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Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
Correspondence
Mitsuo Sakamoto
sakamoto{at}jcm.riken.jp
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60 % DNA–DNA relatedness). The DNA G+C content of the strain is 47.6 mol%. On the basis of these data, strain M-165T represents a novel species of the genus Parabacteroides, for which the name Parabacteroides johnsonii sp. nov. is proposed. The type strain is M-165T (=JCM 13406T=DSM 18315T).
The cellular fatty acid compositions of strain M-165T and closely related species are available in a supplementary table in IJSEM Online.
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). Consequently, some species that were not included in Bacteroides sensu stricto (Shah & Collins, 1989) have been transferred to novel genera, such as Alistipes (Rautio et al., 2003), Dialister (Moore & Moore, 1994), Dichelobacter (Dewhirst et al., 1990) and Tannerella (Sakamoto et al., 2002). More recently, the misclassified species Bacteroides distasonis (Eggerth & Gagnon, 1933), Bacteroides goldsteinii (Song et al., 2005) and Bacteroides merdae (Johnson et al., 1986) were ultimately reclassified as Parabacteroides distasonis, Parabacteroides goldsteinii and Parabacteroides merdae (Sakamoto & Benno, 2006). In the process of collecting P. merdae JCM 13405 (Sakamoto & Benno, 2006), we found a P. merdae-like strain isolated from human faeces that was not identified as P. merdae by a PCR technique using species-specific primers (Liu et al., 2003). The present study was designed to determine the taxonomic status of this strain.
The strains used in the present study were maintained for 2 days at 37 °C under CO2 on Eggerth Gagnon (EG) agar (Merck) supplemented with 5 % (v/v) horse blood. Strain M-165T was isolated from human faeces. Bacteroides bile-aesculin agar (Shah, 1992) was used to check whether the growth of the isolate was inhibited on this medium. A PCR technique involving species-specific primers (Liu et al., 2003) was used to identify the P. merdae-like strain, as described previously (Sakamoto & Benno, 2006). 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 by Holdeman et al. (1977) and were analysed as described previously (Sakamoto et al., 2005). Fatty acid methyl esters were obtained from wet cells (approx. 40 mg) by saponification, methylation and extraction using the method of Miller (1982) with minor modifications (Kuykendall et al., 1988). Cellular fatty acid profiles were determined by using the MIDI microbial identification system (Microbial ID). Isoprenoid quinones were extracted as described by Komagata & Suzuki (1987) and were analysed as described previously (Sakamoto et al., 2002). 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 using methods described previously (Marmur, 1961; Saito & Miura, 1963), but 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). DNA–DNA hybridization experiments were carried out in microplate wells as described by Ezaki et al. (1989). Hybridization was performed at 44 °C for 16 h. 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). A bootstrap resampling analysis (Felsenstein, 1985) was performed to estimate the confidence of the tree topologies.
Strain M-165T was obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative and rod-shaped; it was not identified as P. merdae by the PCR. Growth of the isolate was not inhibited on medium containing 20 % bile. Cells on EG agar were 0.8 µm by 1.7–2.5 µm in size and occurred singly. On EG agar plates, colonies were 1–2 mm in diameter, grey to off-white in colour, circular, entire, slightly convex and smooth. The phenotypic characteristics are given in the species description. The isolate could be differentiated from P. merdae by the ability of the former to ferment L-arabinose and L-rhamnose in the API 20A panel. Catalase production was a key characteristic for differentiating the isolate (and P. distasonis) from P. merdae. Biochemical characteristics of the isolate determined using the Rapid ID 32A kit were similar to those of P. merdae (Sakamoto & Benno, 2006); only pyroglutamic acid arylamidase activity was different from P. merdae.
The major cellular fatty acids of the isolate were anteiso-15 : 0 and iso-17 : 0 3-OH (32 and 21 %, respectively). The amount of 18 : 1
9c (9 %) present was slightly lower than that found in Parabacteroides species (14–16 %), while the amount of 15 : 0 (5.5 %) present was slightly higher than that found in Parabacteroides species (1.3–3.1 %) (see Supplementary Table S1 available in IJSEM Online).
The major menaquinones of the isolate were MK-9 (54 %) and MK-10 (39 %). A small amount of MK-8 (5 %) was also present. These data are in agreement with the description of the genus Parabacteroides (Sakamoto & Benno, 2006). The menaquinone compositions of strain M-165T and P. merdae JCM 9497T were almost the same.
Approximately 1500 bases of the 16S rRNA gene sequence were determined for the isolate. For the phylogenetic analysis, 1340 bp (positions 61–1375; Escherichia coli numbering system) sequences of each strain were used. The 16S rRNA gene sequence analysis showed that strain M-165T represented a species within the genus Parabacteroides (Fig. 1), being closely related to P. merdae (similarities of 97.9 and 98 % for JCM 9497T and JCM 13405, respectively).
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). The levels of DNA–DNA relatedness observed serve to distinguish strain M-165T genomically from P. merdae JCM 9497T and JCM 13405 (relatively high DNA–DNA relatedness, 60 %; Table 1).
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Cells are obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods (0.8x1.7–2.5 µm). On EG agar plates, colonies are 1–2 mm in diameter, grey to off-white in colour, circular, entire, slightly convex and smooth. Indole and urease are not produced. Catalase is produced. Aesculin is hydrolysed. Gelatin is not digested. Grows on medium containing 20 % bile. Acid is produced from L-arabinose, glucose, lactose, maltose, D-mannose, D-raffinose, L-rhamnose, sucrose, D-trehalose and D-xylose but not from D-cellobiose, glycerol, D-mannitol, D-melezitose, salicin or D-sorbitol. Positive Rapid ID 32A reactions are obtained for 
-galactosidase, 
-galactosidase, -glucosidase, -arabinosidase, -glucuronidase, N-acetyl--glucosaminidase, glutamic acid decarboxylase, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase and serine arylamidase. Mannose and raffinose are fermented. All of the other tests (for urease, arginine dihydrolase, 6-phospho--galactosidase, -glucosidase, -fucosidase, nitrate reduction, indole production, proline arylamidase and pyroglutamic acid arylamidase) are negative. The major end products from PYG broth cultures (1 % peptone, 1 % yeast extract, 1 % glucose) are succinic and acetic acids; small amounts of isovaleric acid and propionic acid are also produced. The major cellular fatty acids are anteiso-15 : 0 and iso-17 : 0 3-OH. The predominant respiratory quinones are menaquinones MK-9 and MK-10. The DNA G+C content of the type strain is 47.6 mol%.
The type strain, M-165T (=JCM 13406T=DSM 18315T), was isolated from human faeces.
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Sakamoto, M., Suzuki, M., Umeda, M., Ishikawa, I. & Benno, Y. (2002). Reclassification of Bacteroides forsythus (Tanner et al. 1986) as Tannerella forsythensis corrig., gen. nov., comb. nov. Int J Syst Evol Microbiol 52, 841–849.[Abstract]
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