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1 School of Biology, King George VIth Building, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
2 Department of Microbiology, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK
3 Department of Microbiology, Sunderland Royal Hospital, Kayll Road, Sunderland SR4 7TP, UK
4 Department of Dermatology, Sunderland Royal Hospital, Kayll Road, Sunderland SR4 7TP, UK
Correspondence
Roland J. Koerner
Roland.Koerner{at}chs.northy.nhs.uk
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for actinomycetes previously classified as Rhodococcus maris Nesterenko et al. (1982). At the time of writing, the genus Dietzia consists of five species with validly published names: Dietzia kunjamensis (Mayilraj et al., 2006); Dietzia maris (Rainey et al., 1995), the type species; Dietzia cinnamea (Yassin et al., 2006); Dietzia natronolimnaea (Duckworth et al., 1998); and Dietzia psychralcaliphila (Yumoto et al., 2002). The type strains of these species form a distinct 16S rRNA gene clade within the evolutionary radiation occupied by mycolic-acid-containing actinomycetes, that is, by organisms classified in the suborder Corynebacterineae (Stackebrandt et al., 1997; Butler et al., 2005; Soddell et al., 2006; Yassin et al., 2006).
D. maris strains have been isolated from the skin and intestinal tract of carp, from soil and from deep-sea sediments in the Pacific Ocean (Nesterenko et al., 1982; Rainey et al., 1995; Takami et al., 1997; Colquhoun et al., 1998), D. cinnamea was isolated from a perianal swab of a patient with a bone marrow transplant (Yassin et al., 2006), D. kunjamensis from a cold desert soil (Mayilraj, et al., 2006), D. natronolimnaea from a moderately saline and alkaline East African soda lake (Duckworth et al., 1998) and D. psychralcaliphila was isolated from a drain pool of a fish-egg-processing plant (Yumoto et al., 2002). Species of the genus Dietzia have been reported as potential human pathogens in an immunocompetent patient (Pidoux et al., 2001) and in immunocompromised patients (Bemer-Melchior et al., 1999; Yassin et al., 2006).
The aim of the present investigation was to determine the taxonomic position of an actinomycete that had been isolated from the skin of an immunocompetent patient with confluent and reticulated papillomatosis and presumptively assigned to the genus Dietzia (Natarajan et al., 2005). The isolate was the subject of a polyphasic taxonomic investigation which showed that it warrants recognition as a novel species of the genus Dietzia.
Strain N 1280T was isolated from skin scrapings from a patient suffering from confluent and reticulated papillomatosis, as described by Natarajan et al. (2005). The organism was maintained on glucose-yeast extract agar (GYEA; Gordon & Mihm, 1962) at room temperature and as glycerol suspensions (20 %, v/v) at –20 °C. Biomass required for chemotaxonomic and 16S rRNA gene sequence analyses was obtained by growing the novel strain in shake flasks of glucose-yeast extract (GYE) broth for 5 days at 28 °C; cells were checked for purity and harvested by centrifugation. Cells for chemosystematic studies were washed twice in distilled water and freeze-dried; those for 16S rRNA gene sequencing were washed in NaCl/EDTA buffer (0.1 M EDTA, 0.1 M NaCl, pH 8.0) and stored at –20 °C until required.
The phylogenetic position of strain N 1280T was determined by 16S rRNA gene sequence analysis. Isolation of chromosomal DNA, PCR amplification and direct sequencing of the purified products were carried out after Kim et al. (1998). The resultant 16S rRNA gene sequence (1437 nt) was aligned manually with corresponding sequences of representatives of the suborder Corynebacterineae, retrieved from the DDBJ/EMBL/GenBank databases, using the pairwise alignment option and 16S rRNA secondary structural information held in the program PHYDIT (available at http://plaza.snu.ac.kr/
jchun/phydit/). Phylogenetic trees were inferred using the least-squares (Fitch & Margoliash, 1967), neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) tree-making algorithms from the PHYLIP suite of programs (Felsenstein, 1993); evolutionary distance matrices were prepared after Jukes & Cantor (1969). The topologies of the resultant unrooted trees were evaluated in a bootstrap analysis (Felsenstein, 1985) based on 1000 resamplings of the neighbour-joining dataset using the CONSENSE and SEQBOOT options from the PHYLIP package.
