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1 Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
2 Laboratory of Dairy Science and Technology, Kyodo Milk Industry Co. Ltd, Hinode, Tokyo 190-0182, Japan
Correspondence
Mitsuo Sakamoto
sakamoto{at}jcm.riken.jp
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ABSTRACT |
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Tables showing biochemical properties, fatty acid composition, DNA base composition and DNA–DNA hybridization values of Bacteroides dorei sp. nov. and closely related Bacteroides species, and figures showing maximum-parsimony and UPGMA trees are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Bacteroides species represent predominant organisms in the human colon (Benno et al., 1989). They play a variety of roles as members of the indigenous flora that contribute to normal intestinal physiology and function. Several Bacteroides species, including the type species, Bacteroides fragilis, are important opportunistic pathogens and the most frequently isolated organisms from anaerobic infections (Finegold & George, 1989). Novel phylotypes of yet to be cultured Bacteroides species have been detected by 16S rRNA gene clone libraries of microbiota from the human intestine (Hayashi et al., 2002; Suau et al., 1999). Isolation and characterization of previously unknown Bacteroides species are important as they are related to human and animal health. Recently, Bacteroides coprocola (Kitahara et al., 2005), Bacteroides coprosuis (Whitehead et al., 2005), Bacteroides finegoldii (Bakir et al., 2006b), Bacteroides intestinalis (Bakir et al., 2006a) and Bacteroides plebeius (Kitahara et al., 2005) have been described. During the characterization of bacteria isolated from human faeces, two anaerobic bacteria were recovered on polyamine-deficient medium. The aim of this study was to determine the taxonomic position of the two isolates. The results of phenotypic, chemotaxonomic and 16S rRNA gene sequence analyses, and DNA–DNA homology values provided evidence that these two isolates represent a single species and that these isolates should be classified as a new species of the genus Bacteroides, for which the name Bacteroides dorei is proposed.
Isolates 175T and 219 were recovered from the faeces collected from one healthy, Japanese, 23-year-old male. Polyamine-deficient medium (Noack et al., 1998) with minor modification and a standard dilution plate method were used for isolation and enumeration of faecal bacteria as described previously (Bakir et al., 2006a). AnaeroPack (Mitsubishi Gas) was used for creating anaerobic conditions and the plates were incubated at 37 °C for 72–120 h. Single colonies were picked and streaked out until single cultures were obtained on Eggerth Gagnon (EG) agar (Merck) medium supplemented with 5 % horse blood for 2 days at 37 °C in an anaerobic jar (Hirayama) filled with 100 % CO2. Colony and cell morphology were observed by phase-contrast microscopy (Nikon) with an oil immersion objective lens and by Gram-staining after 48 h of culture on EG agar medium supplemented with 5 % horse blood at 37 °C. The two isolates originating from human faeces, 175T and 219, were anaerobic, non-spore-forming, non-motile, Gram-negative rods. Typical cells were 1.6–4.2 µm by 0.8–1.2 µm. Colonies on EG agar plates after 48 h incubation at 37 °C under 100 % CO2 gas were circular, whitish, raised and convex, and attained a diameter of 2.0 mm. The isolates were incubated under various oxygen conditions (aerobic, microaerophilic, anaerobic) and at different temperatures (25–40 °C) to examine bacterial growth. Strains grew at temperatures from 25 to 40 °C with an optimum of 37 °C. The isolates were grown on Gifu anaerobic medium (GAM; Nissui) agar supplemented with 2 % bacto-oxgall (Difco), which is equivalent to 20 % bile for testing bile resistance. The two isolates were observed to be resistant to 20 % bile. The isolates were stab-inoculated into tubes containing semisolid GAM agar (0.5 %) for motility testing, and were incubated at 37 °C for up to 72 h before a negative result was recorded (McClung & Lindberg, 1957).
