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Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., isolated from human faeces

Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan

Correspondence
Maki Kitahara
kitahara{at}jcm.riken.jp

	ABSTRACT

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Nine strains of Gram-negative, anaerobic rod were isolated from human faeces. Based on phylogenetic analysis and specific phenotypic characteristics, these strains were included within the Bacteroides cluster and were divided into two clusters. Strains from the two clusters showed 16S rRNA gene sequence similarities of 90·4 and 92·7 % to the nearest recognized species, Bacteroides vulgatus. The strains also formed two clusters exhibiting a 16S rRNA gene sequence divergence of approximately 6 %. DNA–DNA hybridization studies confirmed that the two novel strain clusters were distinct from each other. Based on the phenotypic and phylogenetic findings, two novel species, Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov., are proposed, each representing one of the two strain clusters. The DNA G+C content of the type strains were 43·9 mol% for B. plebeius (M12^T=JCM 12973^T=DSM 17135^T) and 42·4 mol% for B. coprocola (M16^T=JCM 12979^T=DSM 17136^T).

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Bacteroides plebeius M12^T and Bacteroides coprocola M16^T are AB200217 and AB200224, respectively.

Tables detailing results using the API 20A and Rapid ID 32A systems, cellular fatty acid contents, and DNA base composition and levels of DNA–DNA relatedness among the new and related strains are available as supplementary material in IJSEM Online.

	MAIN TEXT

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Recently, culture-independent approaches using a 16S rRNA gene sequence clone library have demonstrated that approximately 75 % of the clones from human faecal samples represented novel phylotypes, indicating that our knowledge of the predominant members is very limited (Hayashi et al., 2002a, b; Suau et al., 1999). Bacteroides is one of the predominant genera in human faecal microbiota revealed by culture methods (Benno et al., 1989; Finegold et al., 1983) and 16S rRNA gene sequence clone libraries have shown that many novel phylotypes in the Bacteroides cluster were retrieved from human faecal samples (Hayashi et al., 2002a, b; Suau et al., 1999). Therefore, it is important for our understanding of the composition of human faeces to isolate and identify novel bacterial strains included in the genus Bacteroides. Here, we report two novel species within the genus Bacteroides isolated from human faeces.

Nine strains (M12^T, M14, M35, M122, M131, M132, M16^T, M11 and M156) were isolated from faeces of three healthy Japanese men and women (22 years old, female; 29, male; 31, male) via growth on medium 10 and using the ‘plate-in-bottle’ method (Hayashi et al., 2002a; Mitsuoka et al., 1969). Briefly, after collecting faecal samples, each (0·5 g) was suspended immediately in dilution buffer and samples (50 µl) of faeces diluted to 10^–8 were plated anaerobically on medium 10 by using the ‘plate in bottle’ filled with 100 % CO₂. Isolates were subcultured on Eggerth Gagnon (EG; Merck) agar plates supplemented with 5 % horse blood for 2 days at 37 °C in an anaerobic jar (Hirayama Manufacturing Corp.) filled with 100 % CO₂.

Bile resistance was tested by growing the bacteria on GAM (Nissui) agar plates supplemented with 2 % bacto-oxgall (Difco). Other physiological, biochemical and enzymic-activity tests were performed by inoculation using the API 20A and API Rapid ID 32A systems (bioMérieux) according to the manufacturer's instructions and incubation at 37 °C in an anaerobic jar. Nearly complete (1500 bases) 16S rRNA gene sequences of the strains investigated were amplified by PCR with universal primers 27F (5'-dAGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-dGGTTACCTTGTTACGACTT-3') using a Biometra Thermocycler Tgradient. PCR products were purified by using an Ultraclean PCR Clean-up kit (MO BIO) and were sequenced by using a BigDye Terminator cycle sequencing kit and ABI PRISM 3100 Genetic Analyser (both Applied Biosystems). The closest recognized relatives of the isolates were determined by performing database searches, and sequences of closely related species were retrieved from DDBJ, EMBL and GenBank. Phylogenetic analysis was performed with CLUSTAL X (version 1.83) (Thompson et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987). Topology of the tree was evaluated by a bootstrap analysis with 1000 replicates using the CLUSTAL X software. The virtually complete 16S rRNA gene sequences of Bacteroides helcogenes JCM 6297^T (Benno et al., 1983), Bacteroides pyogenes JCM 6294^T (Benno et al., 1983) and Bacteroides tectus JCM 10003^T (Love et al., 1986) were also determined because their 16S rRNA gene sequences have not been deposited in GenBank/EMBL/DDBJ. For DNA–DNA hybridization experiments, bacterial DNA of the isolated strains and of B. helcogenes JCM 6297^T and Bacteroides vulgatus JCM 5826^T was extracted from cells harvested from EGF broth (Kitahara et al., 2001) after growth for 12 h at 37 °C, as described previously, and purified by using the methods of Saito & Miura (1963). Levels of DNA–DNA hybridization were determined by using the method of Ezaki et al. (1989) using photobiotin and microplates. The DNA G+C content was determined as described previously (Kitahara et al., 2001).

