Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposal of Christensenellaceae fam. nov.

The use of culture-independent molecular methods to analyse the gastrointestinal (GI) microbiota has allowed a more complete and accurate assessment of the biodiversity of this ecosystem (Eckburg et al., 2005; Ley et al. 2008; Turnbaugh et al., 2009; Tap et al., 2009; Qin et al., 2010). Such molecular methods using small-subunit rRNA gene sequence-based technologies are considered fruitful for finding potential links between microbes and a certain health and/or disease status. However, as pointed out by Zoetendal et al. (2008), it has to be realized that the results obtained with such approaches cannot necessarily be extrapolated beyond the description of the microbial diversity, since potential functions of these microbes are not always extracted from small-subunit rRNA gene sequence data. At the moment, although links have been elucidated between the intestinal microbiota and GI tract disorders, causal effects have been determined only for cultured bacterial species such as Helicobacter pylori and Clostridium difficile. This observation, and the fact that the recent study of Eckburg et al. (2005) reported that the proportion of reported cultivable bacteria was only 20 % for the human intestinal microbiota, indicated that cultivation of novel GI-tract inhabitants is indispensable for future studies of links between microbes and GI tract diseases such as inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS).

To understand better the physiological characteristics and function of the majority of the human GI microbiota, we have performed several intensive cultivation trials aimed at isolating so-called ‘unculturable’ or ‘as-yet-uncultured’ bacteria from the human GI tract (Sakon et al., 2008; Morotomi et al., 2008, 2009, 2010, 2011; Nagai et al., 2009, 2010a, b; Watanabe et al., 2010). Among these bacteria, 15 strains of 15 novel species including three new genera were adopted as reference genomes by the Human Microbiome Project () and the assembled and annotated genomic sequences of these bacteria will be submitted to GenBank. In this article, we report the characterization and classification of strain YIT 12065^T, proposing its affiliation to a new family, genus and species.

Faecal samples were collected from two healthy Japanese males (subjects H and K, aged 57 and 27 years, respectively) and transferred immediately to anaerobic conditions. Each sample was weighed and diluted with pre-reduced 0.1 M PBS (pH 7) in an anaerobic glovebox (Coy Laboratory Products), which contained 88 % nitrogen, 7 % hydrogen and 5 % carbon dioxide. Each dilution was then spread on modified Gifu anaerobic medium (GAM; Nissui Pharmaceutical) containing 1.5 % (w/v) agar that was supplemented with bile (2, 4, 6 and 8 % Bacto oxgall; Difco), NaCl (1, 3, 6 and 10 %, w/v) or antibiotics (one of 12 compounds, at three different concentrations) in an attempt to isolate subdominant groups of the intestinal microbiota. The composition of the modified GAM agar was described previously (Sakon et al., 2008). The inoculated plates were incubated at 37 °C for 3 days in an anaerobic glovebox. Subsequently, colonies showing different morphologies were selected and subcultured on GAM agar to obtain pure cultures. Strain YIT 12065^T was isolated from a GAM agar plate (pH 6.8) supplemented with 8 % Bacto oxgall (equivalent to 80 % bile) inoculated with a 10⁻⁶ serially diluted faecal sample from subject H.

The end products of strain YIT 12065^T grown in pre-reduced peptone-yeast extract (PY) medium (Holdeman et al., 1977) supplemented with 1 % glucose (PYG), lactate or succinate were analysed by HPLC as described previously (Chonan et al., 1995). Carbohydrate fermentation tests and other biochemical tests, such as indole production, urease activity and hydrolysis of gelatin and aesculin, were performed by the methods described by Holdeman et al. (1977) and by using the API 20A anaerobic test kit (bioMérieux). Additional enzyme activities were determined in duplicate by using the API Rapid ID32A and API ZYM systems (bioMérieux) according to the manufacturer’s instructions. Oxidase activity was determined with oxidase test strips (Eiken Chemical). Sensitivity to bile and NaCl was determined by comparing growth on modified GAM agar with and without 2 % Bacto oxgall (Difco) or 1, 2, 3, 5, 10 or 20 % NaCl after 5 days of incubation at 37 °C. Growth on modified GAM agar at different temperatures was assessed over time for 14 days at 15, 20, 25, 30, 34, 37, 40, 43 and 45 °C under anaerobic conditions by use of an AnaeroPak system (Mitsubishi Gas Chemical). The effects of pH on growth were determined over the range pH 4.0–10.0 at intervals of 0.5 pH units using HCl and NaOH to adjust the pH after cultures had been incubated at 37 °C for 3 days, under anaerobic conditions with nitrogen to limit undesirable changes in pH during incubation.