The results of phenotypic tests are listed in the species description. Biochemical tests were performed using API 20 A and API rapid ID 32 A strips (bioMérieux), according to the manufacturer's instructions, and incubated at 37 °C as described by Fenner et al. (2005) and Kitahara et al. (2005). Biochemical tests that showed differences from other recognized species of the genus Bacteroides are listed in Table 1. Biochemical test results of the isolates and the close neighbours Bacteroides vulgatus and Bacteroides massiliensis are listed in Supplementary Table S1 (available in IJSEM Online). The two isolates showed identical biochemical profiles. They showed the following differential biochemical test results with B. vulgatus, the most closely related species: 
-glucosidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, histidine arylamidase and serine arylamidase (Supplementary Table S1, available in IJSEM Online). Biochemical properties of the isolates and the close neighbour B. massiliensis are listed in Table 1 and in supplementary Table S1. The isolates demonstrated different biochemical reactions with B. massiliensis for aesculin, arabinose, rhamnose, xylose, sorbitol, 6-phospho--galactosidase, -glucosidase, 
-arabinosidase, -glucoronidase, phenylalanine arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase and serine arylamidase.
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. Fatty acid profiles of the isolates and the closest relative B. vulgatus are provided in Supplementary Table S2 (available in IJSEM Online). The major fatty acids of the isolates and B. vulgatus were anteiso-C15 : 0, iso-C17 : 0 3-OH and C18 : 1
9c. The major cellular fatty acid content of the isolates supports their affiliation as members of the genus Bacteroides, particularly anteiso-C15 : 0 (Miyagawa et al., 1979).
The 16S rRNA genes of the two isolates were amplified by PCR and sequenced to determine their phylogenetic affiliation. The genes were amplified by PCR (Biometra) with universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR products were purified by using a Montage PCR96 filter plate (Millipore) and sequenced directly by the dideoxynucleotide chain-termination method using a DNA sequencer (ABI PRISM 3100; Applied Biosystems/Hitachi) with a BigDye Terminator, version 3.1 cycle sequencing RR-100 kit (Applied Biosystems), according to the manufacturer's instructions. 16S rRNA gene sequences of >1490 bp were obtained. Phylogenetic relatives of the isolates were determined by performing database searches, and sequences of related species were retrieved from DDBJ, EMBL and GenBank nucleotide sequence databases. Sequences were aligned using CLUSTAL X (version 1.81) (Thompson et al., 1997). Alignment gaps and ambiguous bases were removed prior to phylogenetic analysis using MacClade (version 4.03) (Maddison & Maddison, 2002). Neighbour-joining and UPGMA phylogenetic trees were inferred using the software package MEGA version 3.1 (Kumar et al., 2004), according to the Kimura two-parameter model. Parsimony analysis was carried out with maximum-parsimony implemented in the PAUP version 4.0b10 software package (Swofford, 2000). Maximum-parsimony trees were obtained by a heuristic search and by selecting the tree bisection/reconnection branch-swapping option (Dauga, 2002). The topology of phylogenetic trees was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates. Sequence comparison against 16S rRNA gene sequences deposited in the DDBJ/EMBL/GenBank database and treeing analysis preliminarily revealed that the two isolates demonstrated a specific affinity with members of the genus Bacteroides (Fig. 1; Supplementary Figs A and B, available in IJSEM Online). The two isolates appeared to be genetically highly related to each other, displaying 99.5 % 16S rRNA gene sequence similarity (Stackebrandt & Goebel, 1994; Konstantinidis & Tiedje, 2005). The phylogenetic positions of isolates 175T and 219 among recognized members of the genus Bacteroides are shown in Fig. 1. The phylogenetic tree indicated that the isolates represented a new subline within the genus Bacteroides. The isolates displayed phylogenetic affinity with B. vulgatus and the level 16S rRNA gene sequence divergence among them was 4 %. The branching of the isolates at the base of this group was supported by a bootstrap resampling value of 100 % (Fig. 1). Although there is no precise correlation between percentage 16S rRNA gene sequence divergence and species delineation, however, it is generally recognized that divergence values of 3 % or more are significant (Stackebrandt & Goebel, 1994; Whitehead et al., 2005).
