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Meningitis and Special Pathogens Branch; Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
Correspondence
June M. Brown
jmb6{at}cdc.gov
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Present address: Epidemiology and Surveillance Division, National Immunization Program, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. 
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. At present, the genus comprises one species, Cellulosimicrobium cellulans (Schumann et al., 2001). Cellulosimicrobium variable, proposed by Bakalidou et al. (2002), has been re-evaluated, transferred from the genus and reclassified as Isoptericola variabilis (Stackebrandt et al., 2004). In this paper, we describe the morphological, physiological and biochemical characteristics, antimicrobial susceptibility, whole-cell composition, cellular fatty acid and menaquinone content, 16S rRNA gene sequences and DNA–DNA hybridization of 13 clinical isolates (ten of which were epidemiologically related). A comparison was made with the type strains of Cellulosimicrobium cellulans, Cellulomonas fimi, Cellulomonas hominis, Sanguibacter suarezii and Oerskovia turbata. On the basis of the results of this polyphasic study, the clinical isolates represent a novel taxon within the genus Cellulosimicrobium.
Strain W6122T was isolated from the blood of an endocarditis patient and identified in 1975 as O. turbata (Reller et al., 1975; Sottnek et al., 1977). Recently, the clonality of W6122T and three additional blood isolates (strains W2622, W2728 and W2729) from the same patient, as well as six homograft valve isolates (strains W2732, W2734, W2736, W2739, W6120 and W6121), was confirmed (McNeil et al., 2004). In addition, McNeil et al. (2004) found that strain W6122T was more closely related to Cellulosimicrobium cellulans (99·8 % 16S rRNA gene sequence similarity) than to the type strain of O. turbata (95·3 %). Strain W4083, isolated from a patient with acute myelogenous leukaemia with a catheter-related bacteraemia, was also described as O. turbata (LeProwse et al., 1989; McNeil et al., 2004). The two remaining strains, W2796 and W6123, were formerly identified as O. turbata, based primarily on phenotypic characteristics (Sottnek et al., 1977; McNeil et al., 2004). No clinical histories were available for either of these strains, except that strain W2796 was isolated from lung and strain W6123 was isolated from an unknown human source.
All isolates were inoculated onto heart infusion agar with 5 % rabbit blood (BBL Microbiology Systems) and incubated at 25 and 35 °C for 2 days for morphological studies. Gram stain was used to study microscopic morphology. The isolates were examined for the presence of aerial and substrate hyphae at low power (x10 magnification) under a stereomicroscope. Phenotypic characterization was performed using previously described methods (McNeil et al., 2004) and results are given in the species description. Antimicrobial susceptibilities were determined by a previously described broth microdilution method with cation-supplemented Mueller–Hinton broth (NCCLS, 1997). The antimicrobial agents tested were amikacin, amoxycillin–clavulanic acid, ampicillin, ceftriaxone, ciprofloxacin, clarithromycin, imipenem, minocycline, trimethoprim–sulfamethoxazole and vancomycin. Plates were incubated at 25 or 35 °C for 48 h. Since the methods of testing and the break-points for resistance for the genus have not been standardized or approved by the NCCLS, the break-points used for resistance were those of NCCLS for organisms that grow aerobically (NCCLS, 1997). The break-point for resistance for clarithromycin was taken from the recently NCCLS-approved standard for the susceptibility testing of mycobacteria, nocardiae and other aerobic actinomycetes (NCCLS, 2003).
The genomic DNAs of the isolates were purified from lysed protoplasts as described previously (Lasker et al., 1992). DNA labelling and DNA–DNA relatedness experiments were performed with strain W6122T and strains W2796, W4083 and W6123 and the type strains of Cellulosimicrobium cellulans and O. turbata using the hydroxyapatite method described by Brenner et al. (1982). Strain W6122T was related to strains W2796 (76 %), W4083 (78 %) and W6123 (79 %), with a divergence (
Tm) of 1·5, 2·0 and 1·5 %, respectively, but showed low DNA–DNA relatedness to Cellulosimicrobium cellulans ATCC 12830T (47 %) and O. turbata ATCC 25835T (3·0 %).
The DNA G+C content was determined spectrophotometrically by thermal denaturation as described previously (Mandel et al., 1970). The DNA G+C content of strain W6122T was 74·5 mol%. This value was consistent with the DNA G+C content of 74 mol% observed for the genus Cellulosimicrobium (Schumann et al., 2001).
