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1 Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
2 Department of Microbiology, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Yanagido, Gifu 501-1194, Japan
3 The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Ohkubo, Kumamoto 860-8568, Japan
4 Murohara-kai Kikunan Hospital, Surgery, Tsuruhata-machi 685, Kumamoto 861-5513, Japan
Correspondence
Mitsuo Sakamoto
sakamoto{at}jcm.riken.jp
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ABSTRACT |
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9c and anteiso-C15 : 0 fatty acid content). Based on these data, we propose a novel Prevotella species, Prevotella pleuritidis sp. nov., with the type strain GTC 3021T (=JCM 14110T =CCUG 54350T). The G+C content of the type strain is 45.4 mol%.
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). Prevotella species are isolated mainly from the oral cavity, and some species are associated with periodontitis. They are also isolated from the genitourinary tract, vagina and cervix. Recently, a novel species, Prevotella bergensis, was isolated from infections of the skin and soft tissues (Downes et al., 2006). The present study was designed to determine the taxonomic status of an anaerobic Gram-negative rod isolated from a human clinical specimen that was smear-positive but culture-negative following initial examination.
The strains used in the present study were maintained on Eggerth Gagnon (EG) agar (Merck) supplemented with 5 % (v/v) horse blood for 2 days at 37 °C in an atmosphere containing 100 % CO2. Strain GTC 3021T was isolated from pleural fluid of a patient with suppurative pleuritis. Bacteroides bile aesculin agar (Shah, 1992) was used to check whether growth of the isolate was inhibited on this medium.
Physiological reactions were determined with an API 20A anaerobe test kit in duplicate as recommended by the manufacturer (bioMérieux). The metabolic end products were prepared (Holdeman et al., 1977) and analysed (Sakamoto et al., 2005a) as described previously. Fatty acid methyl esters were obtained from about 40 mg wet cells by saponification, methylation and extraction using minor modifications (Kuykendall et al., 1988) of the method of Miller (1982). Cellular fatty acid profiles were determined by the MIDI microbial identification system (Microbial ID). Isoprenoid quinones were extracted (Komagata & Suzuki, 1987) and analysed (Sakamoto et al., 2002) as described previously. Biochemical reactions were determined with the Rapid ID 32A anaerobe identification kit in duplicate as recommended by the manufacturer (bioMérieux). Chromosomal DNA was isolated by previously described methods (Marmur, 1961; Saito & Miura, 1963), with some modifications. The DNA base composition was determined by the HPLC method of Tamaoka & Komagata (1984). The elution solvent was a mixture of 0.02 M NH4H2PO4 and acetonitrile (20 : 1, v/v). The 16S rRNA gene was analysed as described previously (Sakamoto et al., 2002). Related sequences were aligned with the CLUSTAL W program (Thompson et al., 1994) and corrected by manual inspection. Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) after gaps and unknown bases were eliminated. The phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987). Bootstrap resampling analysis (Felsenstein, 1985) was performed to estimate the confidence of tree topologies.
Cells of strain GTC 3021T were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods. Cells on EG agar were 0.8 by 1.7–4.2 µm in size and occurred singly. Colonies were 0.5–1.5 mm in diameter, grey to off-white–grey, circular, entire, slightly convex and smooth on EG agar plates. Growth of the isolate was inhibited on Bacteroides bile aesculin agar. Results of phenotypic characterization are given in the species description. The isolate could be differentiated from Prevotella enoeca JCM 12259T by only D-mannose fermentation with API 20A tests. The biochemical characteristics of the isolate were similar to those of P. enoeca JCM 12259T. Only test results for mannose fermentation and activities of leucine and glutamyl glutamic acid arylamidases were different from P. enoeca JCM 12259T.
The cellular fatty acid composition of Prevotella species has been determined previously (Sakamoto et al., 2004, 2005a, b). In this study, the cellular fatty acid composition of strain GTC 3021T was significantly different from that of P. enoeca JCM 12259T (C18 : 19c and anteiso-C15 : 0 fatty acid content; Table 1). The cellular fatty acid composition of P. enoeca JCM 12259T was different from previously reported data (Moore et al., 1994), especially the anteiso-C15 : 0 fatty acid content.
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, 2005a, b). The major menaquinones of the isolate were MK-11 (42 %) and MK-12 (50 %). Minor menaquinones were MK-10 (3 %) and MK-13 (4 %). P. enoeca JCM 12259T also possessed MK-11 (59 %) and MK-12 (36 %) as the major menaquinones. Although a minor amount of MK-10 (6 %) was present in P. enoeca JCM 12259T, MK-13 was not detected. The DNA G+C content of strain GTC 3021T was 45.4 mol%. This value is lower than that (47.5 mol%) of P. enoeca JCM 12259T.
