SGM Journals
Actinobacteria

Bifidobacterium stercoris sp. nov., isolated from human faeces

Min-Soo Kim, Seong Woon Roh, Jin-Woo Bae

  • Department of Life and Nanopharmaceutical Sciences and Department of Biology, Kyung Hee University, Seoul 130-701, Republic of Korea
  • Correspondence
    Jin-Woo Bae
    baejw{at}khu.ac.kr

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2823–2827 · https://doi.org/10.1099/ijs.0.019943-0

    View at publisher PubMed

    Abstract

    Strain Eg1T, an anaerobic, Gram-stain-positive, non-motile and non-spore-forming bacterium, was isolated from human faeces. The optimal temperature for growth was 37 °C and tests for oxidase and catalase activities gave negative results. Fructose-6-phosphate phosphoketolase activity was detected. Acid was produced during fermentation of several substrates, including glucose. The end products of glucose fermentation were acetic acid and lactic acid, which were produced in a molar ratio of 1.76 : 1 (approximately 3 : 2). The G+C content was 57.8 mol%. Comparative analysis of 16S rRNA gene sequences showed that strain Eg1T was closely related to Bifidobacterium adolescentis YIT 4011T (98.36 % 16S rRNA gene sequence similarity) and Bifidobacterium ruminantium JCM 8222T (97.93 %) and analysis of hsp60 sequences showed that strain Eg1T was closely related to B. adolescentis JCM 1275T (99.35 % hsp60 sequence similarity) and B. ruminantium JCM 8222T (92.13 %). However, despite these degrees of similarity being high enough for strain Eg1T to be included at the same species level as B. adolescentis and B. ruminantium (96.5–100 % for the genus Bifidobacterium), the isolate could be distinguished from B. adolescentis KCTC 3216T and B. ruminantium KCTC 3425T by low levels of DNA–DNA relatedness (41 and 17 %, respectively). Based on phenotypic, genotypic and phylogenetic analyses, we propose that strain Eg1T is classified in a novel species, Bifidobacterium stercoris sp. nov. The type strain is Eg1T (=KCTC 5756T =JCM 15918T).

    • The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and hsp60 sequences of strain Eg1T are FJ611793 and FJ770059, respectively.

    • A neighbour-joining phylogenetic tree based on partial hsp60 sequences is available as supplementary material with the online version of this paper.

    Bifidobacteria are non-motile, non-sporulating, obligately anaerobic, non-gas-producing, saccharoclastic bacteria that belong to the family Bifidobacteriaceae in the class Actinobacteria, which comprises Gram-positive bacteria that have DNA with a high G+C content (Ventura et al., 2007). As bifidobacteria facilitate the fermentation of a large variety of oligosaccharides, they are one of the most well-known probiotic bacteria and are considered the main target organism for inclusion in prebiotic foods, which may provide health benefits (Gibson & Roberfroid, 1995; Saulnier et al., 2009; Ventura et al., 2007). However, despite the physiological importance of bifidobacteria in the intestine, according to culture-independent studies (especially metagenomic studies based on the analysis of genomic DNA and rRNA gene libraries), they comprise only a small part of the diverse bacterial population found in the intestinal microbiota; however, the exact population in the human intestine has not been fully established (Eckburg et al., 2005; Palmer et al., 2007; Turroni et al., 2009; Wang et al., 2005). The general characteristics of the genus Bifidobacterium are the presence of fructose-6-phosphate phosphoketolase (F6PPK) and production of both acetic and lactic acids from glucose fermentation in a molar ratio of 3 : 2 (Scardovi, 1986). To date, 30 species and nine subspecies have been described as belonging to the genus Bifidobacterium.