It is apparent from Fig. 1 that strain N 1280T belongs to the Dietzia 16S rRNA gene clade, an association supported by the three tree-making algorithms and by a 100 % bootstrap value in the neighbour-joining analysis. The novel strain was most closely related to the type strain of D. cinnamea: the two organisms had a 16S rRNA gene sequence similarity of 98.3 %, a value that corresponds to 24 nucleotide differences at 1437 locations. Lower similarity values were recorded for the type strains of D. kunjamensis (95.6 %), D. maris (96.4 %), D. natronolimnaea (95.5 %) and D. psychralcaliphila (94.6 %). DNA–DNA relatedness studies were not carried out between these strains as the type strains of D. kunjamensis and D. maris share a high 16S rRNA gene sequence similarity value, but have a DNA–DNA relatedness value of only 59.2 % (Mayilraj et al., 2006), a figure well below the 70 % guideline recommended for the delineation of bacterial species (Wayne et al., 1987).
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), fatty acids (Sutcliffe, 2000), isoprenoid quinones (Collins, 1994), muramic acid type (Uchida et al., 1999), mycolic acids (Hamid et al., 1993), polar lipids (Minnikin et al., 1984) and whole-organism sugars (Hasegawa et al., 1983). The organism contained meso-A2pm, arabinose and galactose in whole-organism hydrolysates (wall chemotype IV sensu Lechevalier & Lechevalier, 1970), N-acetyl muramic acid, dihydrogenated menaquinones with eight isoprene units [MK-8(H2)] as the predominant isoprenologue and a minor amount of MK-7(H2), straight-chain saturated, unsaturated and tuberculostearic acids (fatty acid type 1b sensu Kroppenstedt, 1985) as major fatty acids, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol as major polar lipids, and mycolic acids that co-migrated with those from D. maris DSM 43672T. This chemotaxonomic profile is consistent with classification of the novel strain in the genus Dietzia (Goodfellow & Maldonado, 2006).
The novel strain and D. cinnamea DSM 44904T, D. kunjamensis DSM 44907T, D. maris DSM 43672T, D. natronolimnaea DSM 44806T and D. psychralcaliphila DSM 44820T were examined for a range of degradative properties using well-established procedures (Goodfellow, 1971; Isik et al., 1999). Aesculin and arbutin hydrolysis were examined following Williams et al. (1983), allantoin hydrolysis was according to Gordon (1967), nitrate reduction was following Gordon & Mihm (1962) and urease production was as described by Rustigan & Stuart (1941). The oxidase reaction was performed on filter paper moistened with a 1 % (w/v) aqueous solution of N,N,N',N'-tetramethyl-p-phenylenediamine and catalase activity was demonstrated using 3 % (v/v) hydrogen peroxide. Acid production from carbohydrates was carried out using media and methods described by Gordon et al. (1974) and utilization of sole carbon and sole carbon/nitrogen sources were determined after Stevenson (1967) and Tsukamura (1966), respectively. Tolerance to pH, temperature and sodium chloride were established using GYEA plates that were incubated for up to 14 days. Resistance to lysozyme was determined after Gordon et al. (1974). It is evident from Table 1 that although the Dietzia strains have many properties in common, they can be distinguished from each other using a combination of phenotypic features.
