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1 Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2 Department of Life Science, Faculty of Agriculture, Tamagawa University, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan
Correspondence
Hidenori Hayashi
h-hayashi{at}maebashi-it.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of P. copri CB7T and P. stercorea CB35T are AB064923 and AB244774, respectively.
API 20A test results, cellular fatty acid compositions, menaquinone compositions and API ZYM and API An-Ident test results of P. copri sp. nov., P. stercorea sp. nov. and related species are available as supplementary material in IJSEM Online.
Present address: Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisatori, Maebashi, Gunma 371-0816, Japan. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Hold et al., 2002; Suau et al., 1999; Wilson & Blitchington, 1996; Zoetendal et al., 1998). A large number of species that have not yet been identified and characterized exists in the human gut. It has been reported that the human faecal microbiota could be analysed by 16S rRNA gene libraries and strictly anaerobic culture-based methods (Hayashi et al., 2002a, b). Many novel operational taxonomic units (OTUs) and isolates that have not yet been characterized were detected and phylogenetic correlation between novel isolated strains and 16S rRNA sequences has been shown. Some novel OTUs and isolates were phylogenetically related to the genus Prevotella. These isolates were the predominant species in human faeces. Suau et al. (1999) and Eckburg et al. (2005) also detected some novel OTUs belonging to the genus Prevotella from 16S rRNA gene library data. Although Prevotella species have been isolated mainly from the oral cavity, these species also inhabit the human gut. Here, two novel species within the genus Prevotella isolated from human faeces are reported.
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 CB7T, CB18, CB23, CB26, CB28 and CB35T were isolated from faeces of a healthy Japanese male (52 years old) using medium 10 and the ‘plate-in-bottle’ method as described previously (Hayashi et al., 2002a; Mitsuoka et al., 1969). Briefly, after collecting samples, each 0.5 g faecal sample was immediately suspended in dilution buffer and 50 µl 108-diluted faecal sample was plated anaerobically on medium 10 by using the ‘plate-in-bottle’ filled with 100 % CO2. Isolates were subcultured on EG agar. Bacteroides bile aesculin agar (Shah, 1992) was used to check whether growth of the isolates was inhibited on this medium.
Physiological, biochemical and enzyme activity tests were performed by inoculation of API 20A, API ZYM and API An-Ident (bioMérieux) test strips according to the manufacturer's instructions followed by incubation at 37 °C in anaerobic jars. The isolates were cultured in PYG broth for analysis of metabolic end products (Sakamoto et al., 2004, 2005a). The metabolic end products were prepared as described by Holdeman et al. (1977) and analysed as described previously (Sakamoto et al., 2004, 2005a). Cellular fatty acid profiles were determined using the MIDI Microbial Identification System (Microbial ID). Saponification, methylation, extraction and determination of cellular fatty acid profiles were conducted as described previously (Sakamoto et al., 2002). Isoprenoid quinones were extracted as described by Komagata & Suzuki (1987) and analysed as described previously (Sakamoto et al., 2004, 2005a). Genomic DNA was extracted from cells harvested from Gifu anaerobic medium (Nissui). Cells were suspended in buffer A (10 mM Tris/HCl and 10 mM EDTA, pH 8.0) containing Triton X-100 (final concentration, 1.5 %) and centrifuged at 19 000 g for 5 min. Cells were then resuspended in buffer A containing proteinase K (final concentration, 2 mg ml–1). After incubation at 37 °C for 10 min, Triton X-100 was added to a final concentration of 1.5 % and the mixture was centrifuged at 19 000 g for 5 min. The following operations were carried out as described previously (Sakka et al., 1989). The G+C content was determined by using the HPLC method (Kitahara et al., 2001; Tamaoka & Komagata, 1984). The 16S rRNA gene was analysed as described previously (Hayashi et al., 2002a). Sequence data were aligned with the CLUSTAL W package (Thompson et al., 1994) and corrected by manual inspection. Nucleotide substitution rates (Knuc values) were calculated and phylogenetic trees were constructed using the neighbour-joining method (Kimura, 1980; Saitou & Nei, 1987). Bootstrap resampling analysis (Felsenstein, 1985) of 100 replicates was performed to estimate the confidence of tree topologies.
