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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
Mohammad Abdul Bakir
bakir{at}jcm.riken.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 199T is AB222699.
API 20A and API rapid ID 32 A test results, cellular fatty acid compositions, DNA base composition and levels of DNA–DNA hybridization of strains 199T and 176 and related strains and a maximum-parsimony phylogenetic tree showing the positions of strains 199T and 176 among representative members of the genera Prevotella, Bacteroides, Porphyromonas and Tannerella are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Finegold et al., 1983). In addition to cultivable species, novel phylotypes of Bacteroides that have not yet been cultivated have been detected by using 16S rRNA gene clone libraries of microbiota of the human intestine (Hayashi et al., 2002; Suau et al., 1999). Recovery of micro-organisms from any sample depends primarily on the isolation media used. Inclusion or exclusion of media ingredients could affect the recovery of micro-organisms. In general, polyamines (putrescine, spermidine and spermine) are ubiquitous and necessary for cell growth (Noack et al., 1998). Micro-organisms cannot grow without polyamines. This growth inhibition can be overcome by simply adding polyamines to the culture medium (Macrae et al., 1998). Bacteria synthesize polyamines by decarboxylation of the amino acids ornithine, arginine and lysine (Macfarlane & Macfarlane, 1997). Members of the genus Bacteroides have been identified as spermidine producers (Noack et al., 1998). The use of polyamine-deficient media facilitates the isolation of polyamine-producing bacteria (Noack et al., 2000). Bacteroides intestinalis was isolated recently using polyamine-deficient medium (Bakir et al., 2006). We isolated the strains described in this study from human faeces.
Two strains, 199T and 176, were isolated from the faeces of a healthy, 23-year-old Japanese male. Polyamine-deficient medium (Noack et al., 1998), with minor modifications, and a standard dilution plate method were used for isolation, as described previously (Bakir et al., 2006). An AnaeroPack (Mitsubishi Gas) was used for creating anaerobic conditions and the incubation period used was 72–120 h at 37 °C. The strains were subcultured on Eggerth–Gagnon (EG) agar (Merck) supplemented with 5 % horse blood for 2 days at 37 °C in an anaerobic jar (Hirayama) filled with 100 % CO2. Colony and cell morphologies were observed by using phase-contrast microscopy (Nikon) with an oil-immersion objective lens and by Gram-staining cultures after growth for 48 h on EG agar supplemented with 5 % horse blood at 37 °C in an anaerobic jar. To examine bacterial growth, the isolates were incubated under various oxygen conditions (aerobic, microaerophilic, anaerobic) and at various temperatures (20, 25, 30, 37 and 40 °C). The strains were grown on Gifu anaerobic medium (GAM) agar (Nissui) supplemented with 2 % bacto-oxgall (Difco) to test for bile resistance. Strains were inoculated by stabbing into tubes containing semisolid GAM agar (0.5 %) to test for motility, and were incubated at 37 °C for up to 72 h before a negative reaction was recorded (McClung & Lindberg, 1957). Results of phenotypic analyses are listed in the species description. Other physiological, biochemical and enzymic activity tests were performed by using API 20A and API rapid ID 32 A kits (bioMérieux), according to the manufacturer's instructions, with incubation at 37 °C in an anaerobic jar. Results from chemotaxonomic analyses that were useful in distinguishing the two novel strains from some other recognized Bacteroides species are summarized in Table 1. The biochemical test results from API 20A and API rapid ID 32 A for the two novel strains and the closely related species Bacteroides thetaiotaomicron and Bacteroides ovatus are listed in Supplementary Table S1 in IJSEM Online. Strains 199T and 176 had identical biochemical profiles. The two strains could be differentiated from their closest relative, B. thetaiotaomicron JCM 5827T, by utilization of trehalose, indole production, acid production from salicin and activities of 
-fucosidase, arginine arylamidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase and serine arylamidase. Biochemical characteristics of strains 199T and 176 that differed from those of their close relative B. ovatus JCM 5824T included indole production, acid production from trehalose and activities of -fucosidase, glutamyl glutamic acid arylamidase and leucine arylamidase.
