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The genus Arthrobacter includes catalase-positive coryneform bacteria with an oxidative metabolism, the cell wall of which contains L-lysine as the diamino acid and cellular fatty acids of the branched type (6). Arthrobacter strains have only recently been recognized as opportunistic pathogens belonging to three species, A. cumminsii, A. woluwensis, and A. creatinolyticus, which hitherto were isolated only from humans (4, 7). Based on phenotypic, chemotaxonomic, and genetic studies, we describe the species identification of five Arthrobacter isolates. Two of them were assigned to the species A. oxydans, and the three others were assigned to two as-yet-undescribed species for which the names Arthrobacter luteolus sp. nov. and Arthrobacter albus sp. nov. are proposed.

Bacterial strains. The five strains and the sites from which they were isolated are as follows: CF25, surgical wound; CF39, blood; CF43, blood; CF44, urine; and CF46, blood. Additional Arthrobacter strains were included for comparison in the study: seven A. cumminsii strains, including two laboratory isolates and the reference strains DSM 10493^T, DSM 10494, CCUG 33745, CCUG 35863, and CCUG 35241, as well as the type strains A. woluwensis DSM 10495, A. oxydans DSM 20119, and A. citreus DSM 20133. They were inoculated on blood agar and incubated at 37°C.

Phenotypic characterization. Most cultural, morphologic, and biochemical properties were investigated as described previously (6, 20). Flagellum staining was performed according to the method of Kodaka et al. (12). Gelatin hydrolysis was detected as outlined by Pitt and Dey (15). Oxidative acid production from carbohydrates was detected on phenol red agar slants with a low peptone content as described for acidification of ethylene glycol (20). Pyrrolidonyl peptidase, -glucosidase, -galactosidase, and N-acetylglucosaminidase were detected by diagnostic tablets (Rosco Diagnostica, Taastrup, Denmark). Susceptibility to desferrioxamine was tested by the method of Lindsay and Riley (13). API Coryne strips (bioMérieux, Marcy l'Etoile, France) were inoculated and interpreted according to the manufacturer's instructions. Carbohydrate assimilations were performed with the API 50 CH system using AUX medium (bioMérieux). Results were recorded after 5 days. API ZYM strips (bioMérieux) were read after 4 h of incubation at 37°C.

Antibiotic susceptibility. To evaluate antibiotic susceptibility, MICs were determined using E-test strips (PDM, Solna, Sweden) on Mueller-Hinton blood agar incubated at 37°C for 24 h. Penicillin, ampicillin, cefotaxime, cephalothin, erythromycin, ciprofloxacin, gentamicin, and vancomycin were tested, and the results were interpreted according to the criteria established for staphylococci in 1997 by the National Committee for Clinical Laboratory Standards (14a).

Chemotaxonomic properties. Cellular fatty acids (CFAs) were assayed by gas-liquid chromatography using a Delsi chromatograph as described previously (19). The amino acid composition of the peptidoglycan was studied by N. Weiss at the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany) by a thin-layer chromatography method as outlined by Schleifer and Kandler (18).

16S rRNA gene sequence determination. Determination of the 16S rRNA gene sequence was performed at the DSMZ by C. Sproër. Approximately 95% of the 16S rRNA gene sequence of the strains was determined by direct sequencing of PCR-amplified 16S ribosomal DNA. Genomic DNA extraction, PCR-mediated amplification of the 16S ribosomal DNA, and purification of the PCR products were carried out as previously described (16). Purified PCR products were sequenced using the ABI Prism dye terminator cycle sequencing ready reaction kit (Applied Biosystems, Weiterstadt, Germany) according to the manufacturer's protocol. Sequencing products were electrophoresed using the Applied Biosystems 373A DNA sequencer. The resulting sequence data were put into the alignment editor ae2 (14), aligned manually, and compared with representative 16S rRNA gene sequences of organisms belonging to the Arthrobacter subgroup of the gram-positive bacteria (14). For comparison, 16S rRNA sequences were obtained from the EMBL database or the Ribosomal Database Project (14). The 16S rRNA gene similarity values were calculated by pairwise comparison of the sequences within the alignment. For construction of a phylogenetic dendrogram, operations of the PHYLIP package (version 3.5.1; J. Felsenstein, Seattle Department of Genetics, University of Washington) were used: pairwise evolutionary distances were computed from percent similarities by the correction method of Jukes and Cantor (10), and based on the evolutionary distance values, the phylogenetic tree was constructed by the neighbor-joining method (17). The root of the tree was determined by including the 16S rRNA gene sequence of Nocardioides simplex in the analysis.

