Anaerostipes butyraticus sp. nov., an anaerobic, butyrate-producing bacterium from Clostridium cluster XIVa isolated from broiler chicken caecal content, and emended description of the genus Anaerostipes

It has been well documented that butyric acid, which is produced in the intestinal lumen by microbial fermentation of dietary carbohydrates (Topping & Clifton, 2001), exerts a wide variety of effects on intestinal function. Indeed, butyrate enhances proliferation of normal intestinal epithelial cells, has anti-inflammatory effects and reinforces the colonic defence barrier by increasing the production of mucins, antimicrobial peptides and tight junction proteins (Mariadason et al., 1997; Barcelo et al., 2000; Schauber et al., 2003; Bordin et al., 2004; Peng et al., 2007). Due to these characteristics, butyrate, and thus certain butyrate-producing bacteria, is considered to have a protective role against several colonic diseases (Raz et al., 2007; Sokol et al., 2008). Many studies on the caecal bacterial community of chickens have shown the predominance of sequences representing bacteria of the phylum Firmicutes (Selim, 2006). Although butyrate-producing bacteria are known to belong to several genera within this phylum, there is limited information about butyrate-producing strains in the chicken gut. During a study on the phylogenetic relationship of butyrate-producing bacteria from the chicken caecum (data not shown), we observed that all isolates belonged to the low-G+C-content Gram-positive bacteria of clostridial cluster XVI, cluster XIV subclusters a and b and cluster IV (Collins et al., 1994). We previously characterized an organism belonging to cluster IV, Butyricicoccus pullicaecorum (Eeckhaut et al., 2008), and showed that it could synthesize considerable amounts of butyrate under anaerobic conditions at pH 6, which is the ambient pH in the chicken caecum (van der Wielen et al., 2000). In the present study, a novel butyrate-producing organism belonging to cluster XIVa is described.

Isolates 4-7, 16-7, 35-7^T and 50-7 were obtained from the caecum of a 4-week-old healthy chicken as follows. Caecal content (1 g) was diluted in 9 ml M2GSC medium (Miyazaki et al., 1997) at pH 6 under anaerobic conditions (Barcenilla et al., 2000). Tenfold dilutions were spread onto agar plates containing M2GSC medium with 1.5 % agar. Plates were incubated for 48 h in an anaerobic chamber (Ruskinn Technology) with 84 % N₂, 8 % CO₂ and 8 % H₂ at 41 °C, which is the temperature of the chicken caecum (van der Wielen et al., 2000). Sixty-one pure colonies were grown overnight in M2GSC broth culture and tested for the production of short-chain fatty acids using GLC (GC14; Shimadzu) as described by Van Nevel & Demeyer (1977). Isolates 4-7, 16-7, 35-7^T and 50-7 were studied in more detail as their supernatants contained high concentrations of butyric acid (approximately 15 mM). Analysis of the supernatants (Table 1⇓) revealed that, besides the production of butyrate, the isolates consumed small amounts of acetate and propionate. H₂ and CO₂ production were analysed by sampling 1 ml of the gas phase with a gas-tight syringe (FM Dual Column GC 700; Avondale) (Van Nevel et al., 1970).

DNA was extracted via an alkaline lysis procedure and used for repetitive-element-primed PCR with the (GTG)₅ primer and randomly amplified polymorphic DNA typing using two distinct primers (5′-TGCGCGCGGG-3′ and 5′-AGCGGGCCAA-3′) as described previously (Versalovic et al., 1994; Coenye et al., 2002). The isolates yielded identical or almost-identical DNA fingerprints in all three assays (data not shown), suggesting that all four originated from a single parent strain that had colonized the intestinal tract of the sampled chicken.

Approximately 1400 bases of the 16S rRNA gene of all four isolates were sequenced after amplification using the universal eubacterial primers fD1 and rD1 (Weisburg et al., 1991). The purified amplicons were sequenced using the BigDye Terminator sequencing kit with primers pD, Gamma*, 3 and O* on an ABI PRISM 310 Genetic Analyzer (Coenye et al., 1999). The sequences obtained were compared with entries in public databases and aligned with reference sequences using the clustal w program (Thompson et al., 1994). A neighbour-joining tree (Saitou & Nei, 1987) was constructed via the phylip package (Felsenstein, 1989) using dnadist for distance analysis (Kimura, 1980) and bootstrap values from 100 replicates.

