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1 Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
2 Division of Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8549, Japan
Correspondence
Mitsuo Sakamoto
sakamoto{at}jcm.riken.jp
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ABSTRACT |
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Published online ahead of print on 5 November 2004 as DOI 10.1099/ijs.0.63451-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of P. multiformis strains PPPA21T, PPPA19, PPPA28 and PPPA30 are AB182483–AB182486.
Photomicrographs of P. multiformis cells from EG agar, an extended phylogenetic tree and tables of phenotypic and biochemical characteristics and cellular fatty acid compositions are available as supplementary material in IJSEM Online.
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), we isolated, from the human oral cavity, a number of strains that were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. These isolates were phenotypically related to the genus Prevotella. Prevotella species are mainly isolated from the oral cavity, and some species are associated with periodontitis. 16S rRNA gene sequence analysis showed that each of the isolates was a member of the genus Prevotella. These strains were divided into four groups: group 1 (one strain) was related to Prevotella loescheii with about 95 % similarity; group 2 (one strain) was related to Prevotella oris with about 94 % similarity; group 3 (four strains) was related to Prevotella denticola with 95 % similarity; and group 4 (six strains) was related to Prevotella dentalis with 89 % similarity. We have already proposed that groups 1 and 2 should be classified as two novel species of the genus Prevotella, Prevotella shahii and Prevotella salivae, respectively (Sakamoto et al., 2004). The present study was designed to determine the taxonomic status of group 3. On the basis of the results presented here, we propose that these strains should be classified as a novel species of the genus Prevotella, Prevotella multiformis sp. nov.
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. Strains PPPA19, PPPA21T, PPPA28 and PPPA30 were isolated, on EG agar, from subgingival plaque from a patient with chronic periodontitis. Growth of the isolates on Bacteroides bile aesculin agar (Shah, 1992) was tested.
Physiological reactions were determined with an API 20A anaerobic test kit in duplicate as recommended by the manufacturer (bioMérieux). The metabolic end-products were prepared as described previously (Holdeman et al., 1977) and were analysed by GLC (GC-7A; Shimadzu) using a 2·1 m glass column (2·8 mm, FAL-M 25 %, Chromosorb W, 80/100 mesh, AW-DMCS H3PO4; Shimadzu). 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 using the MIDI microbial identification system (Microbial ID). Isoprenoid quinones were extracted as described previously by Komagata & Suzuki (1987) and were analysed by HPLC with a Cosmosil 5C18 column (4·6x150 mm; Nacalai Tesque). Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malate dehydrogenase and glutamate dehydrogenase activities were determined spectrophotometrically as described previously (Gharbia & Shah, 1991; Bailey & Love, 1995). API ZYM and API An-Ident enzymic substrate tests were performed in duplicate as recommended by the manufacturer (bioMérieux). Chromosomal DNA was isolated by using previously described methods (Marmur, 1961; Saito & Miura, 1963), with some modifications. The DNA G+C content was determined by using the HPLC method of Tamaoka & Komagata (1984), with some modifications. DNA–DNA hybridization experiments were carried out in microplate wells, as described by Ezaki et al. (1989). Hybridization was performed at 45 °C for 16 h. The 16S rRNA gene was analysed as described previously (Sakamoto et al., 2002b). The previously determined 16S rRNA gene sequences used for comparisons in this study were retrieved from the DDBJ, EMBL and GenBank nucleotide sequence databases. Sequence data were aligned using 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 had been 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.
Strains PPPA19, PPPA21T, PPPA28 and PPPA30 were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods or cocci (coccobacilli). Cells on EG agar were 0·5–0·8x1·6–6·6 µm or 0·8x0·9–1·0 µm and occurred singly (see Supplementary Fig. A available in IJSEM Online). Colonies were 1–2 mm in diameter, grey to light brown, circular, entire, slightly convex and smooth on EG agar plates. Colonies of different sizes were detected on EG agar plates for all four strains. The cells forming large colonies were cocci, whereas those forming small colonies were cocci and rods. These four strains did not hydrolyse aesculin on Bacteroides bile aesculin agar, whereas P. loescheii JCM 8530T, Prevotella oralis JCM 6330, P. oris JCM 8540T, P. salivae JCM 12084T and Prevotella veroralis JCM 6290T hydrolysed aesculin on the same agar plates. Growth of the isolates and other Prevotella species was inhibited on Bacteroides bile aesculin agar. The results of phenotypic tests are given in the species description below, while the phenotypic characteristics of the other Prevotella species are available in Supplementary Table A. The phenotypic characteristics of strains PPA19, PPPA21T, PPPA28 and PPPA30 were similar to those of P. denticola JCM 8528. These strains could be differentiated from P. denticola JCM 8528 by aesculin hydrolysis and D-cellobiose fermentation in API 20A tests.
The cellular fatty acid composition of Bacteroides species has been determined (Mayberry et al., 1982; Miyagawa et al., 1979; Shah & Collins, 1980) and was reviewed for the classification of the genus Bacteroides (Shah & Collins, 1983). In this study, the cellular fatty acid compositions of strains PPA19, PPPA21T, PPPA28 and PPPA30 were found to be very similar to that of P. denticola JCM 8528. These four strains and P. denticola JCM 8528 contain greater relative amounts of anteiso-15 : 0 (
30 %) than do other Prevotella species. The findings are summarized in the species description. In addition, the cellular fatty acid compositions of other Prevotella species are available in Supplementary Table B.