The 16S rRNA genes of strains W6122T, W2796, W4083, W6123 and Cellulosimicrobium cellulans ATCC 12830T were analysed as described by McNeil et al. (2004). A continuous stretch of 1444 nt of the 16S rRNA gene sequence was determined and used for comparative phylogenetic analysis. Related sequences were identified in a BLAST search against the GenBank database. Sequences were aligned and a distance matrix was created with CLUSTAL W. A phylogenetic tree was constructed in TREECON with the neighbour-joining method (Fig. 1) and bootstrapped with 1000 replications (McNeil et al., 2004). The 16S rRNA gene sequence similarity values between strains W6122T, W2796, W4083 and W6123 ranged from 99·8 to 100 %. The sequence similarity values between these strains and Cellulosimicrobium cellulans ATCC 12830T ranged from 99·5 to 99·8 %.
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), cellular fatty acid analysis (Weyant et al., 1996) and quinone analysis (Moss & Guerrant, 1983). Whole-cell analyses of the organisms indicated that they contained neither the meso- nor the L-isomer of diaminopimelic acid. Galactose was the only characteristic whole cell-wall sugar found. The presence of only galactose is not consistent with the cell-wall sugars identified from Cellulosimicrobium cellulans; thus, this characteristic may be useful for species differentiation (Stackebrandt et al., 2004). The strains share a similar respiratory quinone profile that is characterized by large amounts of menaquinone-9 [MK-9(H4)]. Predominant fatty acids are 12-methyltetradecanoic acid (ai-C15 : 0), 14-methylpentadecanoic acid (i-C16 : 0), hexadecanoic acid (C16 : 0) and 13-methyltetradecanoic acid (i-C15 : 0). The fatty acid profile of strain W6122T was similar to that of Cellulosimicrobium cellulans ATCC 12830T.
In contrast to the chemotaxonomic similarities between strains W6122T and Cellulosimicrobium cellulans, the only other species of the genus Cellulosimicrobium, clear differences were seen in the results of phenotypic tests (Table 1). Strains W6122T, W2796, W4083 and W6123 lacked the ability to produce acid from inulin and raffinose and were motile, whereas Cellulosimicrobium cellulans ATCC 12830T produced acid from inulin and raffinose and was non-motile. Table 1 lists additional phenotypic properties that distinguish strains W6122T, W2796, W4083 and W6123 and Cellulosimicrobium cellulans ATCC 12830T from related species and genera. The high levels of DNA–DNA relatedness demonstrate that these isolates represent the same species and the low levels of sequence similarity to other genera support the distinctness of these isolates (Fig. 1
).
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).
Emended description of Cellulosimicrobium Schumann et al. 2001
Cellulosimicrobium (Cell.u.lo.si.mi.cro'bi.um. N.L. n. cellulosa cellulose; Gr. adj. mikros small; Gr. masc. n. bios life; N.L. neut. n. Cellulosimicrobium cellulose microbe).
The Neo Latin noun Cellulosimicrobium is in the neuter gender and not in the feminine gender as cited by Schumann et al., 2001 [all Latin names which end in -um (nominative singular) are assigned neuter gender].
The description is as given by Schumann et al., 2001, with the following modifications. Whole-cell sugars are galactose, rhamnose, glucose, fucose and mannose or galactose and ribose. Motile or non-motile. Nitrate reduction is positive or negative.
Description of Cellulosimicrobium funkei sp. nov.
Cellulosimicrobium funkei (fun'ke.i. N.L. gen. masc. n. funkei of Funke, to honour the contributions of Guido Funke, a distinguished microbiologist, who recognized the heterogeneity within the yellow-pigmented coryneform bacteria).
Cells are short (1 µm), thin, Gram-positive and non-sporulating rods. Motile by polar and/or lateral flagella (Sottnek et al., 1977). Growth occurs at 35 and 45 °C, but not at 25 °C. Pale-yellow pigment. Substrate hyphae are present. Catalase-positive. Aesculin, adenine, casein, gelatin, hypoxanthine, urea and xanthine are decomposed. Nitrate is not reduced to nitrite (Sottnek et al., 1977). Acid is produced from L-arabinose, cellobiose, fructose, D-galactose, D-glucose, glycerol, lactose, maltose, mannose, salicin, sucrose, D-trehalose and D-xylose. No acid production from adonitol, dulcitol, i-erythritol, myo-inositol, inulin, D-mannitol, melezitose, melibiose, raffinose, L-rhamnose or D-sorbitol. DNA G+C content is 74·5 mol%. The characteristic whole-cell sugar is galactose.
The type strain, W6122T (=ATCC BAA-886T=DSM 16025T=CCUG 50705T), was isolated from human blood.
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Stackebrandt, E., Schumann, P. & Cui, X.-L. (2004). Reclassification of Cellulosimicrobium variable Bakalidou et al. 2002 as Isoptericola variabilis gen. nov., comb. nov. Int J Syst Evol Microbiol 54, 685–688.
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