 1974; Trülzsch et al., 2002). Biochemical characteristics were determined using the ID 32 STAPH gallery (bioMérieux). Acid production from glucose, 
-D-fructose, D-mannose, maltose, lactose, D-trehalose, D-mannitol, raffinose, D-ribose, D-cellobiose, D-xylose, L-arabinose, salicin, sucrose, D-galactose, D-turanose, xylitol and melezitose (Sigma-Aldrich) was tested using purple agar base medium (Difco) containing 1 % carbohydrate as described previously (Freney et al., 1999). Catalase was tested using the standard procedure (Freney et al., 1999). Deoxyribonuclease activity was assessed with DNase agar (Becton Dickinson), clumping factor was tested using the Staphaurex Plus latex test (Murex Biotech), cytochrome oxidase was tested using BBL DrySlide (Becton Dickinson) and coagulase production was determined using Bacto Coagulase Plasma (Difco); all procedures were carried out according to the manufacturer's instructions. Urease activity was tested on Christensen agar (Becton Dickinson) as described elsewhere (Freney et al., 1999). Antibiotic susceptibility was determined on Mueller–Hinton agar (Oxoid) by disk-diffusion according to CLSI approved standards M2-A9 and M100-S16.
Biomass for menaquinone and cell wall analyses was obtained by cultivation in trypticase soy yeast extract medium no. 92 (DSMZ, 2001) and biomass for analysis of cellular fatty acids was grown on trypticase soy broth (BBL Microbiology Systems) with agar at 28 °C for 48 h. Cell wall analysis was performed as described previously (Groth et al., 1999). For GC analysis of cellular fatty acids, a 10 mg cell sample was saponified, methylated, extracted and analysed by using the Microbial Identification System as described by Miller (1982). Menaquinones were extracted according to Collins et al. (1977) and were analysed by HPLC as described in detail previously (Stackebrandt et al., 1995).
The 16S rRNA gene sequence was determined as described previously (Becker et al., 2002; Breitkopf et al., 2005). Sequence alignment was performed with nucleotide residues 107–1505 of the 16S rRNA gene sequence of strain B3117T. Alignment of 16S rRNA gene sequences, construction of a phylogenetic tree by the neighbour-joining method and bootstrapping analysis based on 1000 resamplings were performed using the software GENEIOUS 2.5.4 (Biomatters). Partial RNA polymerase B (rpoB) sequencing was performed as reported previously (Mellmann et al., 2006). For sequence analysis, the region from base positions 1444 to 1928 (corresponding to S. aureus rpoB gene positions; GenBank accession no. X64172) of the rpoB gene was used. Sequences were analysed using Ridom TRACEEDITPRO software (version 1.0). For spectroscopic DNA–DNA hybridization, DNA was isolated using the method described by Marmur (1961) and was purified by chromatography on hydroxyapatite according to Cashion et al. (1977). DNA–DNA hybridization was carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huß et al. (1983) in 2x SCC at 63 °C using a UV/vis-spectrophotometer (Cary 100; Bio) equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in situ temperature probe (Varian). Fully automated ribotyping of EcoRI-digested samples was performed with a RiboPrinter system (DuPont Qualicon) as described by Bruce (1996).
Gram staining of the unidentified isolates revealed Gram-positive cocci about 1 µm in diameter that occurred singly, in pairs and in irregular clusters. Biochemical analysis with ID 32 STAPH failed to identify these strains. Further phenotypic analysis revealed that the five strains were non-motile and non-spore-forming. They were able to grow aerobically and anaerobically at 35 °C on Columbia and Schaedler agars. When grown aerobically for 2 days, colonies were 1–2 mm in diameter, circular, smooth, slightly convex, glistening, entire, opaque and unpigmented. However, grown at ambient temperature for 3–5 days, strains B3117T and K6999 produced yellowish pigment, whereas strain A6664 produced no pigment. Anaerobically, colonies reached only pinpoint size after 6 days incubation.
Strains B3117T, K6999, 229 and 230 were catalase-positive, cytochrome oxidase-negative, coagulase-negative in rabbit plasma, deoxyribonuclease-negative, clumping factor-negative and novobiocin-susceptible. They were positive for nitrate reduction, urease, alkaline phosphatase, pyrrolidonyl arylamidase and aerobic production of acid from -D-fructose, sucrose and glucose. All strains produced acid from glucose under anaerobic conditions. Further results of the physiological characterization are given in the species description. According to the biochemical profile, the unidentified bacterial isolates appeared to be most similar to Staphylococcus capitis subsp. capitis and Staphylococcus auricularis (Table 1). However, they differed from S. capitis subsp. capitis by being positive for alkaline phosphatase, urease and pyrrolidonyl arylamidase and by being negative for acid production from D-mannitol and D-mannose. They differed from S. auricularis by being positive for alkaline phosphatase and urease and by being negative for arginine arylamidase and aerobic acid production from maltose and D-trehalose. Strain A6664 differed from the other isolates (B3117T, K6999, 229 and 230) by being positive for acid production from D-trehalose, D-mannose (weakly) and D-mannitol (weakly). Strains B3117T, K6999, 229 and 230 were resistant to fosfomycin and polymyxin B. They were sensitive to vancomycin, rifampicin, fusidic acid, linezolid, novobiocin, gentamicin, tobramicin, amikacin and netilmicin. Some of these four strains showed resistance to penicillin (3/4 resistant, type strain sensitive), oxacillin (3/4 resistant, type strain sensitive), erythromycin (3/4 resistant, type strain sensitive), clindamycin (2/4 resistant, type strain sensitive), mupirocin (1/4 resistant, type strain sensitive), trimethoprim-sulfamethoxazole (2/4 resistant, type strain sensitive), ciprofloxacin (3/4 resistant, type strain sensitive) and moxifloxacin (3/4 resistant, type strain sensitive).