The major menaquinones of the clinical isolates and the other Prevotella species were MK-10 and MK-11. This result supports findings reported previously (Shah & Collins, 1980, 1983). In our study, only the clinical isolates possessed MK-13 (Table 1).
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; Shah, 1992). Bacteroides fragilis and related species possess enzymes of the hexose monophosphate shunt/pentose phosphate pathway, such as G6PDH, 6PGDH, malate dehydrogenase and glutamate dehydrogenase. Shah & Collins (1988, 1990) proposed the new genera Porphyromonas and Prevotella because of the lack of particular enzymes (G6PDH and 6PGDH). In the present study, G6PDH and 6PGDH activities were not detected in extracts of the clinical isolates or the other Prevotella species. On the other hand, both malate dehydrogenase and glutamate dehydrogenase activities were detected in extracts of all strains tested.
The API ZYM and API An-Ident systems have been reported to be useful in the identification of oral and non-oral Gram-negative bacteria (Laughon et al., 1982; Slots, 1981; Tanner et al., 1985). In addition, the RapID-ANA system (Innovative Diagnostics Systems) has been reported to be helpful in the identification of some phenotypically similar bile-inhibited Bacteroides species (Dellinger & Moore, 1986). The biochemical characteristics of the clinical isolates and the other Prevotella species are available in Supplementary Table C. All strains were tested with API ZYM and API An-Ident. The biochemical characteristics of strains PPA19, PPPA21T, PPPA28 and PPPA30 were similar to those of Prevotella bivia JCM 6331T and P. denticola JCM 8528. However, these four strains could be differentiated from P. bivia JCM 6331T and P. denticola JCM 8528 by API An-Ident tests. 
-Galactosidase and arginine and glycine aminopeptidase activities were helpful in the differentiation of these species (Table 2).
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). The levels of DNA relatedness among the isolates were above 81 % (81–98 %). The levels of DNA relatedness observed demonstrate the genomic distinction of these four strains from P. denticola JCM 8528 (relatively low DNA relatedness, 
28 %; Supplementary Table D).
Approximately 1500 bases of the 16S rRNA gene sequence were determined for each of the isolates (strains PPA19, PPPA21T, PPPA28 and PPPA30). For the phylogenetic analysis, 1379 bp (positions 34–1391, Escherichia coli numbering system) sequences of each species were used. 16S rRNA gene sequence analysis showed that each of the isolates was a species of the genus Prevotella (Fig. 1 and Supplementary Fig. B). These four strains were related to P. denticola with about 95 % similarity. The levels of sequence similarity among the four strains were above 99·7 % (99·7–100 %). In addition, the isolates were related to Prevotella sp. oral clone AO036 (Paster et al., 2001) with about 96 % similarity. Moreover, the 16S rRNA gene sequences were determined for the different sizes of colony for each isolate. 16S rRNA gene sequence analysis revealed no differences between the cells forming the large and small colonies. Furthermore, the phenotypic and biochemical characteristics (determined using API 20A, API ZYM and API An-Ident) of colonies of different sizes were found to be the same. These findings indicated that only a single micro-organism was present.
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.
Description of Prevotella multiformis sp. nov.
Prevotella multiformis (mul.ti.for'mis. L. fem. adj. multiformis many-shaped, multiform).
Cells are obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods (0·5–0·8x1·6–6·6 µm) or cocci (coccobacilli) (0·8x0·9–1·0 µm). Colonies are 1–2 mm in diameter, grey to light brown, circular, entire, slightly convex and smooth on EG agar plates. Colonies of different sizes are detected on EG agar plates. Cells forming large colonies are cocci, whereas those forming small colonies are cocci and rods. Growth is inhibited in the presence of 20 % (w/v) bile. Acid is produced from D-cellobiose, glucose, glycerol, lactose, maltose, D-mannose, D-raffinose and sucrose. Acid is not produced from L-arabinose, D-mannitol, D-melezitose, D-rhamnose, salicin, D-sorbitol, D-trehalose or D-xylose. Aesculin is not hydrolysed. Indole is not produced. Gelatin is digested. Catalase and urease are not produced. 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; small amounts of isovaleric acid are also produced. Malate dehydrogenase and glutamate dehydrogenase are present, whereas G6PDH and 6PGDH are absent. Both non-hydroxylated and 3-hydroxylated long-chain fatty acids are present. The major cellular fatty acids are anteiso-15 : 0, iso-15 : 0, iso-3-OH-17 : 0 and 18 : 1
9c. The principal respiratory quinones are menaquinones MK-11 (49–55 %) and MK-12 (29–31 %). Minor menaquinones are MK-8 (1 %), MK-9 (1 %), MK-10 (8–12 %) and MK-13 (2–3 %). The DNA G+C content of the type strain is 51·1 mol%.
The type strain is strain PPPA21T (=JCM 12541T=DSM 16608T), which was isolated from subgingival plaque from a patient with chronic periodontitis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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