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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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9c. On the basis of the data presented, a novel Bacteroides species, Bacteroides coprophilus sp. nov., is proposed, with CB42T (=JCM 13818T=DSM 18228T) as the type strain.
Tables detailing API 20A and Rapid ID 32A test results and the cellular fatty acid composition of strains of B. coprophilus sp. nov. and related species are available as supplementary material with the online version of this paper.
Present address: Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisatori, Maebashi, Gunma 371-0816, Japan. 
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Suau et al., 1999; Wilson & Blitchington, 1996; Zoetendal et al., 1998). We have previously reported that the human faecal microbiota can be analysed based on 16S rRNA gene libraries and strictly anaerobic culture-based methodologies (Hayashi et al., 2002a, b). We have detected many potentially novel species that have not yet been characterized and have demonstrated correlations between novel isolates and 16S rRNA gene sequences. Among the isolates that we have studied are some that have been shown to be representatives of novel species of the genus Bacteroides (Hayashi et al., 2002a, b). Some potential novel species belonging to the genus Bacteroides have also been observed from 16S rRNA gene sequence libraries (Eckburg et al., 2005; Hayashi et al., 2002a; Suau et al., 1999). Bacteroides is one of the predominant genera in the human gut microbiota (Benno et al., 1989; Finegold et al., 1983; Hayashi et al., 2002a, b). Recently, five novel species belonging to the genus Bacteroides (Bacteroides coprocola, B. dorei, B. finegoldii, B. intestinalis and B. plebius) that were isolated from human faeces have been described (Bakir et al., 2006a, b, c; Kitahara et al., 2005). The 16S rRNA gene sequences of these species corresponded to undescribed sequences detected in 16S rRNA gene sequence libraries derived from human faecal samples (Eckburg et al., 2005; Hayashi et al., 2002a). Here we describe a novel species belonging to the genus Bacteroides that was isolated from human faeces by using a strictly anaerobic culture-based method.
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 of 100 % CO2. Strains CB40, CB41 and CB42T were isolated from the faeces of a healthy Japanese male (52 years old) by using medium 10 and the ‘plate in bottle’ method as described by Hayashi et al. (2002a) and Mitsuoka et al. (1969). Briefly, after collecting samples, each 0.5 g faecal sample was immediately suspended in dilution buffer, and 50 µl of 108-diluted faecal samples were plated anaerobically on medium 10 by using the ‘plate in bottle’ filled with 100 % CO2. Bile resistance was tested by growing the bacteria on GAM (Nissui) agar plates supplemented with 2 % Bacto oxgall (Difco). Physiological, biochemical and enzyme activity tests were performed by inoculation of API 20A and Rapid ID 32A (bioMérieux) test strips according to the manufacturer's instructions and incubation at 37 °C in an anaerobic jar. The isolates were cultured in PYG broth for analysis of metabolic end products (Sakamoto et al., 2004, 2005). The metabolic end products were prepared as described by Holdeman et al. (1977) and analysed as described by Sakamoto et al. (2004, 2005). Cellular fatty acid profiles were determined by using the MIDI microbial identification system (Microbial ID). Saponification, methylation, extraction and determination of cellular fatty acid profiles were conducted as described by Sakamoto et al. (2002). Genomic DNA was extracted from cells harvested from GAM broth as described by Sakka et al. (1989). Briefly, cells were suspended in buffer A (10 mM Tris/HCl and 10 mM EDTA, pH 8.0) containing lysozyme (1 mg ml–1). After incubation at 37 °C for 5 min, 0.1 vols 10 % SDS was added, and the suspension was incubated at 60 °C for 30 min. The G+C content of the DNA was determined by using the HPLC method (Kitahara et al., 2001; Tamaoka & Komagata, 1984). The 16S rRNA gene was analysed as described by Hayashi et al. (2002a). Sequence data were aligned with the CLUSTAL W (Thompson et al., 1994) package and corrected by manual inspection. Nucleotide substitution rates (Knuc values) were calculated and phylogenetic trees were constructed by 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 CB40, CB41 and CB42T were obligately anaerobic, non-spore-forming, non-motile, Gram-negative short rods or rods. Cells on EG agar were 0.7–0.8x2.5–4.1 µm in size and occurred singly. Colonies were 0.5–1.2 mm in diameter, dark grey, translucent, lustrous, circular and slightly convex on EG agar plates. The three new strains could be differentiated from Bacteroides barnesiae JCM 13652T and Bacteroides salanitronis JCM 13657T based on fermentation of L-arabinose, D-cellobiose, L-rhamnose, salicin and D-xylose (see Supplementary Table S1 in IJSEM Online). Based on Rapid ID 32A test results, the new strains could be differentiated from B. barnesiae JCM 13652T and B. salanitronis JCM 13657T based on activity data for 
-arabinosidase, N-acetyl-
-glucosaminidase, -fucosidase and arginine, glycine, glutamyl glutamic acid, histidine, leucine, phenylalanine, serine and tyrosine arylamidases (see Supplementary Table S2 in IJSEM Online).