    The bifidobacterial strain Eg1T was isolated from faeces of a healthy, 27-year-old Korean male on Eggerth–Gagnon agar supplemented with 5 % horse blood at 37 °C for 2 days in an anaerobic chamber (Bactron II; Sheldon Manufacturing) containing N2/H2/CO2 (90 : 5 : 5). Strain Eg1T was subcultured on Bifidobacterium medium (BM; DSMZ medium 58) under anaerobic conditions and stored as a suspension in 10 % skimmed milk (BBL) containing 10 % glycerol at −80 °C.

    Bifidobacterium adolescentis KCTC 3216T and Bifidobacterium ruminantium KCTC 3425T were obtained from the Korean Collection for Type Cultures as reference strains. All experiments were performed using bacterial strains cultivated and maintained in BM at pH 6.8 and 37 °C for 2 or 3 days unless stated otherwise. Gram staining was performed using a Gram-staining kit (bioMérieux). Spore staining was performed using malachite green dye. Phase-contrast microscopy (Nikon) was used to observe the morphology of individual cells as well as Gram and spore staining. To determine bile resistance, strain Eg1T was cultivated in PYG medium (DSMZ medium 104) supplemented with 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 1.0 and 1.5 % (w/v) bile salts (Sigma). Catalase and oxidase activities were determined using 3 % (v/v) hydrogen peroxide and 1 % (w/v) p-tetramethylphenylenediamine solutions (bioMérieux), respectively. The motility of strain Eg1T was determined by stabbing into PYG medium containing 0.4 % agar. Indole production and nitrate reduction were tested using indole nitrite medium (BBL). To investigate enzyme activities and acid production from various carbohydrates, API 50CHL, API 20A, API ZYM and Rapid ID 32A (bioMérieux) were used according to the manufacturer's instructions. F6PPK activity of strain Eg1T was determined using a phosphoketolase assay (Orban & Patterson, 2000; Scardovi, 1986). The end products of glucose fermentation in cells cultured in Lactobacillus MRS broth (BBL) at 37 °C for 4 days were analysed by HPLC using an LC-10AT liquid chromatograph and an SPD-10A VP detector (Shimadzu) with an Alltech Prevail organic acid column (Grace Davison Discovery Sciences). The fermentation products were acetic acid and lactic acid in a molar ratio of 1.76 : 1, which closely corresponded to the usual molar ratio of 3 : 2 for acetic and lactic acid production by Bifidobacterium species (Scardovi, 1986). Other results of the phenotypic characterization are given in Table 1⇓ and the species description.

    Table 1.

    Comparison of phenotypic characteristics of strain Eg1T and its closest phylogenetic neighbours

    Strains: 1, Bifidobacterium stercoris sp. nov. Eg1T; 2, B. adolescentis KCTC 3216T; 3, B. ruminantium KCTC 3425T. Data were obtained in this study unless indicated. All strains were anaerobic and Gram-stain-positive, fermented glucose with production of acetic and lactic acids in a molar ratio of 3 : 2 and produced F6PPK (Biavati & Mattarelli, 1991; Gavini et al., 1991; Scardovi, 1986) and were positive for production of acid from d-glucose, maltose, raffinose and salicin and production of acid phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, arginine arylamidase and proline arylamidase. All strains were negative for catalase, urease, gelatin hydrolysis, nitrate reduction, indole formation, d-mannose fermentation and production of alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chymotrypsin, α-fucosidase, N-acetyl-β-glucosaminidase, β-glucuronidase, α-mannosidase, arginine dihydrolase, β-galactosidase-6-phosphate, glutamic acid decarboxylase, alanine arylamidase, cystine arylamidase, glutamyl glutamic acid arylamidase, leucyl glycine arylamidase, pyroglutamic acid arylamidase and valine arylamidase.

