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1 Microbe Division, Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
2 Institute of Biotechnology, Vietnamese Academy of Science and Technology, Hanoi, Vietnam
Correspondence
Mitsuo Sakamoto
sakamoto{at}jcm.riken.jp
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; Zhu et al., 2002). Using a special anaerobic culture technique, referred to as the ‘plate-in-bottle’ method (Mitsuoka et al., 1969), attempts were made to cultivate bacteria from chicken caecum. We found that many of the isolates obtained were members of the order ‘Bacteroidales’. From these isolates, we recently proposed three novel Bacteroides species: Bacteroides barnesiae, Bacteroides gallinarum and Bacteroides salanitronis (Lan et al., 2006). Analyses of the 16S rRNA gene sequences of two other isolates, which could not grow on medium containing 20 % bile, showed that these strains represented a new subline within the family Porphyromonadaceae. These strains shared 100 % 16S rRNA gene sequence similarity with each other and were related to Parabacteroides distasonis (86 % sequence similarity). In the present study, we have attempted to determine the taxonomic status of these strains.
Strains C46T and C47 used in the present study were isolated from chicken caecum (Lan et al., 2002) and were maintained on Eggerth Gagnon (EG) agar (Merck) supplemented with 5 % (v/v) horse blood for 2 days at 37 °C in an atmosphere comprising 100 % CO2. The ability to tolerate bile was tested using Bacteroides bile aesculin agar (Shah, 1992). Physiological reactions were determined with an API 20A anaerobe test kit, in duplicate, as recommended by the manufacturer (bioMérieux). The metabolic end products were prepared as described previously (Holdeman et al., 1977) and were analysed as described by Sakamoto et al. (2005). Fatty acid methyl esters were obtained from about 40 mg wet cells by saponification, methylation and extraction using the method of Miller (1982) but with minor modifications as described by Kuykendall et al. (1988). Cellular fatty acid profiles were determined using the MIDI microbial identification system (Microbial ID). Isoprenoid quinones were extracted (Komagata & Suzuki, 1987) and analysed (Sakamoto et al., 2002) as described previously. Biochemical reactions were determined with the Rapid ID 32A anaerobe identification kit, in duplicate, as recommended by the manufacturer (bioMérieux). Chromosomal DNA was isolated by means of previously described methods (Marmur, 1961; Saito & Miura, 1963), with some modifications. 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 NH4H2PO4 and acetonitrile (20 : 1, v/v). The 16S rRNA gene sequence was analysed as described previously (Sakamoto et al., 2002). Related sequences were aligned with the CLUSTAL W program (Thompson et al., 1994) and corrected by manual inspection. Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) after gaps and unknown bases had been eliminated. The phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap resampling analysis (Felsenstein, 1985) was performed to estimate the confidence of the tree topologies.
Strains C46T and C47 were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods. The growth of these isolates was inhibited on a medium containing 20 % bile. Cells on EG agar were 0.8–1.6x1.7–11 µm in size and occurred singly. Colonies on EG agar plates were 1–2 mm in diameter, grey to off-white–grey, circular, entire, slightly convex and smooth. The physiological and biochemical characteristics are given in the species description.
The major cellular fatty acids of the two novel strains were anteiso-C15 : 0 and iso-C15 : 0 (41–42 % and 16–18 %, respectively; Table 1). The amount of anteiso-C15 : 0 was relatively high in comparison with amounts reported for members of the genera Bacteroides (27–42 %), Parabacteroides (25–32 %) and Prevotella (7–37 %) (Sakamoto & Benno, 2006; Sakamoto et al., 2002, 2005). Although Proteiniphilum acetatigenes also possessed a large proportion of anteiso-C15 : 0 (46.21 %), the proportion of iso-C15 : 0 was low (3.77 %) (Chen & Dong, 2005).
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; Sakamoto et al., 2002). Among the strains isolated from chicken caecum, B. salanitronis BL78T also exhibited MK-11 (43 %) and MK-12 (43 %) as the main components (Lan et al., 2006).
The DNA G+C contents of strains C46T and C47 were in the range 52.0–52.2 mol%. These values are somewhat higher than those for the members of other genera in the family Porphyromonadaceae (Table 2).
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). In addition, the novel strains were related to Parabacteroides distasonis (86 % sequence similarity). For the phylogenetic analysis, 1340 bp (positions 61–1375; Escherichia coli numbering system) sequences of each species were used. 16S rRNA gene sequence analyses of the two novel strains showed that the strains represented a novel subline within the family Porphyromonadaceae (Fig. 1). Distantly related taxa included members of the genera Dysgonomonas (84.0–85.9 % sequence similarity), Paludibacter (84.4 % sequence similarity), Parabacteroides (84.4–86.0 % sequence similarity), Porphyromonas (81.4–84.2 % sequence similarity), Proteiniphilum (83.8 % sequence similarity) and Tannerella (84.1–85.1 % sequence similarity).
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On the basis of the above-mentioned findings and the results of the 16S rRNA gene sequence analysis, strains C46T and C47 represent a novel genus and species, for which the name Barnesiella viscericola gen. nov., sp. nov. is proposed. Differential characteristics of Barnesiella gen. nov. and some related taxa are shown in Table 2
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Description of Barnesiella gen. nov.
Barnesiella (Bar.ne.si.el'la. N.L. dim. fem. n. Barnesiella named after the British microbiologist Ella M. Barnes, who has contributed much to our knowledge of intestinal bacteriology and anaerobic bacteriology in general).
Cells are Gram-negative, obligately anaerobic, non-spore-forming, non-motile, rod-shaped and 0.8–1.6x1.7–11 µm in size. On EG agar plates, colonies are 1–2 mm in diameter, grey to off-white–grey, circular, entire, slightly convex and smooth. Saccharolytic. Growth is inhibited on a medium containing 20 % bile. Aesculin is hydrolysed. Indole is not produced. The DNA G+C content is 52 mol%. The genus Barnesiella is a member of the family Porphyromonadaceae. The type species is Barnesiella viscericola.
Description of Barnesiella viscericola sp. nov.
Barnesiella viscericola [vis.ce.ri'co.la. L. neut. n. viscus, visceris intestine; L. suff. n. -cola (from L. n. incola) inhabitant; N.L. fem. n. viscericola inhabitant of the intestine].
Exhibits the following characteristics in addition to those given in the description of the genus. Urease is not produced. Catalase is not produced. Gelatin is digested. Acid is produced from D-cellobiose, glucose, maltose, D-mannose and sucrose, but not from L-arabinose, glycerol, lactose, D-mannitol, D-melezitose, D-raffinose, L-rhamnose, salicin, D-sorbitol, D-trehalose or D-xylose. Positive reactions are obtained using the Rapid ID 32A tests for 
-galactosidase, 
-galactosidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, glutamic acid decarboxylase, -fucosidase, alkaline phosphatase, leucyl glycine arylamidase and alanine arylamidase. Raffinose is fermented. All of the other tests give negative results. The major end products are acetic acid and succinic acid; lower levels of other acids may be produced. Both non-hydroxylated and 3-hydroxylated long-chain fatty acids are present. The major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0. The predominant respiratory quinones are MK-11 (65–66 %) and MK-12 (21–24 %). MK-10 is present as a minor menaquinone (10–11 %). The DNA G+C content of the type strain is 52 mol%.
The type strain, C46T (=JCM 13660T=DSM 18177T), was isolated from chicken caecum. One additional strain, C47 (=JCM 13661), is included in this species.
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ACKNOWLEDGEMENTS |
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