“Staphylococcus pettenkoferi,” a novel staphylococcal species isolated from clinical specimens

International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1543–1548

DOI 10.1099/ijs.0.64381-0

Staphylococcus pettenkoferi sp. nov., a novel coagulase-negative staphylococcal species isolated from human clinical specimens Konrad Tru¨lzsch,1 Be´atrice Grabein,1 Peter Schumann,3 Alexander Mellmann,4 Uladzimir Antonenka,1 Ju¨rgen Heesemann1 and Karsten Becker2 1

Max von Pettenkofer Institute for Hygiene and Medical Microbiology, University of Munich, Munich, Germany

Correspondence Karsten Becker [email protected]

2

Institute of Medical Microbiology, University of Mu¨nster, Hospital and Clinics, Mu¨nster, Germany

3

DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

4

Institute of Hygiene, University of Mu¨nster, Hospital and Clinics, Mu¨nster, Germany

Five coagulase-negative, novobiocin-susceptible staphylococcal strains were isolated from human blood cultures in different German and Belgian medical facilities. A novel species, ‘Staphylococcus pettenkoferi’ was proposed recently to accommodate two of these strains (B3117T and A6664), although the name was not validly published. All five strains belonged to the genus Staphylococcus because they were non-motile, Gram-positive, catalase-positive cocci with peptidoglycan type (A3a 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 genus Staphylococcus consists of more than 40 species and subspecies that are ubiquitous in nature and are able to colonize or infect a wide range of animals. In addition to coagulase-positive Staphylococcus aureus subsp. aureus, coagulase-negative staphylococci (CoNS) are among the most commonly isolated bacterial species in clinical microbiology laboratories, undoubtedly mostly as skin contaminants. However, CoNS are becoming increasingly important as nosocomial pathogens, especially in patients with indwelling or implanted foreign bodies such as intravenous catheters, prosthetic heart valves and joint Abbreviation: CoNS, coagulase-negative staphylococci.

prostheses (Huebner & Goldmann, 1999; 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; PessoaSilva et al., 2001). Two strains of CoNS of human origin have been isolated and described as ‘Staphylococcus pettenkoferi’ (Tru¨lzsch 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.

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.

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

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culture and wound infection, respectively) at the University of Munich Medical Center, Munich, Germany, in August and November 2000 (designated ‘S. pettenkoferi’; Tru¨lzsch et al., 2002). Strain K6999 was recovered from a blood culture at the Institute of Medical Microbiology, University of Mu¨nster, Mu¨nster, 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 (bioMe´rieux) 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 uC. Pigment production was tested after incubation for 3–5 days at room temperature. All physiological tests were performed at 37 uC (Cowan & Steel, 1974; Kloos et al., 1974; Tru¨lzsch et al., 2002). Biochemical characteristics were determined using the ID 32 STAPH gallery (bioMe´rieux). Acid production from glucose, b-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 uC 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. 1544

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 26 SCC at 63 uC using a UV/vis-spectrophotometer (Cary 100; Bio) equipped with a Peltier-thermostatted 666 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 Grampositive cocci about 1 mm 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 nonmotile and non-spore-forming. They were able to grow aerobically and anaerobically at 35 uC 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 b-Dfructose, 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 International Journal of Systematic and Evolutionary Microbiology 57

Staphylococcus pettenkoferi sp. nov

Table 1. Characteristics that differentiate strain B3117T from closely related staphylococcal species and those with a similar biochemical profile

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).

Taxa: 1, strain B3117T; 2, S. auricularis; 3, S. capitis subsp. capitis; 4, S. caprae; 5, S. cohnii subsp. cohnii; 6, S. cohnii subsp. urealyticus; 7, S. saprophyticus subsp. bovis; 8, S. saprophyticus subsp. saprophyticus. Data for reference taxa were obtained from Freney et al., (1999) and Bannerman, (2003). +, 90 % or more strains positive; 2, 90 % or more strains negative; d, 11–89 % strains are positive; +w, positive to weak reaction; 2w, negative to weak reaction; (), delayed reaction. Characteristic

1

2

3

4 5

6

7

In accordance with affiliation to the genus Staphylococcus, the unidentified bacterial strains B3117T, A6664 and K6999 contained a peptidoglycan that was typical of the genus (Go¨tz et al., 2006) with an interpeptide bridge consisting of 3–5 glycine residues and a single serine residue [L-Lys– Gly2–4–L-Ser–Gly, type A3a 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; Go¨tz 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 %).

