Paraprevotella clara gen. nov., sp. nov. and Paraprevotella xylaniphila sp. nov., members of the family ‘Prevotellaceae’ isolated from human faeces

Comparative 16S rRNA gene sequence analysis has revealed that the large intestine in humans harbours up to 10¹⁴ bacteria, comprising more than 500 species (reviewed by Rajilić-Stojanović et al., 2007). Most of these organisms are members of the phyla Firmicutes and Bacteroidetes (Eckburg et al., 2005; Hayashi et al., 2002; Wang et al., 2003). Species belonging to the families Bacteroidaceae, ‘Prevotellaceae’, ‘Porphyromonadaceae’ and ‘Rikenellaceae’ of the order ‘Bacteroidales’ are common members of the human intestinal and oral microbiota, whereas more than 70 % of the human intestinal ‘Bacteroidales’ phylotypes reported are detected only in cultivation-independent studies (Rajilić-Stojanović et al., 2007). This observation can be attributed (at least to some extent) to the lack of appropriate cultivation techniques and to incomplete cultivation efforts.

To clarify the physiological characteristics and functions of the majority of the members of the human gastrointestinal microbiota, we have performed several intensive cultivation trials aimed at isolating so-called ‘unculturable’ or ‘as-yet-uncultured’ bacteria from the human gastrointestinal tract (Sakon et al., 2008; Morotomi et al., 2008; Nagai et al., 2009). In this article, we report the isolation of two novel members of the family ‘Prevotellaceae’ isolated from human faeces. Although we propose novel taxonomic units (genus and species) based on a single isolate, 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 A and B; aged 54 and 38 years, respectively) and immediately transferred anaerobically. Initial processing and subsequent weighing and dilution of the specimens were carried out under strictly anaerobic conditions by using a modification of the Hungate technique (Holdeman et al., 1977). Each sample was weighed and diluted with pre-reduced 0.1 M PBS (pH 7). Diluted samples were spread onto modified Gifu medium (GAM; Nissui Pharmaceutical) containing agar and supplemented with a mixture of antibiotics (described below) to isolate subdominant groups in the intestinal microbiota. Plates were incubated at 37 °C for 3 days in an anaerobic glove box (Coy Laboratory Products) containing N₂/H₂/CO₂ (88 : 7 : 5). The composition of the modified GAM agar was described in our previous report (Sakon et al., 2008). Strain YIT 11840^T was isolated from a GAM agar plate supplemented with trimethoprim (32 μg ml⁻¹), streptomycin sulfate (16 μg ml⁻¹) and vancomycin hydrochloride (2 μg ml⁻¹) and then inoculated with a 10⁻⁶ serially diluted faecal sample from subject A. Strain YIT 11841^T was isolated from a GAM agar plate supplemented with trimethoprim (32 μg ml⁻¹), streptomycin sulfate (16 μg ml⁻¹), vancomycin hydrochloride (0.5 μg ml⁻¹) and erythromycin (0.25 μg ml⁻¹) and then inoculated with a 10⁻⁶ serially diluted faecal sample from subject B. All of these antibiotics were obtained from Sigma-Aldrich. Single colonies were picked and streaked on the modified GAM agar until single cultures were obtained.

The end products of bacterial metabolism of glucose and xylan, in pre-reduced peptone/yeast extract medium (Holdeman et al., 1977) supplemented with 1 % glucose or 1 % birch-wood xylan (Sigma), respectively, were analysed using HPLC according to a previously described procedure (Chonan et al., 1995). Cellular morphology was determined after Gram-staining of 3-day plates. Bile resistance was tested by growing the bacteria on GAM agar plates supplemented with 2 % Bacto oxgall (Difco). Biochemical characteristics were determined using the API Rapid ID 32A, API ZYM and API 20A systems (bioMérieux) according to the manufacturer's instructions. Fatty acid methyl esters were obtained from lyophilized cells by saponification, methylation and extraction, using the method of Miller (1982) but with minor modifications (Kuykendall et al., 1988). Fatty acid methyl esters were determined with a Shimadzu gas chromatograph (model GC-14A) and a Shimadzu chromatograph-data processor (model C-R5A). Fatty acid methyl ester peaks were identified with a bacterial acid methyl ester mix (Supelco) by using retention-time comparisons against standard compounds. Isoprenoid quinones were extracted as described by Komagata & Suzuki (1987) and were analysed by means of an HPLC-atmospheric pressure chemical ionization-MS/MS system (API3200; Applied Biosystems) with an L-column ODS (2.1×150 mm; Chemicals Evaluation and Research Institute, Japan), using the modified method of Katsuta et al. (2005). The G+C content was determined by hydrolysing the DNA enzymically and quantifying the nucleosides by means of HPLC according to the method of Ezaki et al. (1990).

Closely related sequences were retrieved from the DNA Data Bank of Japan by using the program fasta (Pearson & Lipman, 1985). The sequences were aligned and used to produce an unrooted phylogenetic tree with the neighbour-joining method (Saitou & Nei, 1987), using clustal_x (version 1.83) (Thompson et al., 1997). The stability of the groups was estimated by means of bootstrap analysis (1000 replications) in clustal_x. The trees were visualized by using the program TreeView (version 1.6.6) (Page, 1996). The minimum-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. 16S rRNA gene sequence similarities between YIT 11840^T and YIT 11841^T and between these strains and the type strains of all described species within the family ‘Prevotellaceae’ were calculated using fasta (Pearson & Lipman, 1985) and EzTaxon (Chun et al., 2007).