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). The results are given in Supplementary Table S2 in IJSEM Online. No significant differences in fatty acid profiles were found for strains 199T and 176 and B. thetaiotaomicron JCM 5827T, except that B. ovatus JCM 5824T contained <1 % C16 : 0. The major cellular fatty acid of strains 199T and 176 was anteiso-C15 : 0 (36.2 and 31.8 %, respectively), in agreement with data for the genus Bacteroides as described by Miyagawa et al. (1979). The closely related strains B. thetaiotaomicron JCM 5827T and B. ovatus JCM 5824T also contained anteiso-C15 : 0 (35.4 and 38.9 %, respectively) as the major cellular fatty acid.
Almost complete (1485 and 1488 bases) 16S rRNA gene sequences of strains 199T and 176 were amplified by PCR (Biometra) using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The products were purified by using a Montage PCR96 filter plate (Millipore) and sequenced directly using the dideoxynucleotide chain-termination method with a DNA sequencer (ABI PRISM 3100; Applied Biosystems/Hitachi) and a BigDye Terminator version 3.1 cycle sequencing RR-100 kit (Applied Biosystems), according to the manufacturer's instructions. Strains closely related to the novel strains were determined by retrieval of closely related 16S rRNA gene sequences from DDBJ, EMBL and GenBank. The sequences were aligned using CLUSTAL_X (version 1.81) (Thompson et al., 1997). Alignment gaps and ambiguous bases were removed prior to phylogenetic analysis by using MacClade (version 4.03) (Maddison & Maddison, 2002). A phylogenetic tree was constructed based on the neighbour-joining method (Saitou & Nei, 1987) using PAUP version 4.0b10 (Swofford, 2000). Distance matrices were calculated by using Kimura's 2-parameter distances for neighbour-joining analysis (Gascuel, 1997). Parsimony analysis was carried out with maximum-parsimony implemented in PAUP version 4.0b10. Maximum-parsimony trees were obtained by using 100 random addition, heuristic search replicates and the tree bisection–reconnection branch-swapping option (Dauga, 2002). The topologies of the phylogenetic trees were evaluated by using the bootstrap resampling method of Felsenstein (1985), with 1000 replicates. Analysis of the 16S rRNA gene sequences revealed that strains 199T and 176 were members of the genus Bacteroides. The phylogenetic position of the two strains among Bacteroides species and representative members of the genera Prevotella, Bacteroides, Porphyromonas and Tannerella are shown in Fig. 1 and Supplementary Fig. S1 in IJSEM Online, respectively. The phylogenetic trees clearly showed that strains 199T and 176 formed a single cluster and had a distinct line of descent with B. thetaiotaomicron ATCC 29148T. Close phylogenetic relatives of strains 199T and 176 were B. thetaiotaomicron ATCC 29148T and B. ovatus NCTC 11153T. Sequence similarity values were calculated using the program GENETYX (MAC version 11.2.0; Software Development) (Yumoto et al., 2005). 16S rRNA gene sequence similarities of strain 199T with B. thetaiotaomicron ATCC 29148T and B. ovatus NCTC 11153T were 95.9 and 94.6 %, respectively. Strain 176 showed 96.5 and 95.2 % sequence similarity with B. thetaiotaomicron ATCC 29148T and B. ovatus NCTC 11153T, respectively. The 16S rRNA gene sequence similarity between strains 199T and 176 was 99.2 %.
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) after incubation for 12 h at 37 °C, and purified by using the methods of Saito & Miura (1963). Cells were disrupted with lysozyme (Wako Pure Chemical Industries) and SDS, extracted with phenol, and the DNA was recovered using alcohol precipitation and suspended in TE buffer (1 mM EDTA, 10 mM Tris/HCl, pH 8.0). The DNA was treated with RNase A, RNase T1 (both from Sigma) and proteinase K (Wako Pure Chemical Industries), and extracted with phenol and phenol/choloroform/isoamyl alcohol (25 : 24 : 1, by vol.). The DNA was recovered by alcohol precipitation and dissolved in TE buffer. The DNA concentration was measured by using a UV-spectrophotometer (Shimadzu, UV-2100). DNA base compositions were determined by using HPLC (Tamaoka & Komagata, 1984) after enzymic digestion of the DNA to deoxyribonucleosides. An equimolar mixture of four deoxyribonucleotides from a Yamasa G+C kit (Yamasa Shoyu) was used as a quantitative standard. The G+C contents of strains 199T, 176 and the closely related strains B. thetaiotaomicron JCM 5827T and B. ovatus JCM 5824T were 42, 43, 43 and 42 mol%, respectively (Supplementary Table S3 in IJSEM Online). The G+C contents of strains 199T and 176 supported the affiliation of the novel strains with members of the genus Bacteroides, which have a G+C content of 40–48 mol% (Shah, 1992).
