Slackia equolifaciens sp. nov., a human intestinal bacterium capable of producing equol

Dietary isoflavones such as puerarin, daidzin and daidzein have recently aroused interest because of their potential health benefits (Anderson et al., 1999; Kurzer & Xu, 1997; Barnes, 1998; Setchell, 1998; Magee & Rowland, 2004). After ingestion from edible plants, these three compounds are converted to O-desmethylangolensin and equol as end products (Supplementary Fig. S1, available in IJSEM Online) (Heinonen et al., 1999, 2003). Equol, in particular, has been extensively studied as an effective phytoestrogen. However, only 30–50 % of individuals in the general population are capable of producing equol from daidzein (Rowland et al., 2000; Kelly et al., 1995). Because equol is produced exclusively by the intestinal bacterial metabolism of isoflavones, isolation and characterization of equol-producing bacterial strains has been attempted. Several strains have been reported to date (Maruo et al., 2008; Minamida et al., 2008).

The genus Slackia belongs to the family Coriobacteriaceae and its members are Gram-positive, non-motile, obligate anaerobes (Wade et al., 1999). Four species, Slackia faecicanis (Lawson et al., 2005), Slackia exigua, Slackia heliotrinireducens (Wade et al., 1999) and Slackia isoflavoniconvertens (Matthies et al., 2009), have been described. S. isoflavoniconvertens, which was isolated from human faeces, is able to transform daidzein to equol.

We isolated a strain capable of transforming daidzein to equol from the faeces of a healthy female and reported its metabolic characteristics (Jin et al., 2008). A bacterial suspension from fresh faeces was repeatedly cultured in 2 ml of general anaerobic medium (GAM) broth (Nissui) containing 0.1 mM daidzein as a substrate at 37 °C in an anaerobic incubator. A portion of the culture, possessing metabolic activity, was seeded on GAM agar plates and incubated anaerobically for 72 h at 37 °C. Colonies were repeatedly screened for their activity of transforming daidzein, to obtain a single daidzein-metabolizing bacterium (strain DZE^T). The strains used in the present study were maintained on GAM agar incubated for 3 days at 37 °C in an anaerobic jar (Hirayama) filled with CO₂. Physiological and biochemical reactions were determined with an API 20A anaerobe test kit and a Rapid ID 32A anaerobe identification kit, respectively, as recommended by the manufacturer (bioMérieux). All tests were performed in duplicate. The DNA G+C content was determined by using the HPLC method of Tamaoka & Komagata (1984). The elution solvent was a mixture of 0.02 M (NH₄)H₂PO₄ and acetonitrile (20 : 1, v/v). Fatty acid methyl esters were obtained from approximately 40 mg of wet cells by saponification, methylation and extraction using minor modifications (Kuykendall et al., 1988) of the method of Miller (1982). Cells were cultured anaerobically on GAM agar (0.5 % arginine-hydrochloride) for 96 h at 37 °C. The 16S rRNA gene sequence was analysed as described previously (Sakamoto et al., 2002). Almost 1500 bases of 16S rRNA gene sequence for strain DZE^T were amplified by PCR with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) using a Biometra Thermocycler Tgradient (Biometra). 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 (Applied Biosystems) and ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Phylogenetic relatives of the bacterium were determined by performing database searches, and sequences of related species were retrieved from the DDBJ, EMBL and GenBank nucleotide sequence databases. Sequences were aligned by using clustal x (version 2.0) (Thompson et al., 1997) and a phylogenetic tree was reconstructed according to the neighbour-joining method (Saitou & Nei, 1987). Minimum-evolution and maximum-parsimony phylogenetic trees were inferred by using the software package mega version 4.0 (Tamura et al., 2007). Equol production was examined by incubation of the bacteria with daidzein. GAM broth (2 ml) containing daidzein (final concentration of 0.1 mM) was incubated with a bacterial suspension (100 μl) at 37 °C for 48 h under anaerobic conditions. A 100 μl aliquot was taken out, extracted with 30 μl of ethyl acetate and subjected to TLC, which was carried out on silica gel pre-coated 60 F₂₅₄ plates (0.25 mm, Merck). The products were detected under a UV lamp or by exposure to I₂ vapour.

Analysis of 16S rRNA gene sequences revealed that the novel strain was a member of the family Coriobacteriaceae. Strain DZE^T displayed highest sequence similarity with species of the genus Slackia [S. faecicanis (92.8 %), S. exigua (91.0 %), S. heliotrinireducens (91.1 %) and S. isoflavoniconvertens (90.6 %)] (Fig. 1⇓). However, the strain exhibited substantially lower similarities with other members of the family Coriobacteriaceae [species of the genus Eggerthella (89.1–89.7 %), Denitrobacterium detoxificans (88.8 %) and Asaccharobacter celatus (91.1 %)]. Moreover, strain DZE^T showed 91.4 % similarity with Adlercreutzia equolifaciens, which also converts daidzein to equol. Although it is not possible to distinguish species on the basis of 16S rRNA sequence similarities alone, it is clear that the greater than 7 % gene sequence divergence observed between strain DZE^T and the four currently recognized species of the genus Slackia is consistent with separate species status. It is now generally accepted that organisms showing 3 % or greater 16S rRNA gene sequence divergence are not members of the same species (Lawson et al., 2005). Physiological and biochemical reactions of strain DZE^T were studied with S. faecicanis JCM 14555^T and S. exigua JCM 11022^T as references. The phenotypic and biochemical characteristics that are useful in differentiating strain DZE^T from other members of the genus Slackia are shown in Table 1⇓. The characteristics of strain DZE^T were similar to those of S. exigua. However, the two reference strains, S. faecicanis JCM 14555^T and S. exigua JCM 11022^T, did not produce equol from daidzein. The long-chain cellular fatty acids of strain DZE^T included C_12 : 0 (2.9 %), C_14 : 0 (15.1 %), C_16 : 0 (2.7 %) and C_18 : 1ω9c (29.5 %) (Supplementary Table S1). Based upon phylogenetic and phenotypic findings, we propose the name Slackia equolifaciens sp. nov. for this strain.