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 the genus (Shah & Collins, 1983). The major cellular fatty acids of strains CB40, CB41 and CB42T were anteiso-C15 : 0, C16 : 0, iso-C17 : 0 3-OH and C18 : 19c (see Supplementary Table S3 in IJSEM Online).
The lengths of the 16S rRNA gene sequences of strains CB40, CB41 and CB42T obtained in this study were about 1500 bases, and phylogenetic analysis was based on about 1472 aligned homologous nucleotides (Escherichia coli positions 8–1492). The phylogenetic tree clearly indicated that these strains were related to strains within the genus Bacteroides (Fig. 1). The three novel strains formed a single cluster and a distinct line of descent; their 16S rRNA gene sequences were identical. Highest sequence similarity to strain CB42T was found with B. barnesiae JCM 13652T (93.4 %), indicating that strain CB42T could represent a novel species (similarity <97 % to the closest related species; Stackebrandt & Goebel, 1994). The DNA G+C contents of strains CB40, CB41 and CB42T were 44.2–45.2 mol%.
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analysed the human gut microbiota by real-time PCR using a Bacteroides fragilis group-specific primer set. The B. fragilis group was detected from all subjects at a level of antilog10 9.9±0.3 cells g–1 (wet wt). We detected isolates CB40, CB41 and CB42T at high frequency [antilog10 10.4 cells g–1 (wet wt)] by using a strictly anaerobic culture-based method (Hayashi et al., 2002a). The 16S rRNA gene sequence of the isolates investigated herein has been detected previously from a 16S rRNA gene sequence library (Hayashi et al., 2002a). These isolates may thus represent an important Bacteroides species occurring in human faeces.
On the basis of the results presented, strains CB40, CB41 and CB42T are considered to represent a novel species of the genus Bacteroides, for which the name Bacteroides coprophilus sp. nov. is proposed. Differential characteristics of B. coprophilus and some related Bacteroides species are given in Table 1.
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Cells are anaerobic, non-spore-forming, Gram-negative short rods or rods. Colonies on EG agar plates after 48 h incubation at 37 °C under 100 % CO2 are circular, dark grey, translucent and convex. Grows in the presence of bile. Indole-negative. Aesculin is not hydrolysed. Nitrate is not reduced. No activity is detected for urease or gelatin. Acid is produced from D-glucose, lactose, maltose, D-mannose, D-raffinose and sucrose. Positive reactions are obtained using the API Rapid ID 32A system for N-acetyl--glucosaminidase, alkaline phosphatase, -fucosidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase and alanine, arginine, histidine, leucine and leucyl glycine arylamidases. Negative reactions are obtained for -arabinosidase, arginine dihydrolase, -galactosidase 6-phosphate, -glucuronidase, glutamic acid decarboxylase, nitrate reductase, indole, proline arylamidase and pyroglutamic acid arylamidase. The major end products are succinic and acetic acids; small amounts of isovaleric acid, propionic acid and pyruvic acid are also produced. Major fatty acids are anteiso-C15 : 0 (12–16 %), C16 : 0 (9–12 %), iso-C17 : 0 3-OH (17–19 %) and C18 : 19c (16–18 %). The DNA G+C content of the type strain is 44.7 mol%.
The type strain, CB42T (=JCM 13818T=DSM 18228T), was isolated from human faeces. Strains CB40 (=JCM 13816) and CB41 (=JCM 13817) are included in this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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50 are shown. The sequence of Prevotella melaninogenica ATCC 25845T was used as the outgroup for rooting the tree. Bar, 0.05 substitutions per nucleotide position.