Cells of strains CB7T, CB18, CB23, CB26 and CB28 were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods. Cells of these five strains on EG agar were 0.83x0.91–0.99 µm in size and occurred singly; colonies were 0.3–2.0 mm in diameter, milk-white, circular, entire, slightly convex and smooth on EG agar plates. Cells of strain CB35T were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods. Cells of strain CB35T on EG agar were 0.25–0.42x1.08–1.25 µm in size and occurred singly; colonies were 0.2–1.8 mm in diameter, translucent, circular, entire, slightly convex and smooth on EG agar plates. Physiological and biochemical properties of the six novel strains, Prevotella melaninogenica JCM 6325T, Prevotella loescheii JCM 8530T, Prevotella marshii JCM 13450T, Prevotella oralis JCM 12251T, Prevotella salivae JCM 12084T, Prevotella shahii JCM 12083T and Prevotella veroralis JCM 6290T were determined by API 20A. Five strains (CB7T, CB18, CB23, CB26 and CB28) could be differentiated from P. melaninogenica JCM 6325T, Prevotella oulorum NCTC 11871T, P. salivae JCM 12084T and P. veroralis JCM 6290T by D-mannose and L-rhamnose fermentation (see Supplementary Table S1 available in IJSEM Online). Strain CB35T could be differentiated from P. loescheii JCM 8530T by gelatin digestion and aesculin hydrolysis, from P. marshii JCM 13450T by lactose and sucrose fermentation and gelatin digestion, from P. oralis JCM 12251T by aesculin hydrolysis and D-cellobiose and lactose fermentation and from P. shahii JCM 12083T by gelatin digestion.
The cellular fatty acid composition of Bacteroides species has been determined (Mayberry et al., 1982; Miyagawa et al., 1979; Shah & Collins, 1980) and used to provide a classification for members of the genus Bacteroides (Shah & Collins, 1983). The major cellular fatty acids of strains CB7T, CB18, CB23, CB26 and CB28 were C16 : 0, C18 : 1
9c and anteiso-C15 : 0. In addition, these strains contained dimethyl acetals. Recently, Prevotella multisaccharivorax has been reported to possess dimethyl acetals (Sakamoto et al., 2005b). The major cellular fatty acids of strain CB35T were anteiso-C15 : 0, iso-C15 : 0 and C18 : 19c (see Supplementary Table S2 available in IJSEM Online).
The major menaquinones of strains CB7T, CB18, CB23, CB26 and CB28 were MK-11, MK-12 and MK-13 (see Supplementary Table S3 available in IJSEM Online), whereas the major menaquinones of strain CB35T were MK-12 and MK-13. The major menaquinones of other Prevotella species are generally MK-10 and MK-11 (Sakamoto et al., 2005a).
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). The biochemical characteristics of Prevotella species determined using these two systems are given in Supplementary Table S4 (available in IJSEM Online). Based on the results of API ZYM and API An-Ident, the six strains were divided into two groups, with one group consisting of five strains (CB7T, CB18, CB23, CB26 and CB28) and one strain (CB35T) in the other.
The 16S rRNA gene sequences determined in this study were about 1500 bp long and phylogenetic analysis was based on about 1435 aligned homologous nucleotides (Escherichia coli positions 34–1482). The phylogenetic tree clearly indicated that the isolates were related to strains within the genus Prevotella (Fig. 1). Five strains (CB7T, CB18, CB23, CB26 and CB28) formed a single cluster and a distinct line of descent; their sequence similarity to each other was 99.1–99.5 %. The 16S rRNA gene of CB7T showed highest sequence similarity to that of P. veroralis JCM 6290T (92 %). CB35T formed a single cluster and a distinct line of descent. The 16S rRNA gene of CB35T showed highest sequence similarity to that of P. shahii JCM 12083T (90 %). These results indicated that strains CB7T and CB35T could represent novel species, since 16S rRNA gene sequence similarity to the most closely related species was <97 % (Stackebrandt & Goebel, 1994). The DNA G+C contents of strains CB7T, CB18, CB23, CB26 and CB28 were 44.2–45.9 mol%; that of strain CB35T was 48.2 mol%.
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analysed human gut microbiota (46 healthy adults) by real-time PCR using a Prevotella-specific primer. Prevotella was found in 21 of 46 subjects (46 %), with the log10 of the number of cells per g wet weight being 9.7±0.8. Some OTUs that belong to the genus Prevotella were also detected in 16S rRNA gene libraries (Eckburg et al., 2005; Hayashi et al., 2002a; Suau et al., 1999). These results suggest that members of the genus Prevotella are common in the human gut. The novel Prevotella species described here (represented by CB7T and CB35T) were detected at high frequency using anaerobic culture-based methods (Hayashi et al., 2002a). These isolates represent two species that are important constituents of human gut microbiota.