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) with an interpeptide bridge consisting of 3–5 glycine residues and a single serine residue [L-lys–gly2–4–L-Ser–Gly, type A3 according to Schleifer & Kandler (1972) and variation A11.3 according to the DSMZ (http://www.dsmz.de/species/murein.htm)] (Table 2). The quinone systems of the unidentified bacterial isolates contained the menaquinones MK-7, MK-6 and MK-8 (Table 2), which is similar to the situation reported for members of the genus Staphylococcus (i.e. MK-7 and MK-8; Götz et al., 2006). The fatty acid profile of strain B3117T corresponded in its general composition to those of CoNS (Kotilainen et al., 1991) and consisted of ai-C15 : 0 (37.5 %), ai-C17 : 0 (30.0 %), i-C15 : 0 (11.3 %), i-C17 : 0 (9.1 %), ai-C19 : 0 (4.4 %), i-C19 : 0 (3.1 %), C20 : 0 (1.6 %), C18 : 0 (1.5 %), i-C16 : 0 (0.7 %), C16 : 0 (0.6 %) and i-C18 : 0 (0.2 %).
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used for genus definition (95 % 16S rRNA gene sequence similarity), strain B3117T can be classified as a representative of a member of the genus Staphylococcus. Partial rpoB sequencing revealed a high similarity (99.8 %) between strains B3117T and K6999 with one base pair exchange, whereas strain A6664 showed only 99.2 % similarity compared with the type strain. The intra-species variability that was determined by rpoB sequencing was as shown for other staphylococcal species (Mellmann et al., 2006). The next most closely related species based on rpoB sequencing, S. cohnii subsp. urealyticus, showed a similarity of 87.8 % (compared with strain B3117T), whereas the remaining species of the Staphylococcus saprophyticus cluster and S. auricularis showed similarities of 82.5–87.1 %. Phylogenetic studies using 16S rRNA gene and rpoB gene sequences suggested that strain B3117T represents a taxon that is separate from recognized staphylococcal species and showed that it formed a distinct and deep subline most closely related to S. auricularis and the S. saprophyticus cluster group (Staphylococcus arlettae, S. cohnii, Staphylococcus equorum, Staphylococcus gallinarum, Staphylococcus kloosii, S. saprophyticus, Staphylococcus succinus and Staphylococcus xylosus) (Fig. 1, Fig. 2).
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).
RiboPrint analysis revealed that the patterns of strains K6999, 229 and 230 are nearly identical, exhibiting certain similarity to that of strain B3117T. The patterns of all these strains were completely different from the pattern derived from S. saprophyticus subsp. saprophyticus DSM 20229T (Fig. 3).
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) and of DNA–DNA hybridization, strain A6664 was not included in the description of the novel species.
Description of Staphylococcus pettenkoferi sp. nov.
Staphylococcus pettenkoferi (pett.en'kof.eri. N.L. gen. n. pettenkoferi of Pettenkofer, honouring Max von Pettenkofer, 1818–1901, a German pioneer in the field of hygiene and public health).
Cells are Gram-positive, non-motile, non-spore-forming, facultative anaerobic cocci that occur singly, in pairs and in small clusters. After 2 days growth, colonies are 1–2 mm in diameter, circular, smooth, slightly convex, glistening and opaque with entire margins. Some isolates are yellow pigmented, in particular under ambient temperatures. Catalase-positive and novobiocin-susceptible. Negative for staphylocoagulase, clumping factor and deoxyribonuclease. Produces pyrrolidonyl arylamidase, alkaline phosphatase, urease and nitrate reductase. Produces acid aerobically from glucose, sucrose and fructose. Negative for arginine dihydrolase, ornithine decarboxylase, aesculin hydrolysis, acetoin production, -galactosidase, arginine arylamidase, N-acetylglucosamine and -glucuronidase. Does not produce acid aerobically from mannose, maltose, lactose, trehalose, mannitol, raffinose, D-ribose, D-cellobiose, D-xylose, L-arabinose, salicin, D-galactose, D-turanose, xylitol or melezitose. Menaquinones MK-7, MK-6 and MK-8 predominate. The major cellular fatty acids are ai-C15 : 0, ai-C17 : 0 and i-C15 : 0.
The type strain is B3117T (=CIP 107711T=CCUG 51270T), was isolated from blood of a patient in Germany. Other representative strains are K6999, 229 and 230, all isolated from clinical specimens.
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ACKNOWLEDGEMENTS |
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