The physiological characteristics of strains YIT 11840^T and YIT 11841^T were very similar, except for the production of acid from maltose (YIT 11840^T, weakly positive; YIT 11841^T, negative) and from d-xylose (YIT 11840^T, positive; YIT 11841^T, weakly positive). The cells were Gram-negative, obligately anaerobic, non-motile, pleomorphic coccobacilli (YIT 11840^T, 0.4–1.3×0.7–2.2 μm; YIT 11841^T, 0.4–1.5×0.9–2.2 μm) that occurred singly, in pairs and in short chains. After 3 days of anaerobic incubation on GAM agar, colonies were 1.5–4.0 mm (YIT 11840^T) or 0.3–1.0 mm (YIT 11841^T) in diameter, entire, shiny and translucent with irregular or round margins. Growth of these strains in peptone/yeast extract/glucose broth was weak; small amounts of succinate and acetate were detected as end products of metabolism. However, growth of these strains was stimulated by xylan: large amounts of succinate (YIT 11840^T, 6.7 mM; YIT 11841^T, 18.3 mM) and acetate (YIT 11840^T, 7.0 mM; YIT 11841^T, 13.2 mM) were detected after incubation in peptone/yeast extract/xylan broth. Biochemical characteristics determined using the API Rapid ID 32A, API ZYM and API 20A systems are described in Table 1⇓.

The chemotaxonomic characteristics of strains YIT 11840^T and YIT 11841^T were very similar. The major cellular fatty acids were anteiso-C_15 : 0 (33.0 and 21.8 %, respectively, in strains YIT 11840^T and YIT 11841^T), iso-C_15 : 0 (17.0 and 17.3 %) and C_18 : 1ω9c (20.8 and 38.0 %). The following other fatty acids were detected: C_14 : 0 (5.4 and 2.6 %), C_15 : 0 (8.5 and 2.4 %), C_16 : 0 (2.2 and 2.4 %), C_18 : 2ω9,12c (1.1 and 1.2 %), C_18 : 1ω9t (0.8 and 1.8 %) and unknown fatty acids (11.3 and 12.5 %; peaks with equivalent chain-lengths of 13.6 and 17.3 in both strains and 16.3 in YIT 11840^T or 22.2 in YIT 11841^T). For both strains, the major respiratory quinones were MK-10(H₀) and MK-11(H₀) (Table 1⇑). Although the predominant menaquinones of strains YIT 11840^T and YIT 11841^T were similar to those of Prevotella melaninogenica and Prevotella veroralis, the overall pattern of the cellular fatty acid content and the other biological and biochemical characteristics differed from those of phylogenetically related, phenotypically similar Prevotella species of human oral and intestinal origin (Table 1⇑).

Approximately 1500 bp of the 16S rRNA genes of YIT 11840^T and YIT 11841^T were sequenced. Although YIT 11840^T and YIT 11841^T had very similar phenotypic characteristics, comparison of their partial 16S rRNA gene sequences revealed that these strains were only distantly related (93.8 % sequence similarity) and therefore (on the basis of the phylogenetic species concept) represented two different species. In database searches, the highest levels of similarity were found with respect to Prevotella species (YIT 11840^T, 88.5–84.9 %; YIT 11841^T, 88.6–84.6 %). A phylogenetic analysis of these and other related sequences was performed: it confirmed that strains YIT 11840^T and YIT 11841^T were phylogenetically most closely associated with members of the ‘Prevotellaceae’ (species belonging to the genera Prevotella, Hallela and Xylanibacter) but formed a separate cluster (Fig. 1⇓). In contrast, the 16S rRNA gene sequences most similar to those of YIT 11840^T (98.9–98.6 %) and YIT 11841^T (99.9–99.5 %) were derived from studies of uncultured human faecal or colonic bacteria (Eckburg et al., 2005; Gophna et al., 2006; Mai et al., 2006; Ley et al., 2006; Li et al., 2008; Kassinen et al., 2007) (Fig. 1⇓). These data indicate that these bacteria are common members of the human intestinal microbiota.

Species belonging to the genus Prevotella are common members of the human indigenous intestinal and oral microbiota (for reviews, see Rajilić-Stojanović et al., 2007; Sakamoto et al., 2005). As described above, both the phylogenetic and phenotypic characteristics of strains YIT 11840^T and YIT 11841^T were similar to those of Prevotella species (Willems & Collins, 1995). Characteristics that serve to differentiate strains YIT 11840^T and YIT 11841^T from Prevotella type strains of human oral and intestinal origin are shown in Table 1⇑. On the basis of their phylogenetic distinctiveness, therefore, strains 11840^T and 11841^T represent two novel species in a novel genus of the family ‘Prevotellaceae’, for which we propose the names Paraprevotella clara gen. nov., sp. nov. and Paraprevotella xylaniphila sp. nov., respectively.