Cellular fatty acids, isoprenoid quinones, cell-wall amino acids and whole-cell sugars were prepared from cells grown on PYG agar at 37 °C for 3 days. Cellular fatty acid methyl esters were obtained from lyophilized cells by saponification, methylation and extraction using the method of Miller (1982) with minor modifications (Kuykendall et al., 1988). Fatty acid methyl esters were identified by using the MIDI system with MOORE5 from the MIS Standard Libraries. Isoprenoid quinones were extracted as described by Komagata & Suzuki (1987) and analysed by using an HPLC atmospheric pressure chemical ionization (APCI)-MS/MS system (API 3200; Applied Biosystems) with an L-column ODS (2.1×150 mm; Chemicals Evaluation and Research Institute) and an HPLC-APCI-MS system (Micromass ZQ equipped with 2996 photodiode array detector; Waters) with a Cadenza CD-C18 column (3.0×150 mm; Imtakt), following the modified method of Katsuta et al. (2005). Isolation of the cell-wall fraction and preparation of the cell-wall hydrolysate were carried out by using the method of Schleifer (1985). The amino acid composition of the cell-wall hydrolysate was determined by using an Aquity ultraperformance liquid chromatography system (Waters). Whole-cell sugars were determined by HPLC [Shimadzu LC-20A system; Symmetry 300 TM C18 column (4.6×250 mm)] according to the method of Honda et al. (1989). The DNA G+C content was determined by hydrolysing the DNA enzymically and quantifying nucleosides by HPLC according to the method of Ezaki et al. (1990).

Caldicoprobacter oshimai DSM 21659^T and Tindallia californiensis DSM 14871^T, purchased from the DSMZ (Braunschweig, Germany), and Clostridium ganghwense JCM 13193^T, purchased from the JCM (Wako, Japan), were used as reference strains.

Closely related sequences were retrieved from GenBank/EMBL/DDBJ by using the fasta program (Lipman & Pearson, 1985). Sequences were aligned and used to produce an unrooted phylogenetic tree according to the neighbour-joining method (Saitou & Nei, 1987) using clustal_x (version 1.83) (Thompson et al., 1997). The stability of groupings was estimated by bootstrap analysis (1000 replications). Trees were visualized by using the TreeView program (version 1.6.6) (Page, 1996). Maximum-parsimony (MP) and maximum-likelihood (ML) methods were used to confirm the phylogenetic placement of the aligned sequences. MP analysis was performed using the software package mega4 (Tamura et al., 2007). The ML tree was constructed via the phyml program (Guindon & Gascuel, 2003) using Kimura’s two-parameter nucleotide substitution model (Kimura, 1980). The input file was prepared via the seqboot program in the phylip software package (Felsenstein, 2004).

