Butyricicoccus pullicaecorum gen. nov., sp. nov., an anaerobic, butyrate-producing bacterium isolated from the caecal content of a broiler chicken

In broiler chickens, the gastrointestinal tract harbours numerous bacterial species, of which only 10 % have been characterized: 35 % represent previously unknown species within a known bacterial genus and the remaining 55 % represent bacteria for which even genus allocation is not possible (Apajalahti et al., 2004). A study of the microbiota of chicken caeca conducted by Lan et al. (2002) showed the predominance (94 %) of sequences from within the phylum Firmicutes, the most abundant sequences belonging to the Clostridium coccoides subgroup and the Clostridium leptum subgroup (Collins et al., 1994). Both subgroups contain numerous members that are known to produce butyrate as a product of fermentation (Pryde et al., 2002). There is increased interest in butyrate-producing strains in the context of veterinary medicine, because butyrate reduces invasion and colonization by Salmonella and stimulates intestinal epithelial cell growth (Van Immerseel et al., 2005; Kien et al., 2007). To our knowledge, butyrate-producing bacteria belonging to the phylum Firmicutes have not been isolated from the chicken gastrointestinal tract to date.

Bacteria were obtained from the caecal content of a 4-week-old broiler chicken that had been given a wheat-based, commercial, broiler feed supplemented with 2 % fructo-oligosaccharides (Orafti) from week 3. The chicken was euthanized and a sample of the caecal content was homogenized in anaerobic M2GSC medium (Miyazaki et al., 1997) as described by Barcenilla et al. (2000). Tenfold serial dilutions of this suspension were made and, from each dilution, 0.3 ml was spread onto agar plates containing M2GSC medium with 1.5 % agar. The plates were incubated at 42 °C, which is similar to the body temperature of poultry (Richards, 1970), for 48 h and then 58 single colonies were transferred to tubes containing M2GSC broth (10 ml). All manipulations were performed in an anaerobic workstation (Ruskinn Technology). Short-chain fatty acid concentrations in overnight cultures were analysed using GLC (GC14; Shimadzu) as described by Van Nevel & Demeyer (1977). Several butyrate-producing cultures were obtained. The supernatants of five of these bacterial cultures, i.e. ones obtained using isolates 11-3, 25-3^T, 44-3, 49-3 and 54-3, contained more than 13 mM butyric acid. These isolates were characterized in detail.

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′) were performed as described previously (Versalovic et al., 1994; Coenye et al., 2002) after DNA extraction with an alkaline lysis procedure. The isolates yielded identical, or virtually identical, DNA fingerprints in all three assays (see Supplementary Figs S1 and S2, available in IJSEM Online), suggesting that all five originate from a single strain that colonized the intestinal tract of the sampled chicken.

To elucidate the phylogenetic position of the five isolates, almost-complete 16S rRNA gene sequences (corresponding to positions 8–1541 in the Escherichia coli numbering system) were determined for each isolate by 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 closest match to the deduced sequences was found using the fasta program (Pearson & Lipman, 1988). The nucleotide sequences were aligned with reference 16S rRNA gene sequences by using the clustal w program (Thompson et al., 1994) and a phylogenetic tree was constructed using the neighbour-joining method (Saitou & Nei, 1987) via the phylip package (Felsenstein, 1989), using dnadist for distance analysis (Kimura, 1980) (Fig. 1⇓).

Fig. 1⇑ shows the phylogenetic position of the five isolates among their closest relatives, i.e. members of the C. leptum subgroup. The isolates shared virtually identical (98–99 %) 16S rRNA gene sequences and were most closely related to a butyrate-producing bacterium from the human gut (Barcenilla et al., 2000) (94 % sequence similarity) and to Eubacterium desmolans ATCC 43058^T (Morris et al., 1986) (93 %).

Determination of the genomic DNA G+C content of isolate 25-3^T was performed by using a Waters Breeze HPLC system and an XBridge Shield RP18 column maintained at 37 °C. This gave a value of 54.5 mol%.

All five isolates comprised Gram-positive coccoid cells that were 1.2–1.5 μm long. A scanning electron micrograph of isolate 25-3^T is shown in Supplementary Fig. S3. Following overnight incubation on M2GSC medium at 42 °C, colonies were white in colour and 1–2 mm in diameter.

Characterization of the biochemical properties of the strains was done using the commercially available API 20A microtest system (bioMérieux) according to the manufacturer's instructions, except that incubation was performed at 42 °C instead of 36±2 °C. All five isolates showed hydrolysis of aesculin and fermentation of glucose, salicin, sucrose, maltose, xylose, cellobiose, mannose and trehalose. No urease activity was detected and hydrolysis of gelatin and fermentation of mannitol, lactose, arabinose, glycerol, melezitose, raffinose, sorbitol and rhamnose were absent. The ability of the isolates to degrade starch was determined by culturing them on M2GSC agar, which contains 0.2 % starch. After overnight anaerobic incubation at 42 °C, the plates were flooded with a commercial Lugol solution (Sigma). Clear zones around areas of cell growth indicated hydrolysis of starch.

When plates inoculated with isolate 25-3^T were exposed to air for more than 3.5 h, subsequent anaerobic growth seemed to be prevented. The isolates produced more than 13 mM butyrate and utilized acetate; three out of the five isolates consumed small amounts of propionate. Four strains were also tested for H₂ and CO₂ production. For this purpose, 1 ml gas phase was sampled with a gas-tight syringe and analysed for H₂ and CO₂ (FM Dual Column GC 700; Avondale) (Van Nevel et al., 1970) (Table 1⇓).

In conclusion, in the course of a study on the butyrate-producing microbiota from the caecal content of a 4-week-old broiler chicken, five isolates were obtained that produced large amounts of butyrate in overnight cultures of M2GSC broth. The five isolates were virtually indistinguishable in biochemical and genetic terms, suggesting that they were derived from a single bacterial clone colonizing this habitat. A phylogenetic analysis demonstrated that the five isolates represented a unique lineage within the phylum Firmicutes, with E. desmolans ATCC 43058^T as the closest phylogenetic neighbour. These data indicate that the five novel isolates represent a single novel species within a novel genus, for which the name Butyricicoccus pullicaecorum gen. nov., sp. nov. is proposed.

The closest phylogenetic neighbour, E. desmolans, can be readily distinguished from B. pullicaecorum sp. nov. on the basis of biochemical criteria. In contrast to B. pullicaecorum, E. desmolans does not ferment cellobiose, aesculin, glucose, maltose, mannose or trehalose. Reclassification of E. desmolans as a member of the genus Butyricicoccus cannot be considered, as there is only 93 % 16S rRNA gene sequence similarity between the two type strains and their G+C contents are very different (35 mol% for E. desmolans ATCC 43058^T versus 54.5 mol% for B. pullicaecorum 23-5^T).