DNA-DNA hybridization. Hybridization was carried out at the DSMZ. DNA was isolated by chromatography on hydroxyapatite by the procedure of Cashion et al. (1). DNA-DNA hybridization was performed as described by De Ley et al. (2) with the modification described by Huss et al. (8) and Escara and Hutton (3), using a Gilford System model 2600 spectrometer equipped with a Gilford model 2527-R thermoprogrammer and plotter. Renaturation rates were computed with the TRANSFER.BAS program of Jahnke (9).

Results and discussion. The five strains exhibited the general characteristics of the genus Arthrobacter. They were gram-positive coryneform bacteria with a nonfermentative metabolism. The CFAs were mainly of the branched type, with a predominant amount of anteiso 15:0 (Table 1). L-Lysine was the diamino acid of the peptidoglycan. Phenotypic, chemotaxonomic, and genetic studies based on 16S rRNA gene sequence determination and DNA-DNA hybridization allowed us to define three clusters among the five isolates.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Unrooted tree showing the phylogenetic positions of A. oxydans (CF46), A. luteolus sp. nov. (CF25), and A. albus sp. nov. (CF43) within the genus Arthrobacter and related taxa. The scale bar indicates 10 nucleotide substitutions per 100 nucleotides.

Description of A. luteolus sp. nov. Cells of A. luteolus (lu-te'-o-lus. N.L. adj. meaning yellowish, because of the yellow-pigmented colonies) are gram-positive coryneform bacteria. No spores are formed. They are motile by peritrichous flagella. Growth is obligately aerobic. Colonies are slightly yellow, smooth, and ~1.5 mm in diameter after 24 h of incubation at 37°C on blood agar. Catalase is positive. Nitrate is reduced. No urease is detected. Gelatin and tyrosine are hydrolyzed, but not esculin. Simmons citrate agar is alkalinized. There is no susceptibility to desferrioxamine. Tween esterase is negative, but DNase is positive. Pyrrolidonyl peptidase is not detected. Acid is produced oxidatively from glucose, maltose, sucrose, and xylose, but not from mannitol and lactose. Glycerol, ribose, D-xylose, D-glucose, D-fructose, D-mannitol, rhamnose, cellobiose, maltose, saccharose, trehalose, xylitol, L-fucose, and 5-keto-gluconate are utilized. Erythritol, D-arabinose, L-arabinose, L-xylose, adonitol, -methylxyloside, galactose, L-sorbose, dulcitol, inositol, mannitol, sorbitol, -methyl-D-mannoside, -methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, lactose, melibiose, inulin, melezitose, D-raffinose, starch, glycogen, geniobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, and 2-keto-gluconate are not utilized. The following enzymatic activities are detected on API ZYM strips: alkaline and acid phosphatase, esterase, esterase-lipase, leucine arylamidase, trypsin, phosphoamidase, and -glucosidase. Not present are lipase, valine arylamidase, cystine arylamidase, chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, N-acetylglucosaminidase, -mannosidase, and -fucosidase. The major CFA is anteiso C_15:0, and the peptidoglycan type is L-Lys-L-Thr-L-Ala₂. The type strain, CF25, has been deposited in the DSMZ as strain DSM 13067. It was isolated from an infected surgical wound.

Description of A. albus sp. nov. Cells of A. albus sp. nov. (al'-bus. L.N. adj. meaning white, because of the white colonies of the organism) are small coryneform bacteria, nonmotile and without spore formation. Colonies are white and are 1 mm in diameter after 48 h of incubation at 37°C on blood agar. Metabolism is obligately aerobic. Catalase is positive. Urease and esculin are not hydrolyzed, and nitrates are not reduced. Gelatin is slowly and weakly liquefied, and there is no DNase activity. Tyrosine is not hydrolyzed. Simmons citrate is negative. Pyrrolidonyl peptidase is positive. The organism is resistant to desferrioxamine. No acid is produced from carbohydrates, and there is no utilization of these substrates in the API 50 CH system. The following enzymatic activities are detected: alkaline and acid phosphatase, phosphoamidase, esterase, esterase-lipase, leucine arylamidase, and trypsin. The following activities are not present: lipase, valine arylamidase, cystine arylamidase, chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetylglucosaminidase, -mannosidase, and -fucosidase. The main CFA is anteiso C_15:0, and the peptidoglycan type is L-Lys-L-Ala-L-Glu. The strains were isolated from human clinical specimens. The type strain, CF43, has been deposited in the DSMZ as strain DSM 13068. It was isolated from human blood.

Nucleotide sequence accession numbers. The 16S rRNA gene sequences of the following strains are deposited in the EMBL Data Library under the indicated accession numbers: CF25 (DSM 13067^T), no. AJ243422; CF43 (DSM 13068^T), no. AJ243421; and CF46 (DSM 13066), no. AJ243423.