Cells of strain YIT 12065^T were Gram-reaction-negative, non-motile, short, straight rods with tapered ends (0.4×0.8–1.9 µm). Electron microscopy of ultrathin sections revealed a Gram-negative cell-wall structure (Fig. 1). Growth was observed under strict anaerobic conditions but not aerobic or microaerobic conditions created by use of the CampyPak Plus system (with 5–15 % O₂ and 5–12 % CO₂; BBL). After 4 days of anaerobic incubation at 37 °C on modified GAM agar, Wilkins–Chalgren anaerobe agar [Wilkins–Chalgren anaerobe broth (Oxoid) with 1.5 % agar] or Schaedler anaerobe agar [Schaedler anaerobe broth (Oxoid) with 1.5 % agar], colonies were punctiform, 0.1 mm in diameter, circular and beige. Strain YIT 12065^T was saccharolytic. Tests for aesculin hydrolysis, gelatin hydrolysis, indole production, nitrate reduction and catalase, oxidase and urease activities were negative. Cells were resistant to 20 % bile. Under anaerobic conditions, growth was most rapid at 37–40 °C, slightly weak at 34 °C, weak at 25–30 °C and scant at 43 °C. No growth was observed below 20 °C or at 45 °C. The pH range for growth was pH 6.0–9.0, with optimal growth at pH 7.5. Numbers of colonies grown on medium containing 1, 2 and 3 % NaCl were 80, 27 and 6 %, respectively, compared with control medium without NaCl. No growth was observed on medium containing ≥5 % NaCl. Growth of strain YIT 12065^T in liquid media was very weak. Small amounts of acetic (3.6 mM) and butyric (0.3 mM) acids were produced as end products of glucose fermentation in PYG broth. Addition of lactate or succinate did not enhance growth or the production of short-chain fatty acids. In the API Rapid ID 32A and API ZYM test systems, strain YIT 12065^T was positive for α-arabinosidase, β-galactosidase, β-glucosidase, glutamic acid decarboxylase and naphthol-AS-BI-phosphohydrolase. Other biochemical characteristics obtained by using the API systems (API Rapid ID32A, API ZYM and API 20A) are included in the species description.

Cellular fatty acid profiles of strain YIT 12065^T and the type strains of phylogenetically related species are shown in Supplementary Table S1, available in IJSEM Online. Respiratory quinones were not detected. The cell wall contained glutamic acid, serine, alanine and ll-diaminopimelic acid (ll-A₂pm). Although ll-A₂pm is found mainly in species of Gram-positive bacteria, some Gram-negative bacteria such as Syntrophomonas species (Zhang et al., 2004, 2005) and Desulfosporosinus species (Stackebrandt et al., 2003; Ramamoorthy et al., 2006; Vatsurina et al., 2008) have been reported to possess ll-A₂pm as a diagnostic peptidoglycan diamino acid. All of these species are members of the order Clostridiales. The whole-cell sugars were ribose, rhamnose, galactose and glucose. The DNA G+C content of strain YIT 12065^T was 51.3 mol%.

A 1497 bp region of the 16S rRNA gene of strain YIT 12065^T was sequenced. Database searches revealed that strain YIT 12065^T was most closely related (≥86 % 16S rRNA gene sequence similarity) to Caldicoprobacter oshimai JW/HY-331^T (86.9 %), Tindallia californiensis DSM 14871^T (86.3 %) and Clostridium ganghwense JCM 13193^T (86.1 %). Both Tindallia californiensis and Clostridium ganghwense belong to the family Clostridiaceae, whereas Caldicoprobacter oshimai is proposed to belong to a novel family Caldicoprobacteraceae, consisting of a single genus and species, within the order Clostridiales. Although Caldicoprobacter oshimai, Clostridium ganghwense and Tindallia californiensis are phylogenetically most closely related to strain YIT 12065^T, they are significantly different from strain YIT 12065^T in their physiological characteristics (Table 1). Strain YIT 12065^T showed even lower similarities (79.2–85.0 %) to members of all other type genera of the families within the order Clostridiales (Fig. 2).

The 16S rRNA gene sequence of strain YIT 12065^T shared highest sequence similarities with uncultured faecal bacteria derived from studies of human (uncultured clone RL385_aao81d07, collected in the USA, GenBank accession no. DQ804839, 98.6 % similarity; Ley et al., 2006) and dugong (uncultured clone dgD-165 from Dugong dugon, 96.7 % similarity, GenBank accession no. AB264080; Tsukinowa et al., 2008) (Fig. 2). Based on their similar sources of isolation and 16S rRNA gene sequences, strain YIT 12065^T and unidentified clone RL385_aao81d07 belong to the same species, which presumably prevails in the human intestine. Phylogenetic analysis of these and other related sequences was performed and confirmed that strain YIT 12065^T was phylogenetically most closely associated with members of the families of the order Clostridiales, but formed a separate cluster (Fig. 2).

On the basis of these phylogenetic and physiological data, strain YIT 12065^T is proposed as the type strain of the novel taxon Christensenella minuta gen. nov., sp. nov. and Christensenella gen. nov. is proposed as the type genus of the novel family Christensenellaceae fam. nov. within the order Clostridiales of the phylum Firmicutes.