Dialister succinatiphilus sp. nov. and Barnesiella intestinihominis sp. nov., isolated from human faeces

Many novel bacteria have been detected in the human gastrointestinal (GI) tract and faeces using culture-independent approaches based on small subunit (16S and 18S) rRNA diversity (for review see Rajilić-Stojanović et al., 2007). The recent study of Eckburg et al. (2005) indicated that the proportion of reported cultivable bacteria was only 20 % for faecal and mucosal samples of three healthy individuals. To better understand the physiological characteristics and function of the majority of human GI microbiota, several intensive cultivation trials aimed at isolating so-called ‘unculturable’ or ‘as-yet-uncultured’ bacteria from the human GI tract have been performed (Sakon et al., 2008). In this article, the isolation of two novel species from human faeces is reported. Although novel taxonomic units (species) based on a single isolate are proposed, these isolates displayed >98 % 16S rRNA gene sequence similarity to some of the human intestinal uncultured clones reported by several groups in the USA and other countries, as described below, indicating that these bacteria are common members of the human intestinal microbiota.

Faecal samples were collected from two healthy Japanese males (subjects C and M, 38 and 29 years old, respectively) and immediately transferred anaerobically. Each sample was weighed and diluted with pre-reduced 0.1 M PBS (0.145 M NaCl, 0.15 M sodium phosphate; pH 7) in an anaerobic glove box (Coy Laboratory Products) containing 88 % nitrogen, 7 % hydrogen and 5 % carbon dioxide. Then, each dilution was spread on modified Gifu anaerobic medium (GAM) agar (Nissui Pharmaceutical) and anaerobe basal agar (Oxoid). The composition of the modified GAM agar has been described in our previous report (Sakon et al., 2008). Plates were incubated at 37 °C for 3 days in the same anaerobic cabinet. Strain YIT 11850^T was isolated from the GAM agar plate inoculated with a 10⁻⁸ serially diluted faecal sample from subject C. Strain YIT 11860^T was isolated from an anaerobe basal agar plate inoculated with a 10⁻⁶ serially diluted faecal sample from subject M. Single colonies were picked and streaked out until single cultures were obtained on modified GAM agar. The end products of bacterial metabolism of glucose, lactate or succinate in pre-reduced peptone-yeast extract (PY) medium (Holdeman et al., 1977) supplemented with glucose (PYG medium), lactate or succinate, respectively, were analysed by HPLC according to a previously described procedure (Chonan et al., 1995). Cellular morphology was recorded after Gram-staining of 3 day plate or 1 day broth cultures. Biochemical characteristics were determined using the API Rapid ID 32A, API ZYM and API 20A systems (bioMérieux) according to the manufacturer's instructions. The DNA G+C content was determined by hydrolysing the DNA enzymically and quantifying the nucleosides by HPLC according to the method of Ezaki et al. (1990). Closely related sequences were retrieved from the DDBJ (DNA DataBank of Japan) using the fasta program (Lipman & Pearson, 1985). Sequences were aligned and used to produce an unrooted phylogenetic tree by the neighbour-joining method (Saitou & Nei, 1987) using clustal_x (version 1.83) (Thompson et al., 1997). The stability of the groupings was estimated by bootstrap analysis (1000 replications) in clustal_x. Trees were visualized by using the treeview program (version 1.6.6) (Page, 1996). The minimal evolution method (1000 bootstrap replicates) in mega4 (Tamura et al., 2007) and the maximum-likelihood method from the phylip program package (Felsenstein, 1993) were used to confirm the phylogenetic placement of the aligned sequences.