The nine isolates investigated were obligately anaerobic, non-spore-forming, non-motile, Gram-negative short rods or rods.

Physiological and biochemical properties of the nine strains, B. helcogenes 6427^T and B. vulgatus JCM 5826^T were determined by using the API 20A and Rapid ID 32A systems. Based on results using the API 20A system, the nine strains were divided into two groups, with one group consisting of six strains (M12^T, M14, M35, M122, M131, M132) and the other group consisting of three strains (M12^T, M14, M35) (see Supplementary Table S1 in IJSEM Online). Based on the Rapid ID 32A results, the cluster of six strains could be further divided into two subgroups, comprising strains M12^T, M14 and M35, and strains M122, M131 and M132 (see Supplementary Table S2 in IJSEM Online). Strains M16^T, M11 and M156 showed the same results using the Rapid ID 32A system.

The cellular fatty acid profiles of Bacteroides species have been determined (Shah & Collins, 1980) and used to provide a classification for the genus (Shah & Collins, 1983). The major cellular fatty acids of the nine strains investigated here included anteiso-branched C_{15 : 0} (11·0–25·8 % of total) and significant amounts of C_{16 : 0} 3-OH (9·8–21·8 %), C_{16 : 0} (7·5–22·5 %), iso-C_{17 : 0} 3-OH (2·6–18·2 %) and C_{18 : 0}9c (9·6–13·6 %) (see Supplementary Table S3 in IJSEM Online).

Approximately 1500 bases of the 16S rRNA gene sequences were determined for the isolated strains, and also for B. helcogenes JCM 6427^T, B. pyogenes JCM 6294^T and B. tectus JCM 10003^T (the phylogenetic relationships of these three species with other species of the genus Bacteroides were not clear in previous studies). The phylogenetic tree thus constructed indicated that the isolates were related to strains in the genus Bacteroides (Fig. 1). Six strains (M12^T, M14, M35, M122, M131 and M132) formed a single cluster and a distinct line of descent, with 16S rRNA gene sequence similarities of between 98·3 and 100 %. Highest sequence similarity to M12^T was found with M16^T (94·0 %), Bacteroides massiliensis, a recently described species (Fenner et al., 2005) (90·9 %), and B. vulgatus (90·4 %). Strains M16^T, M11 and M156 also formed a single cluster and a distinct line of descent, with levels of sequence similarity among the three strains of between 98·9 and 99·9 %. Highest sequence similarity to M16^T was found with M12^T (94·0 %), B. massiliensis (91·2 %) and B. vulgatus (92·7 %). These results indicated that strains M12^T and M16^T each represent a novel species, as the level of 16S rRNA gene sequence similarity to the closest related species was <97 % (Stackebrandt & Goebel, 1994). B. pyogenes JCM 6294^T and B. tectus JCM 10003^T were related closely to ‘Bacteroides denticanum’ (J. M. Hardham, K. W. King, K. Dreier, C. Strietzel, C. Sfintescu & R. T. Evans, unpublished data).

View larger version (46K): [in this window] [in a new window]   Fig. 1. Phylogenetic tree showing the relationship between Bacteroides plebeius sp. nov., Bacteroides coprocola sp. nov. and related Bacteroides species. The tree was constructed by the neighbour-joining method based on 16S rRNA gene sequences. The numbers at nodes indicate bootstrap values greater than 50 %. Bar, 1 % sequence divergence.

Cells cultivated on EG blood agar plates are strictly anaerobic, non-spore-forming, non-motile and Gram-negative. The short rods or rod-shaped cells are 0·8 µm in width and variable in length, generally in the range 1–5 µm. Colonies grown on EG blood agar plates are 1–3 mm in diameter, disc-shaped, greyish-white and translucent. Optimum temperature for growth is around 37 °C. All strains grow in the presence of bile. All strains produce acid from L-arabinose, D-cellobiose, glucose, lactose, maltose, D-mannose, D-raffinose, L-rhamnose, sucrose and D-xylose. None produce acid from glycerol, D-mannitol, D-melezitose, D-sorbitol or D-trehalose. Aesculin is hydrolysed. Gelatin is not hydrolysed. Indole is not produced. Catalase and urease are not produced. Using Rapid ID 32A, all strains are positive for -galactosidase, -galactosidase, -galactosidase-6-phosphate, -glucosidase, -glucosidase, -arabinosidase, -glucuronidase, N-acetyl--glucosaminidase, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, glycine arylamidase, glutamyl glutamic acid arylamidase, glutamic acid decarboxylase and -fucosidase. All strains have positive reactions on urease, arginine dihydrolase, alkaline phosphatase, proline arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase and serine arylamidase. Arginine arylamidase, phenylalanine arylamidase, leucine arylamidase and histidine arylamidase are variable. Major fatty acids are anteiso-C_{15 : 0}, C_{16 : 0} 3-OH, C_{16 : 0}, iso-C_{17 : 0} 3-OH and C_{18 : 1}9c.

