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Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41012 Sevilla, Spain
Correspondence
Antonio Ventosa
ventosa{at}us.es
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), Massilia dura, Massilia albidiflava, Massilia plicata and Massilia lutea (Zhang et al., 2006), with M. timonae as the type species. The genus Massilia was described by La Scola et al. (1998) on the basis of a single isolate from the blood of an immunocompromised patient. Moreover, strains related to M. timonae have been isolated from human patients (Lindquist et al., 2003). However, the isolation from soil samples of phenanthrene-degrading (Bodour et al., 2003), protease-producing (Wery et al., 2003) and N-acyl homoserine lactone-producing (d'Angelo-Picard et al., 2005) strains related to the genus Massilia, as well as the recently described four novel species of the genus Massilia isolated from different soil samples from south-east China (Zhang et al., 2006), showed that species of this genus can be isolated from environmental samples. To monitor the quality of the drinking water of Seville (Spain), we carried out several microbiological studies from April 2003 to January 2004. Samples (25 l) of drinking water were concentrated by using a tangential flow filtration system (Filtron; Pall), plated on plate count agar (PCA; Difco) and incubated at 28 °C for 7 days. Colonies with different morphologies were subsequently plated, using the same isolation medium, in order to obtain pure cultures. Strain AP13T was isolated during the sampling campaign of April 2003 and was studied phylogenetically, phenotypically and genotypically. On the basis of the results of these studies, we propose that strain AP13T represents a novel species of the genus Massilia.
Chromosomal DNA was isolated and purified according to the method described by Marmur (1961). The 16S rRNA gene was amplified using the universal primers 16F27 and 16R1488, as described by Mellado et al. (1995). The almost-complete nucleotide sequence was determined by NBT-Newbiotechnic (Seville, Spain) using an automated DNA sequencer (model 3100; Applied Biosystems). A sequence analysis was subsequently conducted by using the ARB program package (Ludwig et al., 2004). According to the recommendations of Ludwig et al. (1998), alternative treeing methods (maximum parsimony, distance matrix and maximum likelihood) were carried out. A comparison using 16S rRNA gene sequences from databases revealed that the sequence of strain AP13T displays the highest levels of similarity with those from Massilia species. The sequence similarities with respect to Massilia species were 
96.9 %, M. timonae being the most closely related species. The phylogenetic tree obtained with the maximum-parsimony method placed strain AP13T within the branch constituted by Massilia species (Fig. 1). These results were consistent with those obtained with other algorithms. According to the phylogenetic data, the novel isolate belongs to the genus Massilia, but, as it shows relatively low similarity with other species in the genus, strain AP13T could be considered a novel species (Stackebrandt & Goebel, 1994).
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), using the equation of Owen & Hill (1979), as previously described in detail by Ventosa et al. (1999). The DNA G+C content was found to be 66.0 mol%, which is within the range for the genus Massilia (La Scola et al., 1998; Zhang et al., 2006). DNA–DNA hybridization studies were performed using the competition procedure of the membrane method (Johnson, 1994), which was described in detail by Mormile et al. (1999). The hybridization temperature was 59 °C, which is within the limit of validity for the filter method (De Ley & Tijtgat, 1970), and the degree of hybridization (%) was calculated according to Johnson (1994). The experiments were carried out in triplicate. The level of DNA–DNA hybridization between strain AP13T and M. timonae DSM 16850T was 13 %, confirming that strain AP13T constitutes a novel species (Stackebrandt & Goebel, 1994).
The shape and motility of the bacterial cells were observed under a phase-contrast microscope (at x1000) from a 24 h liquid culture (nutrient broth). Growth at different temperatures (4–40 °C), pH values (pH 4–9) and NaCl concentrations (0–5 % NaCl) was tested on PCA medium. The isolate was also tested for its ability to grow on nutrient agar medium, trypticase soy agar (TSA; Difco), R2A (Difco) and MacConkey agar (Scharlau). H2S production was determined on Kligler iron agar (Difco). Oxidase activity was detected using a 1 % solution of tetramethyl-p-phenylenediamine (Difco) (Kovács, 1956). Catalase activity was tested by picking a young colony and smearing it in a drop of H2O2. The methyl red and Voges–Proskauer reactions were tested on Clark–Lubs' medium (Scharlau). Indole production was determined with Kovács' reagent on 1 % tryptone broth. A citrate test was performed on Simmons' citrate agar (Sigma). For the determination of acid production from different carbohydrates, a medium containing 0.5 % peptone, 0.5 % NaCl and 0.001 % phenol red was used (Cowan & Steel, 1974). The reduction of nitrate and nitrite was tested on nitrate broth containing 0.2 % KNO3 (Skerman, 1967). Urease activity was studied in Christensen's medium (Christensen, 1946). The hydrolysis of gelatin, starch and DNA was tested on the corresponding agar media (Scharlau) (Cowan & Steel, 1974). Tween 80 hydrolysis was tested in PCA medium containing 1 % Tween 80 and 0.02 % CaCl2 (Sierra, 1957). Casein hydrolysis was tested in PCA medium supplemented with 2 % skim milk (Difco) (Cowan & Steel, 1974).
