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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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], with about 99 % similarity. The strains were obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rods. The isolates could be differentiated from other Prevotella species by D-mannitol, D-melezitose, D-sorbitol and D-trehalose fermentation in API 20A tests. The cellular fatty acid composition of strains PPPA16, PPPA20T, PPPA24, PPPA31, EPPA6 and EPPA7 was significantly different from that of other Prevotella species. Compared with other Prevotella species, only these six strains contained dimethyl acetals. The major menaquinones of the clinical isolates were MK-12 and MK-13, whereas the major menaquinones of other Prevotella species were MK-10 and MK-11. On the basis of these data, a novel Prevotella species, Prevotella multisaccharivorax sp. nov., is proposed, with PPPA20T (=JCM 12954T=DSM 17128T) as the type strain.
Published online ahead of print on 15 April 2005 as DOI 10.1099/ijs.0.63739-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of P. multisaccharivorax strain PPPA20T is AB200414.
Phenotypic characteristics, cellular fatty acid composition and API ZYM and API An-Ident test results are available as supplementary material in IJSEM Online.
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) associated with periodontitis, such attempts have not yet been successful. In the process of this research, we isolated, from the human oral cavity, a number of strains that were obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rods. These isolates were provisionally identified as belonging to the genus Prevotella. Prevotella species are mainly isolated from the oral cavity, and some species are associated with periodontitis. In addition, it was reported recently that advanced dental caries provides a unique environment for a complex array of novel and uncultured Prevotella and Prevotella-like bacteria (Nadkarni et al., 2004). Prevotella species have also been isolated from the genitourinary tract, vagina and cervix. 16S rRNA gene sequence analysis has previously shown that each isolate was a member of the genus Prevotella and that the strains could be divided into four groups. Among these groups, we have already proposed that groups 1, 2 and 3 should be classified as representing three novel species of the genus Prevotella, Prevotella shahii sp. nov. (Sakamoto et al., 2004), Prevotella salivae sp. nov. (Sakamoto et al., 2004) and Prevotella multiformis sp. nov. (Sakamoto et al., 2005), respectively. Group 4 (6 strains) was related to Prevotella dentalis with 89 % similarity. The present study was designed to determine the taxonomic status of this group. On the basis of the results presented here, we propose that the strains of group 4 should be classified as representing a novel species of the genus Prevotella, Prevotella multisaccharivorax 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 PPPA16, PPPA20T, PPPA24, PPPA31, EPPA6 and EPPA7 were isolated, on EG agar, from subgingival plaque of patients with chronic periodontitis. Bacteroides bile aesculin agar (Shah, 1992) was used to check whether the growth of the isolates 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 as described by Holdeman et al. (1977), and analysed as described previously (Sakamoto et al., 2004, 2005). 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 using the MIDI microbial identification system (Microbial ID). Isoprenoid quinones were extracted as described by Komagata & Suzuki (1987), and analysed as described previously (Sakamoto et al., 2004, 2005). Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malate dehydrogenase and glutamate dehydrogenase activities were determined spectrophotometrically, as described by Gharbia & Shah (1991) and 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 as described by Marmur (1961) and Saito & Miura (1963), with some modifications. The DNA base composition was determined by using the HPLC method of Tamaoka & Komagata (1984), with some modifications. The 16S rRNA gene was analysed as described previously (Sakamoto et al., 2002b). 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 had been eliminated. The phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap resampling analysis (Felsenstein, 1985) was performed to estimate the confidence of tree topologies.
Strains PPPA16, PPPA20T, PPPA24, PPPA31, EPPA6 and EPPA7 were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods. Cells on EG agar were 0·8x2·5–8·3 µm in size and occurred singly. Colonies were 0·5–0·7 mm in diameter, grey to off-white-grey, circular, entire, slightly convex and smooth on EG agar plates. Growth of these six strains and other Prevotella species was inhibited on Bacteroides bile aesculin agar. The phenotypic characteristics are given in the species description (phenotypic characteristics of other Prevotella species are available as Supplementary Table S1 in IJSEM Online). The isolates could be differentiated from other Prevotella species by D-mannitol, D-melezitose, D-sorbitol and D-trehalose 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 reviewed for the classification of the genus Bacteroides (Shah & Collins, 1983). In this study, the cellular fatty acid composition of strains PPPA16, PPPA20T, PPPA24, PPPA31, EPPA6 and EPPA7 was found to be significantly different from those of other Prevotella species. Compared with other Prevotella species, only these six strains contained dimethyl acetals. The summarized findings are given in the species description. In addition, the cellular fatty acid compositions of other Prevotella species are available as Supplementary Table S2 in IJSEM Online.
The major menaquinones of the clinical isolates were MK-12 and MK-13 (Table 1). In contrast, the major menaquinones of other Prevotella species were MK-10 and MK-11 (Sakamoto et al., 2005).
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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 the 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 tested. On the other hand, malate dehydrogenase and glutamate dehydrogenase activities were both 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 other Prevotella species are available as Supplementary Table S3 in IJSEM Online. All strains were tested using API ZYM and API An-Ident.
The DNA G+C contents of strains PPPA16, PPPA20T, PPPA24, PPPA31, EPPA6 and EPPA7 were 48·5–49·9 mol%. These values are somewhat lower than those of Prevotella baroniae (Downes et al., 2005) and P. dentalis, which were phylogenetically related (see below) to the clinical isolates (Table 2).
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). The level of sequence similarity among the six strains was 100 %. The six strains were related to P. dentalis with about 89 % similarity. Although the level of sequence divergence (approximately 11 %) is somewhat high, this level of sequence similarity is similar to the values found when various Prevotella species were compared (Willems & Collins, 1995). In addition, the six strains were related to P. baroniae, a species that was proposed recently by Downes et al. (2005), with about 86 % similarity. Furthermore, the isolates were related to Prevotella sp. oral clone IDR-CEC-0032 (Nadkarni et al., 2004) with about 99 % similarity. Nadkarni et al. (2004) suggested that the numerically dominant cluster VI in the carious dentine lesions, which contains Prevotella sp. oral clone IDR-CEC-0032, represents a novel genus, which was most closely related to uncharacterized Prevotella-like rumen bacteria (93–94 % similarity). However, there is no simple way of subdividing the genus Prevotella at the present time.
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. One significant difference is the absence of menaquinones in P. dentalis.
Description of Prevotella multisaccharivorax sp. nov.
Prevotella multisaccharivorax (mul.ti.sac.cha.ri.vo'rax. L. adj. multus many/much; L. n. saccharum sugar; L. adj. vorax liking to eat; N.L. part. adj. multisaccharivorax liking to eat many sugars).
Cells are obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods (0·8x2·5–8·3 µm). Colonies are 0·5–0·7 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 L-arabinose (variable), D-cellobiose, glucose, glycerol, lactose, maltose, D-mannitol, D-mannose, D-melezitose, D-raffinose, D-rhamnose, salicin (variable), D-sorbitol, sucrose, D-trehalose and D-xylose. Aesculin is 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 C18 : 1
9c and C16 : 0. Dimethyl acetals (mainly C16 : 0 dimethyl aldehyde) are present. The principal respiratory quinones are menaquinones MK-12 (40–55 %) and MK-13 (40–45 %). Minor menaquinones are MK-10 (1–3 %) and MK-11 (8–10 %). The G+C content of the type strain is 49·9 mol%.
The type strain is PPPA20T (=JCM 12954T=DSM 17128T), isolated from subgingival plaque from patients with chronic periodontitis.
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ACKNOWLEDGEMENTS |
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