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) after incubation for 5 days at 30 °C. Smears from this plate were prepared and Gram stained (Hucker's modification; Society for American Bacteriologists, 1957); they were also stained using a modification of the Ziehl-Neelson method (Gordon, 1967) to determine acid–alcohol-fastness. The isolate produced orange-pigmented colonies and was Gram-positive and non-acid–alcohol-fast. The cells required for determination of cellular morphology of the organism were grown in shake flasks of GYE broth for 24, 48 and 72 h at 28 °C; cells were checked for purity at each of these times and harvested by centrifugation. The resultant preparations were fixed in 2 % glutaraldehyde in Sorenson's phosphate buffer for 4 h proceeded by washing three times with 1x phosphate buffer solution. Cell suspensions were inoculated on to separate coverslips coated with 0.025 % poly-L-lysine, dehydrated in a graduated ethanol series (25–100 %, v/v), critical-point-dried in CO2, fixed on a specimen mount with Acheson Silver DAG, gold coated and examined using a Cambridge Stereoscan S40 scanning electron microscope. Strain N 1280T exhibited a rod–coccus life cycle: younger cultures exhibited snapping division and V-forms (1.0–1.4x0.2–0.4 µm in size).
It can be concluded from the genotypic and phenotypic data that strain N 1280T can be readily distinguished from the recognized Dietzia species and hence should be classified as a representative of a novel species in the genus Dietzia. The name proposed for this taxon is Dietzia papillomatosis sp. nov.
Description of Dietzia papillomatosis sp. nov.
Dietzia papillomatosis (pa.pil.lo.ma.to'sis. N.L. gen. n. papillomatosis of papillomatosis).
Aerobic, Gram-positive, non-motile, non-spore-forming, non-acid–alcohol-fast actinomycete that shows snapping division and V-forms and a rod–coccus life cycle. Circular, convex, shiny, orange-pigmented colonies are formed on modified Bennett's agar after growth for 5 days at 30 °C. Neither aerial hyphae nor diffusible pigments are formed. Degrades Tweens 20 and 80, but not adenine. Utilizes isoamyl alcohol as a sole carbon source for energy and growth (at 1 %, v/v). Similarly, fumaric acid, m-hydroxybenzoic acid, DL-β-hydroxybutyric acid, sodium acetate, sodium benzoate, sodium n-butyrate, sodium propionate, sodium pyruvate and sodium DL-malate are used as sole carbon sources, but not 3,3-dimethylglutaric acid, sodium azelate, sodium citrate, sodium pimelate or sodium sebacate (all at 0.1 %, w/v). Growth occurs in the presence of filter paper discs soaked in cephalexin (30 µg ml–1), clindamycin hydrochloride (2 µg ml–1), colistin (25 µg ml–1), erythromycin (5 µg ml–1), nalidixic acid (30 µg ml–1), novobiocin (5 µg ml–1) and tetracycline hydrochloride (10 µg ml–1), but not in the presence of bacitracin (10 U), ciprofloxacin (5 µg ml–1), cotrimoxazole (25 µg ml–1), fusidic acid (10 µg ml–1) or penicillin (1 µg ml–1). Additional phenotypic properties are shown in Table 1
. The cell-wall amino acid is meso-diaminopimelic acid and the major cell-wall sugars are arabinose and galactose. The glycan moiety of the cell wall contains N-acetyl residues (N-acetylmuramic acid). Whole-cell fatty acids consist of predominantly straight-chain saturated and unsaturated components, namely pentadecanoic acid (C15 : 0; 5.4 %), hexadecanoic acid (C16 : 0; 21.1 %), monounsaturated hexadecenoic acid (C16 : 1; 3.0 %), septadecanoic acid (C17 : 0; 6.1 %), monounsaturated septadecenoic acid (C17 : 1; 2.7 %), monounsaturated octadecenoic acid (C18 : 1; 9.0 %), tuberculostearic acid (22.1 %), nonadecanoic acid (C19 : 0; 2.6 %) and unidentified peaks with the retention times of 19.99 (10.6 %), 21.61 (5.6 %) and 21.88 (5.7 %). The polar lipid profile consists of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. MK-8(H2) is the major menaquinone and MK-7(H2) is the minor one.
The type strain, N 1280T (=DSM 44961T=NCIMB 14145T), was isolated from the skin of an immunocompetent patient with confluent and reticulated papillomatosis.
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ACKNOWLEDGEMENTS |
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