Characterization of Phascolarctobacterium succinatutens sp. nov., an asaccharolytic, succinate-utilizing bacterium isolated from human feces

Abstract

Isolation, cultivation, and characterization of the intestinal microorganisms are important for understanding the comprehensive physiology of the human gastrointestinal (GI) tract microbiota. Here, we isolated two novel bacterial strains, YIT 12067(T) and YIT 12068, from the feces of healthy human adults. Phylogenetic analysis indicated that they belonged to the same species and were most closely related to Phascolarctobacterium faecium ACM 3679(T), with 91.4% to 91.5% 16S rRNA gene sequence similarities, respectively. Substrate availability tests revealed that the isolates used only succinate; they did not ferment any other short-chain fatty acids or carbohydrates tested. When these strains were cocultured with the xylan-utilizing and succinate-producing bacterium Paraprevotella xylaniphila YIT 11841(T), in medium supplemented with xylan but not succinate, their cell numbers became 2 to 3 orders of magnitude higher than those of the monoculture; succinate became undetectable, and propionate was formed. Database analysis revealed that over 200 uncultured bacterial clones from the feces of humans and other mammals showed high sequence identity (>98.7%) to YIT 12067(T). Real-time PCR analysis also revealed that YIT 12067(T)-like bacteria were present in 21% of human fecal samples, at an average level of 3.34 × 10(8) cells/g feces. These results indicate that YIT 12067(T)-like bacteria are distributed broadly in the GI tract as subdominant members that may adapt to the intestinal environment by specializing to utilize the succinate generated by other bacterial species. The phylogenetic and physiological properties of YIT 12067(T) and YIT 12068 suggest that these strains represent a novel species, which we have designated Phascolarctobacterium succinatutens sp. nov.

Fig 1

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strains YIT 12067^T and YIT 12068 among related taxa. Bootstrap values (>50%) are given at the branch nodes. Filled circles indicate that the corresponding nodes were also recovered in the tree generated with the maximum-parsimony and maximum-likelihood algorithms. Bar, 0.1 substitutions per nucleotide position.

Fig 2

Phase-contrast (A) and scanning electron (B) micrographs of strain YIT 12067^T grown in PYS broth for 2 days. Bars, 5 μm.

Fig 3

Coculture of the succinate-producing bacterium P. xylaniphila YIT 11841^T and strain YIT 12067^T or YIT 12068 after 72 h of growth in PY medium supplemented with 1% birchwood xylan. (A) Concentrations of succinate, acetate, and propionate, as determined by using HPLC. Triplicate experiments were performed. (B) CFU of strains YIT 12067^T and YIT 12068 and P. xylaniphila YIT 11841^T. Diluted inocula were plated on the appropriate selective agar plates. Strains YIT 12067^T and YIT 12068 were enumerated on PYS agar plates, and P. xylaniphila YIT 11841^T was enumerated on PY agar plates with 1% glucose and 25 μg/ml of streptomycin. Plates were incubated for 48 h at 37°C under anaerobic conditions. Triplicate experiments were performed. The initial cell numbers of strains YIT 12067^Tand 12068 and of P. xylaniphila YIT 11841^T were 3.85 ×10³, 8.44 × 10⁴, and 7.67 × 10⁵ CFU ml⁻¹, respectively.