Approximately 1500 bases of the 16S rRNA gene sequence were determined for the isolate. For the phylogenetic analysis, 1379 bp (positions 34–1391; Escherichia coli numbering system) of each sequence were used. 16S rRNA gene sequence analysis showed that the isolate was a member of the genus Prevotella (Fig. 1). Strain GTC 3021T was related to P. enoeca ATCC 51261T with about 92 % sequence similarity. Strain GTC 3021T formed a subcluster with P. enoeca ATCC 51261T. According to Stackebrandt & Goebel (1994), strains showing 16S rRNA gene sequence similarity of less than 97 % will not show DNA–DNA reassociation of more than 60 % and will thus represent different species.
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Cells are obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods (0.8x1.7–4.2 µm). Colonies are 0.5–1.5 mm in diameter, grey to off-white–grey, circular, entire, slightly convex and smooth on EG agar plates. Growth is inhibited in the presence of 20 % (w/v) bile. Acid is produced from glucose, lactose, maltose and D-mannose but not from L-arabinose, D-cellobiose, glycerol, D-mannitol, D-melezitose, D-raffinose, L-rhamnose, salicin, D-sorbitol, sucrose, D-trehalose or D-xylose. Aesculin is not hydrolysed. Indole is not produced. Gelatin is digested. Catalase and urease are not produced. Positive reactions are obtained using Rapid ID 32A for 
-galactosidase, 
-glucosidase, N-acetyl--glucosaminidase, -fucosidase, alkaline phosphatase, leucyl glycine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, alanine arylamidase and glutamyl glutamic acid arylamidase. Mannose is fermented. Raffinose is not fermented. All the other tests (urease, arginine dihydrolase, -galactosidase, 6-phospho--galactosidase, -glucosidase, -arabinosidase, -glucuronidase, glutamic acid decarboxylase, nitrate reduction, indole production, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase and serine arylamidase) are negative. The major end products [from 1 % (w/v) peptone/1 % (w/v) yeast extract/1 % (w/v) glucose broth cultures] are succinic and acetic acids. Both non-hydroxylated and 3-hydroxylated long-chain fatty acids are present. The major cellular fatty acids are anteiso-C15 : 0, C16 : 0 and C18 : 19c. The principal respiratory quinones are menaquinones MK-11 (42 %) and MK-12 (50 %). Minor menaquinones are MK-10 (3 %) and MK-13 (4 %). The G+C content of the type strain is 45.4 mol%.
The type strain is GTC 3021T (=JCM 14110T =CCUG 54350T), isolated from pleural fluid of a patient with suppurative pleuritis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Felsenstein, J. (1985). Confidence limits of phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic Institute and State University.
Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161–207.
Kuykendall, L. D., Roy, M. A., O'Neill, J. J. & Devine, T. E. (1988). Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
Miller, L. T. (1982). Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 16, 584–586.
Moore, L. V. H., Johnson, J. L. & Moore, W. E. C. (1994). Descriptions of Prevotella tannerae sp. nov. and Prevotella enoeca sp. nov. from the human gingival crevice and emendation of the description of Prevotella zoogleoformans. Int J Syst Bacteriol 44, 599–602.
Saito, H. & Miura, K. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sakamoto, M., Suzuki, M., Umeda, M., Ishikawa, I. & Benno, Y. (2002). Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov. Int J Syst Evol Microbiol 52, 841–849.[Abstract]
Sakamoto, M., Suzuki, M., Huang, Y., Umeda, M., Ishikawa, I. & Benno, Y. (2004). Prevotella shahii sp. nov. and Prevotella salivae sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 54, 877–883.
Sakamoto, M., Huang, Y., Umeda, M., Ishikawa, I. & Benno, Y. (2005a). Prevotella multiformis sp. nov., isolated from human subgingival plaque. Int J Syst Evol Microbiol 55, 815–819.
Sakamoto, M., Umeda, M., Ishikawa, I. & Benno, Y. (2005b). Prevotella multisaccharivorax sp. nov., isolated from human subgingival plaque. Int J Syst Evol Microbiol 55, 1839–1843.
Shah, H. N. (1992). The genus Bacteroides and related taxa. In The Prokaryotes, 2nd edn, pp. 3593–3607. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Shah, H. N. & Collins, M. D. (1990). Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 40, 205–208.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.[CrossRef]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
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