DNA–DNA relatedness was determined based on pairwise 16S rRNA gene sequence similarity values of strain 199T with strain 176, B. thetaiotaomicron and B. ovatus. For the DNA–DNA hybridization experiments, DNA of the two isolates and B. thetaiotaomicron JCM 5827T and B. ovatus JCM 5824T was extracted from cells harvested from EGF broth (Kitahara et al., 2001). DNA–DNA hybridization was performed by using the photobiotin-labelling method of Ezaki et al. (1989), using a microplate reader (Fluoroskan-Ascent; Labsystems). The hybridization temperature used was 42 °C. The DNA–DNA relatedness value between strains 199T and 176 was 89 %. The two strains also had identical biochemical profiles and >97 % 16S rRNA gene sequence similarity. Therefore, they can be considered to represent a single species. Strain 199T had DNA–DNA relatedness values of 20 and 23 % with its close neighbours B. thetaiotaomicron JCM 5827T and B. ovatus JCM 5824T, respectively (see Supplementary Table S3 in IJSEM Online). DNA–DNA hybridization values of <70 % with the closest Bacteroides species confirmed that strain 199T represented a novel species (Stackebrandt & Goebel, 1994). That strain 199T was distinct from other species of the genus Bacteroides with validly published names was also evident from the phenotypic analyses. On the basis of the results presented in this study, strain 199T should be classified as the type strain of a novel species of the genus Bacteroides, for which we propose the name Bacteroides finegoldii sp. nov. An additional strain (176) is also included in this species.
Description of Bacteroides finegoldii sp. nov.
Bacteroides finegoldii (fi.ne.gol'di.i. L. gen. n. finegoldii of Finegold, in honour of Dr Sydney M. Finegold, a contemporary researcher in anaerobic bacteriology and an infectious diseases clinician).
Cells are strictly anaerobic, non-spore-forming, non-motile, Gram-negative rods, about 0.80 µm wide and 1.5–4.5 µm long, and occur singly. Surface colonies on EG blood agar plates after 2 days are 1–2 mm in diameter, circular, translucent–whitish, raised and convex. The optimum temperature for growth is about 37 °C. Grows in the presence of bile. Indole-negative but is able to hydrolyse aesculin. Nitrate is not reduced. No activity is detected for urease and gelatin is not hydrolysed. Acid is produced from glucose, lactose, sucrose, maltose, salicin, xylose, arabinose, cellobiose, mannose, raffinose and rhamnose, but not from mannitol, glycerol, melezitose, sorbitol or trehalose. Positive reactions are obtained using API rapid ID 32 A for -galactosidase, 
-galactosidase, -glucosidase, -glucosidase, -arabinosidase, N-acetyl--glucosoaminidase, glutamic acid decarboxylase, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase and glutamyl glutamic acid arylamidase. Negative reactions are obtained for arginine dihydrolase, 6-phospho--galactosidase, -glucuronidase, -fucosidase, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, glycine arylamidase, histidine arylamidase and serine arylamidase. The major fatty acids are anteiso-C15 : 0 (31.8–36.2 %) and iso-C17 : 0 3-OH (13.1–14.5 %). The DNA G+C content is 42.4–43.0 mol%.
The type strain is 199T (=JCM 13345T=DSM 17565T), isolated from human faeces. Strain 176 (=JCM 13346) 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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Benno, Y., Endo, K., Mizutani, T., Namba, Y., Komori, T. & Mitsuoka, T. (1989). Comparison of fecal microflora of elderly persons in rural and urban areas of Japan. Appl Environ Microbiol 55, 1100–1105.