Cells of YIT 11850^T stained Gram-negative and were obligately anaerobic, non-motile coccobacilli (0.4–0.9×0.8–2.0 μm), occurring singly, in pairs and in short chains. Colonies after 3 days anaerobic incubation on ATCC medium 1257 (ETSA medium) were translucent, entire, circular, convex and pin-point. They were asaccharolytic in API test systems. Although the growth of YIT 11850^T in PYG broth produced no visible turbidity, trace amounts of lactate and propionate were detected as end products of metabolism. In the API Rapid ID 32A and API ZYM test systems, strain YIT 11850^T was very unreactive; apart from activities for esterase C4, naphthol-AS-BI-phosphohydrolase, acid phosphatase and weak alkaline phosphatase, all other tests were negative. Addition of succinate, but not lactate, enhanced the growth of YIT 11850^T and subsequent HPLC analysis revealed that this strain produced a large amount of propionate from PY medium supplemented with succinate. Strain YIT 11850^T was able to decarboxylate succinate to propionate, similar to Dialister propionicifaciens (Jumas-Bilak et al., 2005) and members of the genus Veillonella. Enzymic profiles obtained with Rapid ID 32A highlighted the phenotypic differences within the genus Dialister, which are summarized in Table 1⇓.

Approximately 1500 bp of the 16S rRNA gene of strain YIT 11850^T was sequenced and database searches revealed highest sequence relatedness to that of the type strain of D. propionicifaciens (95.1 % sequence similarity; see supplementary Table S1 in IJSEM Online). Phylogenetic analysis of these and other related sequences was performed and data confirmed that strain YIT 11850^T was phylogenetically most closely associated with D. propionicifaciens and Dialister invisus (Fig. 1⇓). Currently, only four species of the genus Dialister are known: Dialister pneumosintes, formerly classified as Bacteroides pneumosintes and originally isolated from nasopharyngeal secretions of patients with influenza (Willems & Collins, 1995); D. invisus, isolated from the human oral cavity (Downes et al., 2003); and Dialister micraerophilus and D. propionicifaciens, isolated from human clinical samples (Jumas-Bilak et al., 2005). Although there is no evidence that species of the genus Dialister have been isolated from human faeces as common members of human indigenous microbiota, many uncultured human intestinal bacterial clones with 16S rRNA gene sequences that are highly similar to that of strain YIT 11850^T have been deposited in the DDBJ (Ley et al., 2006; Gill et al., 2006) (Fig. 1⇓). The DNA G+C content of strain YIT 11850^T was 51.9 mol%, whereas that of the recognized species of the genus Dialister is 35–46 mol% (Downes et al., 2003). It is evident from the results of the taxonomic study that the Gram-negative staining, coccobacillus strain YIT 11850^T recovered from human faeces represents a hitherto unknown species.

Cells of YIT 11860^T stained Gram-negative and were obligately anaerobic, non-motile, pleomorphic rods (0.4–1.0×1.3–13.0 μm) that hydrolysed aesculin but were negative for indole production. Colonies after 3 days anaerobic incubation on anaerobe basal agar were 1–3 mm in diameter, translucent to pale orange–yellow in colour with irregular margins and a low convex shape. Analysis of metabolic end products by HPLC from PYG broth revealed succinic and acetic acids. Results based on the API Rapid ID 32A, 20A and API ZYM systems are summarized in Table 2⇓. Many of the characteristics were similar to those of Barnesiella viscericola (Sakamoto et al., 2007).

An almost-complete 16S rRNA gene sequence of strain YIT 11860^T was determined (1484 bp). The most similar 16S rRNA gene sequences (98.6–99.9 % similarity) were derived from studies of uncultured colonic bacteria (Ley et al., 2006; Eckburg et al., 2005; Mai et al., 2006) (Fig. 2⇓). The cultured bacterium most closely related to strain YIT 11860^T was B. viscericola (96.0 % similarity). B. viscericola, isolated from chicken caecum, is currently the sole recognized species of the genus Barnesiella. A tree showing the phylogenetic relationships of the unknown bacterium is depicted in Fig. 2⇓ and reveals that strain YIT 11860^T is related to B. viscericola. The DNA G+C content of strain YIT 11860^T was 45.5 mol%.

In this report, the isolation of two novel species from human faeces is described. Their phenotypic criteria corresponded to earlier studies of the genera Dialister and Barnesiella, respectively. Based on their phylogenetic distinctiveness, it is considered that strain YIT 11850^T represents a novel species in the genus Dialister and, on the basis of the presented findings, it is proposed that the unknown species from human faeces be assigned as a novel species, Dialister succinatiphilus sp. nov. Likewise, on the basis of the presented findings, it is proposed that strain YIT 11860^T be assigned to the genus Barnesiella as a novel species, Barnesiella intestinihominis sp. nov.