A 16S rRNA gene sequence tree showing the phylogenetic relationships of the four isolates is depicted in Fig. 1⇓ and shows that the isolates are related to the genus Anaerostipes within the phylum Firmicutes, in the lineage Clostridia, Clostridiales, Lachnospiraceae. The isolates shared ≥99.3 % sequence similarity with each other. Comparison of the 16S rRNA gene sequences with entries in public databases revealed the highest similarity with sequences of uncultured bacteria from human faeces (GenBank accession numbers DQ794058 and DQ793763; Ley et al., 2006), with sequence similarity values of 97.9 and 96.3 %, respectively. The cultured bacteria most closely related to strain 35-7^T were two butyrate-producing bacteria from the human gut (strains SS2/1 and SSC/2; Louis et al., 2004), 94.3 % sequence similarity for both, and an unclassified Anaerostipes strain isolated from the rat gut (IE4; Gourgue-Jeannot et al., 2006) with 94.3 % sequence similarity.

After growth on solid M2GSC medium at pH 6 and 41 °C, cells of strain 35-7^T were Gram-positive-staining, non-motile, anaerobic, large rods, 5–15 μm long and approximately 0.7 μm in diameter. Cells occurred in filaments containing oval spores which were visualized by staining with malachite green and light microscopy (Supplementary Fig. S1, available in IJSEM Online). Colonies after overnight incubation were 1.5–2.5 mm in diameter, circular, smooth, convex, white and shiny.

Growth of strain 35-7^T at ten different temperatures (from 29 to 49 °C) and at nine different pH values (ranging from pH 4.5 to 8.5) was determined in brain heart infusion (BHI) broth supplemented with 1 mg cysteine hydrochloride ml⁻¹. The broth was adjusted to the different pH values, or adjusted to pH 6 for determination of the optimal temperature, and inoculated with an overnight-grown culture (500-fold diluted) of strain 35-7^T. Growth rates were determined in triplicate by measuring the increase in OD₆₀₀ over time with a spectrophotometer. Growth occurred at pH 5.5–8 (optimum pH 6) and 35–47 °C (optimum 37–41 °C).

The biochemical properties of the novel isolates were characterized using API 20A, API Rapid ID 32A and API ZYM systems (bioMérieux), according to the manufacturer's instructions except that the galleries were incubated at 41 °C instead of 36±2 °C. All four isolates produced identical profiles. Aesculin was hydrolysed and acid was produced from cellobiose, glucose, maltose, mannitol, mannose, raffinose, salicin, sorbitol, sucrose and trehalose. Urease activity was not detected and hydrolysis of gelatin and fermentation of arabinose, glycerol, lactose, melezitose, raffinose, rhamnose and xylose were absent. Using the API ZYM system, positive reactions were obtained only for esterase (C4), acid phosphatase and α-glucosidase, with all other tests being negative. With the Rapid ID32A system, activity was detected for phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, glycine arylamidase and histidine arylamidase.

The DNA G+C content of strain 35-7^T was determined by using a Waters Breeze HPLC system and an Xbridge Shield RP18 column maintained at 37 °C, yielding a value of 44 mol%.

In conclusion, in the course of a study on the butyrate-producing caecal microbiota of healthy broiler chickens, four bacterial isolates represented a distinct taxon, with Anaerostipes caccae L1-92^T as the nearest neighbour with a validly published name. Although there is about 6 % 16S rRNA gene sequence divergence between strain 35-7^T and A. caccae L1-92^T, they have similar G+C contents (44 and 45.5–46.0 mol%, respectively; Schwiertz et al., 2002). Strain 35-7^T is also phenotypically compatible with the genus Anaerostipes in terms of colony morphology and end products of glucose metabolism: production of butyrate and utilization of acetate and lactate are observed when A. caccae and strain 35-7^T are grown in PYG medium supplemented with dl-lactate and acetate. Strain 35-7^T can be distinguished from A. caccae by the production of spores and, although most biochemical characteristics of strain 35-7^T resemble those of A. caccae, the two can also be differentiated by acidification of cellobiose, raffinose and trehalose, activity of esterase (C4), production of α-glucosidase, arginine dihydrolase, α-galactosidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, glycine arylamidase and histidine arylamidase, the reduction of nitrate and the production of indole.

Therefore, we conclude that it is most appropriate to assign strain 35-7^T to a novel species in the genus Anaerostipes, for which the name Anaerostipes butyraticus sp. nov. is proposed.