Dauga, C. (2002). Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. Int J Syst Evol Microbiol 52, 531–547.[Abstract]
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid- deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Fenner, L., Roux, V., Mallet, M.-N. & Raoult, D. (2005). Bacteroides massiliensis sp. nov., isolated from blood culture of a newborn. Int J Syst Evol Microbiol 55, 1335–1337.
Finegold, S. M., Sutter, V. L. & Mathisen, G. E. (1983). Normal indigenous flora. In Human Intestinal Microflora in Health and Disease, pp. 3–31. Edited by D. J. Hentges. New York: Academic Press.
Gascuel, O. (1997). BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol 14, 685–695.[Abstract]
Hayashi, H., Sakamoto, M. & Benno, Y. (2002). Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods. Microbiol Immunol 46, 535–548.[Medline]
Jousimies-Somer, H. R., Summanen, P. H., Wexler, H., Finegold, S. M., Gharbia, S. E. & Shah, H. N. (2003). Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and other anaerobic Gram-negative bacteria. In Manual of Clinical Microbiology, 8th edn, pp. 880–901. Edited by P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller & R. H. Yolken. Washington, DC: American Society for Microbiology.
Kitahara, M., Takamine, F., Imamura, T. & Benno, Y. (2001). Clostridium hiranonis sp. nov., a human intestinal bacterium with bile acid 7-dehydroxylating activity. Int J Syst Evol Microbiol 51, 39–44.[Abstract]
Kitahara, M., Sakamoto, M., Ike, M., Sakata, S. & Benno, Y. (2005). Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., isolated from human faeces. Int J Syst Evol Microbiol 55, 2143–2147.
Macfarlane, G. T. & Macfarlane, S. (1997). Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria. Scand J Gastroenterol Suppl 222, 3–9.
Macrae, M., Kramer, D. L. & Coffino, P. (1998). Developmental effect of polyamine depletion in Caenorhabditis elegans. Biochem J 333, 309–315.
Maddison, D. R. & Maddison, W. P. (2002). MacClade 4 – Analysis of Phylogeny and Character Evolution, version 4.0. Sunderland, MA: Sinauer Associates.
McClung, L. S. & Lindberg, R. B. (1957). The study of obligately anaerobic bacteria. In Manual of Microbiological Methods, pp. 120–131. Edited by M. J. Pelczar and others. New York: McGraw-Hill.
Miyagawa, E., Azuma, R. & Suto, T. (1979). Cellular fatty acid composition in Gram-negative obligately anaerobic rods. J Gen Appl Microbiol 25, 41–51.
Noack, J., Kleessen, B., Proll, J., Dongowski, G. & Blaut, M. (1998). Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats. J Nutr 128, 1385–1391.
Noack, J., Dongowski, G., Hartmann, L. & Blaut, M. (2000). The human gut bacteria Bacteroides thetaiotaomicron and Fusobacterium varium produce putrescine and spermidine in cecum of pectin-fed gnotobiotic rats. J Nutr 130, 1225–1231.
Saito, H. & Miura, K. I. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629.[Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sakamoto, M., Suzuki, M., Umeda, M., Ishikawa, I. & Benno, Y. (2002). Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov. Int J Syst Evol Microbiol 52, 841–849.[Abstract]
Shah, H. N. (1992). The genus Bacteroides and related taxa. In The Prokaryotes, 2nd edn, pp. 3593–3607. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Song, Y. L., Liu, C. X., McTeague, M. & Finegold, S. M. (2004). "Bacteroides nordii" sp. nov. and "Bacteroides salyersae" sp. nov. isolated from clinical specimens of human intestinal origin. J Clin Microbiol 42, 5565–5570.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Suau, A., Bonnet, R., Sutren, M., Godon, J. J., Gibson, G. R., Collins, M. D. & Doré, J. (1999). Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 65, 4799–4807.
Swofford, D. L. (2000). PAUP* – Phylogenetic Analysis Using Parsimony (*and other methods), version 4. Sunderland, MA: Sinauer Associates.
Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882.
Yumoto, I., Hirota, K., Goto, T., Nodasaka, Y. & Nakajima, K. (2005). Bacillus oshimensis sp. nov., a moderately halophilic, non-motile alkaliphile. Int J Syst Evol Microbiol 55, 907–911.
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