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Roseomonas Stagni Sp. Nov., Isolated from Pond Water in Japan

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Roseomonas Stagni Sp. Nov., Isolated from Pond Water in Japan J. Gen. Appl. Microbiol., 54, 167‒171 (2008) Short Communication Roseomonas stagni sp. nov., isolated from pond water in Japan Katsunori Furuhata,1,* Hiroshi Miyamoto,2 Keiichi Goto,3 Yuko Kato,3 Motonobu Hara,4 and Masafumi Fukuyama1 1School of Life and Environmental Science, Azabu University, Sagamihara, Kanagawa 229‒8501, Japan 2Faculty of Medicine, Saga University, Saga, Saga 849‒8501, Japan 3Food Research Laboratories, Mitsui Norin Co., Ltd., Fujieda, Shizuoka 426‒0133, Japan 4School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229‒8501, Japan (Received December 26, 2007; Accepted February 28, 2008) Key Words——Acetobacteraceae; pink-pigment; Roseomonas stagni sp. nov. Genus Roseomonas was proposed by Rihs et al. broth containing 20% (w/v) glycerol and stored at (1993), and 6 species and 2 subspecies have been －80°C. recognized so far (Euzéby, 2007). Roseomonas spe- The almost complete sequence of the 16S rRNA cies have been isolated from clinical specimens (Han gene was determined using a MicroSeq 16S rRNA et al., 2003; Lewis et al., 1997; Sandoe et al., 1997; gene bacterial identification kit (Applied Biosystems). Subudhi et al., 2001; Vasallo et al., 1998) and some Multiple sequence alignment analysis was performed species are considered to cause opportunistic infec- using the CLUSTAL W program (Thompson et al., tions in humans. Recently, Roseomonas species have 1994) and gap and unidentified base positions were also been isolated frequently from various natural en- deleted using the BioEdit (Hall, 1999) software pack- vironments, including aqueous environments (Gallego age. Evolutionary distances were calculated by Kimu- et al., 2006), indicating that members of the genus ra’s two-parameter model (Kimura, 1980) without Roseomonas are widely distributed in nature (Jiang et alignment gaps and unidentified base positions taken al., 2006). In this report, we describe the phenotypic into account during distance calculations. A phyloge- and phylogenetic characteristics of a Roseomonas netic tree was constructed using the neighbor-joining strain, HS-69T, isolated from pond water, and propose method (Saitou and Nei, 1987), and bootstrap values that HS-69T is classified as a novel species. were calculated on the basis of 1,000 replications Strain HS-69T was isolated from pond water of No- (Felsenstein, 1985). mori no Ike in Shizuoka, Japan, by a plating method The G+C content, fatty acid composition, and qui- with R2A medium (Nihon Pharmaceutical, Co., Ltd., none analyses were performed by the Japan Food Re- Tokyo) at 30°C for 7 days. The pink-pigmented colony search Laboratories (Tokyo, Japan). The DNA G+C was purified and routinely grown on R2A agar at 30°C contents was determined using the method of Tamao- for 7 days. The isolated strain was preserved in R2A ka and Komagata (1984) with the modification that the DNA was hydrolyzed using nuclease P1 and the resul- tant nucleotides were analyzed by reverse-phase * Address reprint requests to: Dr. Katsunori Furuhata, School of Life and Environmental Science, Azabu University, 1‒17‒71 HPLC. Genomic DNA was extracted from cultured Fuchinobe, Sagamihara, Kanagawa 229‒8501, Japan. cells by a phenol extraction method, and nucleosides Fax: +81‒42‒754‒6215 were obtained from DNA by hydrolysis using phos- E-mail: [email protected] phatase. The various peaks of nucleosides were iso- 168 FURUHATA et al. Vol. 54 lated and detected by HPLC, and the G+C content 20NE, API 50CH, and API ZYM identification systems was calculated. For cellular fatty acids analysis, cells (bioMérieux Japan, Ltd., Tokyo). API ZYM strips were were cultured on R2A agar at 30°C for 7 days and then read after 48 h incubation at 30°C. The API 20NE and the fatty acids were extracted and methylated. The API 50CH were read after 7 days incubation at 30°C. fatty acid methyl esters were analyzed by gas chroma- AUX medium was used as the API 50CH test basal me- tography (GC-1700, Shimadzu Co., Kyoto) as de- dium, and positive reactions were judged based on scribed previously (Furuhata et al., 2007). Quinones the turbidity of the medium after incubation. were extracted from freeze-dried cells and analyzed Antibiotic susceptibility was determined using Etest using an HPLC system (Komagata and Suzuki, 1987). (Aska Diagnostics, Inc., Tokyo) according to the man- The shape and motility of bacterial cells were ob- ufacturer’s instructions. The drugs tested were ampi- served using a phase-contrast microscope (×1,000) cillin (ABPC), piperacillin (PIPC), cefotaxime (CTX), from a 5-day liquid culture in R2A medium. The Gram cefuroxime (CXM), ceftazidime (CAZ), amikacin (AMK), reaction was determined using a Nissui Gram stain kit gentamicin (GM), azithromycin (AZM), erythromycin (Tokyo, Japan) according to the manufacturer’s in- (EM), minocycline (MINO), tetracycline (TC), chloram- structions. Growth at various temperatures (4°C and phenicol (CP), vancomycin (VCM), imipenem (IPM), 15‒45°C; increments of 5°C) was determined in R2A meropenem (MEPM), ciprofloxacin (CPFX), ofloxacin broth (100 ml). Growth at various NaCl concentrations (OFLX), sparfloxacin (SPFX), fosfomycin (FOM), and (0.1, 0.3, 0.5, and 1.0‒5.0%; increments of 1.0%) was rifampicin (RFP) (total 20 drugs). The bacterial cell investigated in R2A broth. The pH range for growth suspension (0.3 ml) was spread onto 60 ml R2A agar was determined in R2A broth that had been adjusted in a 150-mm dish (Corning, Inc., USA) using a Conradi to various pH values (pH 4.0‒10.0; increments of 1.0 stick, and Etest strips were securely attached to the pH units) prior to sterilization by the addition of HCl or medium. The plates were cultured at 30°C for 7 days, NaOH. Growth at various NaCl concentrations and pH and the growth inhibition zone formed around the strip values was tested at 30°C. The turbidity of each culture was recorded. MIC values were judged by macro- was measured after incubation for 2, 5, and 7 days. scopically reading the graduation at which the end of From the rise in turbidity, we judged cultures to be the growth inhibition zone and the strip crossed. positive. The isolate was also tested for its ability to The almost complete 16S rRNA gene sequence grow on horse blood agar, MacConkey agar, and (1,439 bp) of strain HS-69T was determined (acces- BCYEα agar. sion number: AB369258). A neighbor-joining tree (Fig. Biochemical tests were performed by using API 1) showed that strain HS-69T was closely related to the Fig. 1. Phylogenetic tree constructed using the neighbor-joining method based on 16S rRNA gene sequences (1,349 bp) of strain HS-69T (accession number: AB369258) and related bacteria. Methylobacterium organophilum JCM 2833T (AB175639) was served as an out group. The data set was resampled 1,000 times by using the bootstrap option and the percentage values are given at the nodes. The scale bar indicates the number of substitu- tions per nucleotide position. 2008 Roseomonas stagni sp. nov. 169 Table 1. Differential characteristics of strain HS-69T and members of the genus Roseomonas species. Characteristics 1 2a 3b 4c 5d 6d 7e 8e Motility ＋ － － － ＋ ND v ＋ Optimum growth temperature (°C) 30 25 28 30 35 35 35 35 Optimum growth pH 8 7‒8 7 7 ND ND ND ND NaCl (%) range for growth ≦0.5 ≦1 ＜2 ≦6.5 ND ND ≦6 ≦6 Reduction of nitrate － － ＋ ＋ － － v － Utilization of: D-Glucose － － － － ＋ v v － L-Arabinose － － － ＋ ＋ ＋ v v Citrate － － － － ＋ ＋ ＋ v D-Fructose － － － － ND ND ＋ ＋ Ribose － － － ＋ ND ND ND ND D-Galactose － － － ＋ ND ND v － D-Xylose － － － ＋ ND ND v v Glycerol － － － － ND ND ＋ － Enzyme activity (API ZYM) Acid phosphatase － ＋ ＋ ND ND ND ND ND Naphthol-AS-BI-phosphohydrolase － ＋ ＋ ND ND ND ND ND Susceptibility to: Ampicillin S R ND S R ND ND ND Chloramphenicol S S R S ND ND ND ND Tetracycline S S R S ND ND ND ND Isolation source Pond water Soil Potable water Freshwater lake sediment Blood Blood Potable water Cervix DNA G＋C content (mol%) 72.0 69.3 68.6 71.9 ND ND 67.6 70.4 1, HS-69T; 2, R. terrae DS-48T; 3, R. aquatica TR53T; 4, R. lacus TH-G33T; 5, R. mucosa ATCC BAA-692T; 6, R. gilardii subsp. rosea ATCC BAA-691T; 7, R. gilardii subsp. gilardii ATCC 49956T; 8, R. cervicalis ATCC 49957T. ND, Not determined; v, variable; ＋, positive; －, negative; S, sensitive; R, resistant. Data from Yoon et al. (2007)a, Gallego et al. (2006)b, Jiang et al. (2006)c, Han et al. (2003)d, Rihs et al. (1993)e and this study. type strains of the 7 recognized species of the genus result for the urease reaction only, while other exami- Roseomonas, and these 10 strains formed a distinct nation items were all negative (API code number clade in the phylogenetic tree with significant boot- 0200004). API 50CH strips for the sole carbon source strap support (100%). The values of 16S rRNA gene all showed negative results for D-glucose, L-arabinose, sequences similarity between strain HS-69T and the citrate, D-fructose, ribose, D-galactose, D-xylose, and closest relatives were 94.6% (R. gilardii subsp. rosea glycerol (Table 1). Differential phenotypic characteris- MDA 5605T), 94.4% (R. gilardii subsp. gilardii ATCC tics of strain HS-69T and other Roseomonas species 49956T), 94.2% (R. mucosa ATCC-BAA 692T), 93.8% are summarized in Table 1. (R. aquatica TR53T), 93.7% (R. cervicalis ATCC Alkaline phosphatase, esterase (C4), and esterase 49957T), and 92.4% (R. terrae DS-48T and R. lacus TH- lipase (C8) were present in the cells, but the results G33T). were negative for lipase (C14), leucine arylamidase, Cells of strain HS-69T were Gram-negative, non- valine arylamidase, cystine arylamidase, trypsin, spore-forming rods, and the cells were motile.
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