    To measure DNA–DNA relatedness and G+C content, genomic DNA was extracted from strain Eg1T and the two reference strains using a G-spin Genomic DNA extraction kit (iNtRON Biotechnology). DNA–DNA relatedness was determined using the fluorometric method described by Ezaki et al. (1989) with some modifications (Hirayama et al., 1996). Strain Eg1T showed 41 % DNA–DNA relatedness to B. adolescentis KCTC 3216T and 17 % DNA–DNA relatedness to B. ruminantium KCTC 3425T. As reported by Wayne et al. (1987), a bacterial species should include strains with >70 % DNA–DNA relatedness, so the results of this study suggested that strain Eg1T was a member of a novel species of the genus Bifidobacterium. The G+C content of strain Eg1T was determined using a fluorimetric method using SYBR Green I and real-time PCR (Gonzalez & Saiz-Jimenez, 2002). Genomic DNA from Escherichia coli K-12 and the reference strains was used for calibration (Gonzalez & Saiz-Jimenez, 2002). The G+C content of strain Eg1T was estimated to be 57.8 mol%, which is consistent with the high G+C content (55–67 mol%) found in the family Bifidobacteriaceae (Jian et al., 2001).

    The 16S rRNA gene sequence of strain Eg1T was amplified by colony PCR with four bacteria-specific primers (8F, 968F, 518R and 1492R; Baker et al., 2003). The amplification products were purified using a QIAquick PCR Purification kit (Qiagen) and sequenced using a BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems), according to the manufacturers' instructions. The reaction mixtures were analysed using an automated DNA analyser system (PRISM 3730XL DNA Analyzer; Applied Biosystems) and the 16S rRNA gene sequence fragments were assembled using SeqMan software (dnastar). The nearly full-length 16S rRNA gene sequence obtained from strain Eg1T was compared with other sequences in the GenBank database. Strain Eg1T was related to members of the genus Bifidobacterium in the phylum Actinobacteria and exhibited highest 16S rRNA gene sequence similarity to B. adolescentis YIT 4011T and B. ruminantium JCM 8222T (98.36 and 97.93 %, respectively). A total of 38 nearly full-length 16S rRNA gene sequences from reference strains were aligned with that of strain Eg1T using the multiple sequence alignment program clustal x version 1.83 (Thompson et al., 1997) and the alignment was trimmed and converted to mega format for phylogenetic analysis. Phylogenetic consensus trees using neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Kluge & Farris, 1969) algorithms were constructed and visualized using mega4 (Tamura et al., 2007) and tested by randomly selecting 1000 bootstrap replicates. Phylogenetic consensus trees were visualized with mega4 (Tamura et al., 2007). Phylogenetic analysis of 16S rRNA gene sequences showed that strain Eg1T fell within the bifidobacteria cluster and was most closely related to B. adolescentis YIT 4011T and B. ruminantium JCM 8222T (Fig. 1⇓).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining phylogenetic consensus tree based on 16S rRNA gene sequences showing the phylogenetic position of strain Eg1T in the genus Bifidobacterium. Filled diamonds indicate that the corresponding nodes were also recovered in the tree generated using the maximum-parsimony algorithm. Bootstrap values (>50 %) based on 1000 replicates for the neighbour-joining/maximum-parsimony methods are shown at branch nodes; –, bootstrap support ≤50 %. Bar, 1 substitution per 100 nucleotide positions.

    A housekeeping gene (hsp60, encoding the 60 kDa heat-shock protein) was used as an additional phylogenetic marker to identify bifidobacterial species (Jian et al., 2001; Zhu et al., 2003). The partial hsp60 sequence of strain Eg1T was obtained and analysed in a similar way to that for the 16S rRNA gene sequence, using the primers and method described by Okamoto et al. (2008) and the neighbour-joining algorithm. A 616-bp fragment of the hsp60 sequence was obtained and analysed with 31 hsp60 sequences from type strains of species of the genus Bifidobacterium (Supplementary Fig. S1, available in IJSEM Online). The hsp60 sequence of strain Eg1T showed 99.35 % sequence similarity to that of B. adolescentis JCM 1275T, which indicated relatedness at the species level (96.5–100 %), and 92.13 % sequence similarity to that of B. ruminantium JCM 8222T, indicating interspecies relatedness (80–96 %; Jian et al., 2001; Zhu et al., 2003). Even though analysis of the hsp60 sequence is a more accurate tool for the identification of species of the genus Bifidobacterium than 16S rRNA gene sequence analysis, the low DNA–DNA relatedness (41 %) between strain Eg1T and B. adolescentis KCTC 3216T confirmed that these two strains belonged to different species.