Tests for carbon-source utilization, sugar fermentation and enzymes (qualitative) were carried out using API 20NE, API ID 32E and API ZYM kits (bioMérieux) inoculated according to the manufacturer's instructions and incubated at 28 °C. An API 50 CH strip was inoculated as described by Kersters et al. (1984). Antibiotic susceptibility was determined according to the conventional Kirby–Bauer method (Bauer et al., 1966). The results of the phenotypic analysis are summarized in the species description. Several phenotypic characteristics that can be used to differentiate strain AP13T from Massilia species are summarized in Table 1.
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and Kämpfer & Kroppenstedt (1996). The predominant fatty acids of strain AP13T were C16 : 1
7c and C16 : 0. The fatty acid profile of strain AP13T is very similar to those of recently described Massilia species, with C16 : 0 and C16 : 17c and/or iso-C 15 : 0 2-OH as the predominant fatty acids (Table 2). Like members of the genus Massilia, strain AP13T contains ubiquinone Q-8 as the major quinone.
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Description of Massilia aurea sp. nov.
Massilia aurea (au're.a. L. fem adj. aurea golden, referring to the yellowish pigment that the bacterium produces).
Cells are Gram-negative, straight rods 1.0x1.6–3.0 µm in size that occur singly or in pairs (on PCA medium at 28 °C after 48 h). Cells are motile, non-spore-forming and strictly aerobic. Colonies are circular, translucent, yellow-pigmented and 0.6–1.0 mm in diameter on PCA agar after 2 days incubation. Good growth occurs on TSA, R2A and nutrient agar medium. Does not grow on MacConkey agar. Cells have a tendency to form pellicles on the surface of static liquid cultures. Does not grow in the presence of 2 % NaCl. Growth occurs at 4–30 °C (optimum temperature, 28 °C) and at pH 4.0–9.0 (optimum pH, 6.0–7.0). Catalase-positive. Weakly oxidase-positive. Urease-negative. Negative for indole production, hydrogen sulfide production and nitrate reduction, and in the methyl red and Voges–Proskauer tests. Glucose is not fermented. Simmons' citrate test is positive. Starch, gelatin, casein and DNA are hydrolysed but Tween 80 is not. Acid is not produced oxidatively from D-galactose, D-mannose, D-glucose, D-fructose, D-maltose, glycerol, D-mannitol, D-trehalose, D-xylose or lactose. Alkaline and acid phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase and 
-glucosidase are present. Negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, lipase (C14), cysteine arylamidase, trypsin, -chymotrypsin, -galactosidase, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase and -fucosidase. Utilizes D-xylose, D-glucose, D-fructose, D-mannose, L-rhamnose, N-acetylglucosamine, amygdalin, aesculin, D-cellobiose, D-maltose, starch, glycogen, gentiobiose, adipic acid, malic acid and trisodium citrate as sole carbon and energy sources, but not glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, L-xylose, D-adonitol, methyl -D-xylopyranoside, D-galactose, L-sorbose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl -D-mannopyranoside, methyl -D-glucopyranoside, arbutin, salicin, D-lactose (bovine origin), D-melibiose, sucrose, D-trehalose, inulin, D-melezitose, D-raffinose, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium 5-ketogluconate, L-arabinose, capric acid or phenylacetic acid. Resistant to the following antibiotics: penicillin (10 U per disc), bacitracin (10 U per disc) and cephalothin (30 µg per disc). Sensitive to the following antibiotics (µg per disc): tetracycline (30), rifampicin (30), streptomycin (10), neomycin (10), erythromycin (15), kanamycin (30), vancomycin (30), nalidixic acid (30), novobiocin (30) and chloranphenicol (30). The predominant cellular fatty acids are C10 : 0 (0.4 %), C10 : 0 3-OH (4.8 %), C12 : 0 (4.6 %), C12 : 0 2-OH (1.9 %), C14 : 0 (0.6 %), C16 : 17c and/or iso-C15 : 0 2-OH (48.3 %), C16 : 0 (36.8 %) and C18 : 17c (2.5 %). The predominant ubiquinone is Q-8. The DNA G+C content of the type strain is 66.0 mol% (Tm).
The type strain, AP13T (=CECT 7142T=CCM 7363T=DSM 18055T=JCM 13879T), was isolated from drinking water.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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