International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1247–1251 Printed in Great Britain Characterization of a Rothia-like organism NOTE from a mouse: description of Rothia nasimurium sp. nov. and reclassification of Stomatococcus mucilaginosus as Rothia mucilaginosa comb. nov. M. D. Collins,1 R. A. Hutson,1 V. Ba/ verud2 and E. Falsen3 Author for correspondence: M. D. Collins. Tel: j44 118 935 7226. Fax: j44 118 926 7917. e-mail: m.d.collins!reading.ac.uk 1 Department of Food An unknown, Gram-positive, ovoid-shaped bacterium isolated from the nose of Science and Technology, a mouse was subjected to a polyphasic taxonomic analysis. Comparative 16S University of Reading, Reading RG6 6AP, UK rRNA gene sequencing demonstrated that the unknown organism was a member of the family Micrococcaceae and possessed a specific phylogenetic 2 National Veterinary Institute, S-75007 Uppsala, association with Rothia dentocariosa and Stomatococcus mucilaginosus. Sweden Phenotypically, the bacterium closely resembled R. dentocariosa and S. 3 Culture Collection, mucilaginosus but could be distinguished from these species by biochemical Department of Clinical tests and electrophoretic analysis of whole-cell proteins. Based on both Bacteriology, University of phylogenetic and phenotypic evidence, it is proposed that the unknown Go$ teborg, Sweden bacterium be classified in the genus Rothia,asRothia nasimurium sp. nov. In addition, it is proposed that S. mucilaginosus be reclassified in the genus Rothia,asRothia mucilaginosa comb. nov. Keywords: Rothia, Stomatococcus, taxonomy, phylogeny, 16S rRNA Rothia dentocariosa is found in the oral cavity and chemical and serological heterogeneity within the pharynx of man where it forms part of the normal species (e.g. Lesher et al., 1974; Schofield & Schaal, microflora (Schaal, 1992). This species is also now 1981; Fotos et al., 1984; Kronvall et al., 1998). In recognized as an opportunistic pathogen causing addition, the existence of a second genomovar of R. septicaemia and endocarditis, as well as other serious dentocariosa has recently been described from humans infections (e.g. Schafer et al., 1979; Pape et al., 1979; (Kronvall et al., 1998). It is currently not known Pers et al., 1987; Minato & Abiko, 1984; Schiff & whether R. dentocariosa or similar organisms occur in Kaplan, 1987). The taxonomic affinities of R. dento- other animal species (Schaal, 1992). During the course cariosa have always been controversial. Primarily of a study of unusual Actinobacteria from animals, we because of its cellular morphology, the species has have characterized a novel Gram-positive, faculta- historically been associated with Actinomyces and tively anaerobic coccus from the nose of a healthy related genera (Schaal, 1992). In recent years it has mouse which phenotypically resembles the genus become apparent, however, that it is only remotely Rothia. Based on the results of a polyphasic taxonomic related to these taxa, and is phylogenetically a member study, we propose a new species, Rothia nasimurium, of the family Micrococcaceae (Stackebrandt et al., for this bacterium. 1997), exhibiting a specific association with Stomato- T coccus mucilaginosus. Like R. dentocariosa, S. muci- Strain CCUG 35957 was isolated from the nose of a laginosus is a resident of the human oral cavity and healthy mouse. The unidentified organism was cul- pharynx. R. dentocariosa is presently the only recog- tured on Columbia blood agar base supplemented nized species of the genus Rothia. There is, however, a with 5% defribrinated horse blood at 37 mC in air plus considerable body of evidence indicating some bio- 5% CO#. The strain was biochemically characterized by using the API rapid ID32 STREP, API CORYNE ................................................................................................................................................. and API ZYM systems according to the manu- The GenBank accession number for the 16S rRNA sequence of Rothia facturer’s instructions (API bioMe! rieux). Preparation nasimurium CCUG 35957T is AJ 131121. of cellular protein extracts for PAGE analysis, densito- 01253 # 2000 IUMS 1247 M. D. Collins and others ‘ ‘ ................................................................................................................................................................................................................................................................................................................. Fig. 1. Dendogram derived from the unweighted pair group average linkage of correlation coefficients (expressed as percentage values) between whole-cell protein patterns of R. nasimurium sp. nov. and some relatives. Duplicate cultures of strain CCUG 20499 were run to