    On the basis of our phenotypic, genotypic and phylogenetic studies, we propose that strain Eg1T is included in the genus Bifidobacterium as a member of a novel species, Bifidobacterium stercoris sp. nov.

    Description of Bifidobacterium stercoris sp. nov.

    Bifidobacterium stercoris (ster′co.ris. L. gen. n. stercoris of dung, excrement, ordure).

    Anaerobic, Gram-stain-positive, non-motile and non-spore-forming. Cells are club-like or branch-shaped, 4.0–6.0 μm long and 1.0 μm wide, and are generally observed as single cells. Colonies on BM agar at 37 °C after 2 days are 1.0–1.5 mm in diameter, white and circular with a glistening surface, an umbonate side view and a mucoid texture. The optimal temperature for growth is 37 °C. Cells survive 0.05 and 0.1 % bile salts, but not 0.15 % bile salts. Oxidase- and catalase-negative. F6PPK activity is present. The end products of glucose fermentation are acetic acid and lactic acid in a molar ratio of 1.76 : 1 (about 3 : 2). Indole formation and nitrate reduction are negative. With API 20A, aesculin hydrolysis is positive, but gelatin hydrolysis and urease activity are negative. With API 50CHL, aesculin hydrolysis is positive and acid is produced from amygdalin, l-arabinose, cellobiose, d-glucose, methyl α-d-glucoside, glycogen, lactose, maltose, d-mannitol, melibiose, raffinose, d-ribose, salicin, starch, trehalose, turanose and d-xylose, but not from N-acetylglucosamine, d-adonitol, d-arabinose, d- or l-arabitol, arbutin, dulcitol, erythritol, d-fructose, d- or l-fucose, d-galactose, gentiobiose, gluconate, 2-ketogluconate, 5-ketogluconate, glycerol, inositol, inulin, d-lyxose, d-mannose, methyl α-d-mannoside, melezitose, l-rhamnose, d-sorbitol, l-sorbose, sucrose, d-tagatose, xylitol, l-xylose or methyl β-d-xyloside. With API Rapid ID 32A, positive for raffinose fermentation, α- and β-galactosidases, α- and β-glucosidases, arginine arylamidase, glycine arylamidase, histidine arylamidase, leucine arylamidase, phenylalanine arylamidase, proline arylamidase, serine arylamidase and tyrosine arylamidase, but negative for d-mannose fermentation, nitrate reduction, indole production, alkaline phosphatase, arginine dihydrolase, β-galactosidase-6-phosphate, α-arabinosidase, α-fucosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, alanine arylamidase, glutamyl glutamic acid arylamidase, leucyl glycine arylamidase, pyroglutamic acid arylamidase and urease. With API ZYM, positive for acid phosphatase, α- and β-galactosidases, α- and β-glucosidases, leucine arylamidase and naphthol-AS-BI-phosphohydrolase, but negative for alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chymotrypsin, α-fucosidase, N-acetyl-β-glucosaminidase, β-glucuronidase, α-mannosidase, cystine arylamidase and valine arylamidase. The G+C content of the type strain is 57.8 mol%.

    The type strain, Eg1T (=KCTC 5756T =JCM 15918T), was isolated from human faeces.

    Acknowledgments

    We thank Dr J. P. Euzéby (Ecole Nationale Vétérinaire, Toulouse, France) for etymological advice. This work was supported in 2009 by the Korea Food & Drug Administration (grant no. 09172KFDA996).

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