The DNA G+C content of the type strain is 43·9 %. The type strain, M12^T (=JCM 12973^T=DSM 17135^T), was isolated from faeces of a healthy human. Five additional strains [M14 (=JCM 12974), M35 (=JCM 12975), M122 (=JCM 12976), M131 (=JCM 12977) and M132 (=JCM 12978)] are included in this species.

Description of Bacteroides coprocola sp. nov.
Bacteroides coprocola [cop.ro'co.la. Gr. n. kopros faeces; L. suffix -cola inhabitant of; N.L. masc. n. (in apposition) coprocola inhabitant of faeces].

Cells cultivated on EG blood agar plates are strictly anaerobic, non-spore-forming, non-motile and Gram-negative. The short rods or rod-shaped cells are 0·8 µm in width and variable in length, generally in the range 1–4 µm. Colonies on EG blood agar plates are 1·0 to approximately 3·0 mm in diameter, disc-shaped and greyish-white. Optimum temperature for growth is around 37 °C. All strains grow in the presence of bile. All strains produce acid from D-cellobiose, glucose, lactose, maltose, D-mannose, D-raffinose, L-rhamnose, salicin, sucrose and D-xylose. None produce acid from L-arabinose, glycerol, D-mannitol, D-melezitose, D-sorbitol or D-trehalose. Aesculin is hydrolysed. Gelatin is not hydrolysed. Indole is not produced. Catalase and urease are not produced. Using Rapid ID 32A, all strains have positive reactions for -galactosidase, -galactosidase, -galactosidase-6-phosphate, -glucosidase, -glucosidase, -arabinosidase, N-acetyl--glucosaminidase, alkaline phosphatase, leucyl glycine arylamidase, alanine arylamidase, glutamyl glutamic acid arylamidase, glutamic acid decarboxylase and -fucosidase. All strains have negative reactions on urease, arginine dihydrolase, -glucuronidase, 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-C_{15 : 0}, C_{16 : 0} 3-OH, C_{16 : 0}, iso-C_{17 : 0} 3-OH and C_{18 : 1}9c.

The DNA G+C content of the type strain is 42·4 %. The type strain, M16^T (=JCM 12979^T=DSM 17136^T), was isolated from faeces of a healthy human. Two additional strains [M11 (=JCM 12980) and M156 (=JCM 12981)] are included in this species.

	ACKNOWLEDGEMENTS

 
We are grateful to Professor H. Trüper, University of Bonn, Germany, for his suggestions regarding nomenclature.

	REFERENCES

TOP ABSTRACT MAIN TEXT REFERENCES

 
Benno, Y., Watabe, J. & Mitsuoka, T. (1983). Bacteroides pyogenes sp. nov., Bacteroides suis sp. nov., and Bacteroides helcogenes sp. nov., new species from abscesses and feces of pigs. Syst Appl Microbiol 4, 396–407.

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Finegold, S. M., Sutter, V. L. & Mathisen, G. E. (1983). Normal indigenous intestinal flora. In Human Intestinal Microflora in Health and Disease, pp. 3–31. Edited by D. J. Hentges. New York: Academic Press.

Hayashi, H., Sakamoto, M. & Benno, Y. (2002a). Phylogenetic analysis of the human gut microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based methods. Microbiol Immunol 46, 535–548.[Medline]

Hayashi, H., Sakamoto, M. & Benno, Y. (2002b). Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation. Microbiol Immunol 46, 819–831.[Medline]

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]

Love, D. N., Johnson, J. L., Jones, R. F., Bailey, M. & Calverley, A. (1986). Bacteroides tectum sp. nov. and characteristics of other nonpigmented Bacteroides isolates from soft-tissue infections from cats and dogs. Int J Syst Bacteriol 36, 123–128.

Mitsuoka, T., Morishita, Y., Terada, A. & Yamamoto, S. (1969). A simple method ("plate-in-bottle method") for the cultivation of fastidious anaerobes. Jpn J Microbiol 13, 383–385.[Medline]

Saito, H. & Miura, K. (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]

Shah, H. N. & Collins, M. D. (1980). Fatty acid and isoprenoid quinone composition in the classification of Bacteroides melaninogenicus and related taxa. J Appl Bacteriol 48, 75–87.[Medline]

Shah, H. N. & Collins, M. D. (1983). Genus Bacteroides. A chemotaxonomical perspective. J Appl Bacteriol 55, 403–416.[Medline]

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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

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 25, 4876–4882.[Abstract/Free Full Text]

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