8

Alkaline phosphatase + 2 2 + 2 +w 2 2 Arginine dihydrolase 2 d d + 2 2w 2 2w b-Galactosidase 2 (d) 2 2 2 + + d Arginine arylamidase 2 + 2 2 2 2 2 2 Pyrrolidonyl arylamidase + d 2 d 2 d + 2 Urease + 2 2 + 2 + + + Reduction of nitrate + (d) d + 2 2 + 2 Production of acetoin 2 d d + d d d + Aerobic acid production from: b-D-Fructose + + + 2 + + + + D-Mannose 2 2 + + (d) + 2 2 Maltose 2 (+) 2 d (d) (+) + + Lactose 2 2 2 + 2 + 2 d D-Trehalose 2 (+) 2 + + + + + D-Mannitol 2 2 + d d d d d Sucrose + d (+) 2 2 2 + + D-Galactose 2 2 2 + 2 d (+) 2 D-Turanose 2 (d) 2 2 2 2 + + Xylitol 2 2 2 2 (d) (d) d d Novobiocin resistance 2 2 2 2 + + + +

Except for one base pair exchange in strain K6999 (T instead of C in position 85), the 16S rRNA gene sequences of the five strains tested were identical and showed highest binary similarity to S. auricularis ATCC 33753T (97.9 %) and Staphylococcus cohnii subsp. urealyticus GTC 728T (97.7 %). According to the criterion of Ludwig et al. (1998) 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

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 T D-mannitol (weakly). Strains B3117 , 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

Table 2. Chemotaxonomic characterization of strains B3117T, K6999 and A6664 Characteristic Menaquinones Peptidoglycan molar amino acid ratio Peptidoglycan type*

B3117T

K6999

A6664

MK-7, MK-6, MK-8 (74 : 11 : 10) 0.7 Lys, 2.4 Ala, 3.0 Gly, 0.7 Ser, 1.0 Glu A3a LysrGly2rSerrGly A11.3

MK-7, MK-8, MK-6 (78 : 10 : 8) 0.7 Lys, 2.5 Ala, 4.1 Gly, 0.4 Ser, 1.0 Glu A3a LysrGly3rSerrGly A11.3

MK-6, MK-7, MK-8 (60 : 30 : 2) 0.9 Lys, 2.8 Ala, 4.8 Gly, 0.3 Ser, 1.0 Glu A3a LysrGly4rSerrGly A11.3

*According to DSMZ, 2001 (http://www.dsmz.de/species/murein.htm). http://ijs.sgmjournals.org

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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). Using spectroscopic analysis, 100.0 % DNA–DNA hybridization was found between strains B3117T and K6999. In contrast, only 60.5 % (rerun, 65.6 %) DNA–DNA hybridization was found between strains B3117T and A6664, suggesting that they belong to different species. The levels of DNA–DNA relatedness between strain B3117T and type strains of the phylogenetically most closely related species S. auricularis DSM 20609T, S. kloosii DSM 20676T and S. saprophyticus subsp. saprophyticus DSM 20229T were 31.0, 36.0 and 67.7 %, respectively, which is below the 70 % threshold value for the delineation of genospecies (Wayne et al., 1987).

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). The morphological and biochemical properties of the unidentified Gram-positive coccus were found to be consistent with its assignment to the genus Staphylococcus, although it could not be identified as a member of a hitherto established species of this genus. Furthermore, results of DNA–DNA hybridization experiments showed that the staphylococcal strains B3117T, K6999, 229 and 230 merit classification as a distinct species. Thus, based on phylogenetic and phenotypic evidence, it is proposed that these four unidentified micro-organisms recovered from human blood and wound infection should be assigned to the genus Staphylococcus as members of the species Staphylococcus pettenkoferi sp. nov. Due to differences in the results of physiological and

Fig. 1. Unrooted neighbour-joining tree based on alignment of almost complete 16S rRNA gene sequences (1398 bp) of strains B3117T and K6999 with other species of the genus Staphylococcus. Numbers at nodes (shown if greater than 50 %) indicate the percentage of bootstrap support based on analysis of 1000 resampled datasets. Bar, 0.6 % sequence divergence. 1546

International Journal of Systematic and Evolutionary Microbiology 57

Staphylococcus pettenkoferi sp. nov

Fig. 2. Unrooted neighbour-joining tree based on partial rpoB gene sequences (485 bp) showing the relationship of strains B3117T and K6999 with other species within the genus Staphylococcus. The scale bar indicates the evolutionary distance between sequences determined by measuring the lengths of the horizontal lines connecting two organisms. Numbers at the nodes are bootstrap values (1000 replicates). Bar, 2 % sequence divergence.

chemotaxonomic investigations (e.g. major menaquinone; Table 2) 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.en9kof.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, b-galactosidase, arginine arylamidase, N-acetylglucosamine and b-glucuronidase. Does not produce acid aerobically from mannose, maltose, lactose, trehalose, mannitol, raffinose, D-ribose, D-cellobiose, Dxylose, 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.

Fig. 3. Diversity of normalized RiboPrint patterns of strains of Staphylococcus pettenkoferi sp. nov. obtained by EcoRI digestion. The band patterns were compared by using BioNumerics software (Applied Maths). http://ijs.sgmjournals.org

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Acknowledgements The authors are grateful to K. Niedung, M. Schulte, A. Hassing and B. Schuhen for excellent technical assistance. We thank Dr. B. Gualtieri and Pr. G. Wauters for providing the Belgian S. pettenkoferi strains.

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