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[2.4 g Lab-Lemco powder (Oxoid), 10 g Proteose Peptone No. 3 (Difco), 5 g yeast extract, 4 g Na2HPO4, 5 g glucose, 0.5 g soluble starch, 0.5 g L-cysteine.HCl.H2O and 1 l distilled water; pH adjusted to 7.6] after 12 h at 37 °C and purified by the methods of Saito & Miura (1963). Lysozyme (Wako Pure Chemical Industries) and SDS were used for the disruption of cells, followed by phenol extraction. DNA was recovered with alcohol precipitation and suspended in TE (1 mM EDTA, 10 mM Tris/HCl, pH8.0) buffer. DNA was treated with RNase A and RNase T1 (both from Sigma), and then extracted with phenol and phenol/chloroform/isoamyl alcohol (25 : 24 : 1). DNA was obtained by alcohol precipitation and dissolved in TE buffer. The concentration of DNA was measured by using a UV spectrophotometer (UV-2100; Shimadzu). DNA base compositions were determined using HPLC (Tamaoka & Komagata, 1984) after enzymic digestion of DNA to deoxyribonucleosides. An equimolar mixture of four deoxyribonucleotides from the Yamasa GC kit (Yamasa Shoyu) was used as the quantitative standard. The DNA G+C contents of the isolates were determined as 43 mol%. The DNA G+C content of the nearest neighbour B. vulgatus was 41 mol% (Supplementary Table S3, available in IJSEM Online). The G+C content of the isolates also supports the affiliation of the isolates in the genus Bacteroides, which have G+C contents between 40 and 48 mol% (Shah, 1992).
DNA–DNA relatedness was determined based on close pairwise 16S rRNA gene sequence similarity values of strain 175T with 219 and B. vulgatus. For DNA–DNA hybridization experiments, bacterial DNA was extracted from cells harvested from EGF broth (Kitahara et al., 2001). DNA–DNA hybridization was performed by the photobiotin-labelling method of Ezaki et al. (1989) using a microplate reader (Spectroan FL-2575; Towa Scientific). The hybridization temperature was 42 °C. The DNA–DNA homology value between isolates 175T and 219 was 
73 %. The two isolates also showed identical biochemical profiles and a 99.5 % 16S rRNA gene sequence similarity value. Therefore, it was confirmed that they belonged to a single species. The level of DNA–DNA relatedness between strain 175T and the closest neighbour, B. vulgatus was 
46 % (Supplementary Table S3, available in IJSEM Online). DNA–DNA hybridization values of <70 % with the closest Bacteroides species confirmed the novelty of isolate 175T (Wayne et al., 1987; Stackebrandt & Goebel, 1994). Support for the distinctiveness of the isolate from other valid species of the genus Bacteroides was also very evident from phenotypic analyses. Based on the phenotypic and genotypic evidence presented, these two isolates should be assigned to the genus Bacteroides as a single species for which the name Bacteroides dorei sp. nov. is proposed
Description of Bacteroides dorei sp. nov.
Bacteroides dorei [do.re'i. N.L. gen. masc. n. dorei of Doré, in honour of the French microbiologist Joel Doré, in recognition of his many contributions to intestinal (gut) microbiology].
Cells are Gram-negative rods, anaerobic, non-motile and non-spore-forming. Typical cells are 1.6–4.2 µm by 0.8–1.2 µm and occur singly. Colonies on EG agar plates after 48 h incubation at 37 °C under 100 % CO2 gas are circular, whitish, raised and convex, and attain a diameter of 2.0 mm. Optimum temperature for growth is 37 °C. Grows in the presence of bile. Indole-negative and aesculin is not hydrolysed. Nitrate is not reduced. No activity detected for urease and gelatin. Acid is produced from glucose, sucrose, xylose, rhamnose, lactose, maltose, arabinose, mannose and raffinose. Acid is not produced from cellobiose, salicin, trehalose, mannitol, glycerol, melezitose and sorbitol. Positive reactions obtained using API rapid ID 32 A for -fucosidase, -galactosidase, -galactosidase, 6-phospho--galactosidase, -glucosidase, -glucosidase, -arabinosidase, -glucuronidase, N-acetyl--glucosoaminidase, glutamic acid decarboxylase, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase and serine arylamidase. Negative reactions obtained for arginine dihydrolase, proline arylamidase and pyroglutamic acid arylamidase. Major fatty acids are anteiso-C15 : 0 (26–32 %), iso-C17 : 0 3-OH (17–19 %) and C18 : 19c (9–12 %). DNA G+C content is 43 mol%. The type strain is 175T (=JCM 13471T=DSM 17855T). Strain 219 (=JCM 13472) is included in this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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