assess reproducibility. metric analysis, normalization of the protein profiles amplified by PCR and directly sequenced using a Taq and numerical analyses were performed as described DyeDeoxy Terminator Cycle Sequencing kit (Applied by Pot et al. (1994) using the GelCompar 3.0 software Biosystems) and an automatic DNA sequencer (model package (Applied Maths). The similarity between all 373A, Applied Biosystems). The closest known pairs of traces was expressed by the Pearson product relatives of the new isolate were determined by moment correlation coefficient, converted for con- performing database searches. These sequences and venience to a percentage similarity value. Long-chain those of other known related strains were retrieved cellular fatty acids were examined using the MIDI from the GenBank or Ribosomal Database Project system. Cells for fatty acid analysis were prepared on (RDP) libraries and aligned with the newly determined blood agar at 37 mC. The DNA GjC content was sequence using the program (Devereux et al., determined by thermal denaturation as described by 1984). The resulting multiple sequence alignment was Garvie (1978). The 16S rRNA genes of the isolate were corrected manually and a distance matrix was calcu- 1248 International Journal of Systematic and Evolutionary Microbiology 50 Rothia nasimurium sp. nov. lated using the programs and (using dentocariosa and S. mucilaginosus (Fig. 1). To ascertain the Kimura-2 correction parameter) (Felsenstein, the phylogenetic position of the unknown isolate, its 1989). A phylogenetic tree was constructed according almost complete 16S rRNA gene sequence was de- to the neighbour-joining method with the program termined and subjected to a comparative analysis. (Felsenstein, 1989). The stability of the Sequence database searches revealed that the unknown groupings was estimated by bootstrap analysis (500 bacterium was phylogenetically a member of the replications) using the programs , , Actinobacteria and displayed a specific association and (Felsenstein, 1989). The with members of the family Micrococcaceae GenBank accession number of the 16S rRNA gene (Stackebrandt et al., 1997). Highest sequence similarity T sequence of strain CCUG 35957 is AJ 131121. was shown with R. dentocariosa (96n6%), S. muci- laginosus (96n6%), Kocuria erythromyxa (96n6%), The unidentified isolate consisted of Gram-positive, Kocuria rosea (96n6%), Kocuria kristinae (95n3%), non-motile, ovoid-shaped cells. The organism grew on Kocuria palustris (95n5%), with other members of the blood agar producing a weak α-haemolytic reaction Micrococcaceae displaying somewhat lower levels of and was non-pigmented. It was facultatively anaerobic relatedness (approx. 92–95%; data not shown). A and catalase-positive. Using commercially available tree constructed by the neighbour-joining method API systems, the isolate produced acid from glucose, depicting the phylogenetic relationships of the lactose, maltose, methyl β--glucopyranoside, sucrose unidentified organism is shown in Fig. 2 and unequi- and trehalose, but failed to produce acid from - vocally demonstrates that it represents a new species arabinose, -arabitol, cyclodextrin, glycogen, man- within the Micrococcaceae. The bacterium formed a nitol, melibiose, melezitose, N-acetylglucosamine, distinct line branching from the base of a cluster which pullulan, raffinose, ribose, sorbitol, tagatose or - embraced R. dentocariosa (genomovar I and II) and S. xylose. Reactions for alanine phenylalanine proline mucilaginosus. arylamidase, cystine arylamidase, ester lipase C-8, β- galacturonidase, α-glucosidase, leucine arylamidase, The polyphasic taxonomic analysis has shown that the valine arylamidase, pyrazinamidase and trypsin were unknown ovoid-shaped bacterium isolated from the positive, whereas those for alkaline phosphatase, nose of a mouse represents a hitherto unrecognized arginine dihydrolase, esterase C-4, α-fucosidase, α- species within the Micrococcaceae. Phylogenetically, galactosidase, β-glucosidase, β-glucuronidase, lipase the bacterium forms a distinct subline and exhibits a C14, α-mannosidase, β-mannosidase and urease were specific association with the Rothia–Stomatococcus negative. The organism hydrolysed aesculin, reduced group of organisms. The clustering of the unknown nitrate and was Voges–Proskauer-negative. In terms mouse bacterium with this latter group was supported of its overall biochemical characteristics, the unidenti- by a bootstrap resampling value of 100% (Fig. 2). The fied bacterium closely resembled R. dentocariosa and close relationship between the unknown organism and S. mucilaginosus. However, it differed from these Rothia–Stomatococcus is supported by their close species in producing acid from lactose and by its biochemical