On the basis of the results presented in this study, these strains could be classified as representatives of two novel species of the genus Prevotella. The names Prevotella copri sp. nov. and Prevotella stercorea sp. nov. are proposed for the five strains (CB7T, CB18, CB23, CB26 and CB28) and for strain CB35T, respectively. Differential characteristics of P. copri sp. nov., P. stercorea sp. nov. and some related Prevotella species are shown in Table 1.
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Cells are Gram-negative rods that are anaerobic and non-spore-forming. Colonies on EG agar plates after 48 h incubation at 37 °C under 100 % CO2 are white, circular and convex. Optimum temperature for growth is 37 °C. Growth is inhibited on Bacteroides bile aesculin agar. Indole-negative and aesculin is hydrolysed. No activity is detected for urease and gelatin is not hydrolysed. Acid is produced from glucose, lactose, sucrose, maltose, raffinose, salicin, xylose, L-arabinose, cellobiose and L-rhamnose. Positive reactions are obtained using API ZYM for alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-galactosidase, 
-galactosidase, -glucosidase and -glucosidase; negative reactions are obtained for lipase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, -gluconidase, N-acetyl--D-glucosaminidase, -mannosidase and -fucosidase. Positive reactions are obtained using An-Ident for -glucosidase, -arabinofuranosidase, -gluconidase, alkaline phosphatase, -galactosidase, indoxylacetate hydrolase and arginine and alanine aminopeptidases; negative reactions are obtained for indole, N-acetyl--D-glucosaminidase, -L-fucosidase, -galactosidase, pyroglutamic acid arylamidase, leucine, proline, tyrosine, arginine, histidine, phenylalanine and glycine aminopeptidases and catalase. The major metabolic end products are succinic and acetic acids. Major fatty acids are C16 : 0 (12–13 %), C18 : 19c (13–18 %) and anteiso-C15 : 0 (20–27 %). The principal respiratory quinones are menaquinones MK-10 (8–10 %), MK-11 (23–26 %), MK-12 (43–45 %) and MK-13 (15–19 %). Minor menaquinones are MK-8 (0–1 %) and MK-9 (1–4 %).
The type strain is CB7T (=JCM 13464T=DSM 18205T), isolated from human faeces. Strains CB18 (=JCM 13465), CB23 (=JCM 13466), CB26 (=JCM 13467) and CB28 (=JCM 13468) are included in this species. The DNA G+C contents of the five known strains are 44.2–45.9 mol%.
Description of Prevotella stercorea sp. nov.
Prevotella stercorea (ster.co're.a. L. fem. adj. stercorea pertaining to faeces).
Cells are Gram-negative rods that are anaerobic and non-spore-forming. Colonies on EG agar plates after 48 h incubation at 37 °C under 100 % CO2 gas are translucent, circular, entire, slightly convex and smooth. Optimum temperature for growth is 37 °C. Growth is inhibited on Bacteroides bile aesculin agar. Negative for indole, does not hydrolyse aesculin or gelatin and no urease activity is detected. Acid is produced from glucose, lactose, sucrose, maltose, mannose and raffinose. Positive reactions are obtained using API ZYM for alkaline phosphatase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, N-acetyl--glucosaminidase and -L-fucosidase; negative reactions are obtained for esterase (C4), esterase lipase (C8), lipase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, -gluconidase and -glucosidase. Positive reactions are obtained using An-Ident for N-acetyl--D-glucosaminidase, -glucosidase, -L-fucosidase, alkaline phosphatase, -galactosidase, indoxylacetate hydrolase and alanine aminopeptidase; negative reactions are obtained for indole, -arabinofuranosidase, -gluconidase, -galactosidase, pyroglutamic acid arylamidase, arginine, leucine, proline, tyrosine, histidine, phenylalanine and glycine aminopeptidases and catalase. The major metabolic end products are succinic and acetic acids; small amounts of iso-valeric acid are also produced. Major fatty acids are C18 : 19c (15 %), iso-C15 : 0 (24 %) and anteiso-C15 : 0 (26 %). The principal respiratory quinones are menaquinones MK-12 (26 %) and MK-13 (50 %); minor menaquinones are MK-9 (1 %), MK-10 (5 %) and MK-11 (7 %).
The type strain is CB35T (=JCM 13469T=DSM 18206T), isolated from human faeces. The DNA G+C content of the type strain is 48.2 mol%.
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ACKNOWLEDGEMENTS |
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