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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
| ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of P. distasonis strains JCM 5825T, JCM 13400, JCM 13401, JCM 13402, JCM 13403 and JCM 13404 and P. merdae strains JCM 9497T and JCM 13405 are AB238922AB238929, respectively.
The cellular fatty acid content and biochemical characteristics of Parabacteroides distasonis gen. nov., comb. nov. and Parabacteroides merdae comb. nov. and a phylogenetic tree based on sequences of 16S23S rRNA gene ITS regions are available as supplementary material in IJSEM Online.
| MAIN TEXT |
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The strains used in this study were maintained on Eggerth Gagnon (EG) agar (Merck), supplemented with 5 % (v/v) horse blood, for 2 days at 37 °C in an atmosphere of 100 % CO2. Strains JCM 13400, JCM 13401, JCM 13402, JCM 13403, JCM 13404 and JCM 13405 were isolated from human faeces. Bacteroides bile aesculin agar (Shah, 1992
) was used to check whether the growth of the isolates was inhibited on this medium. A multiplex-PCR technique using species-specific primers (Liu et al., 2003
) was used to identify B. distasonis and B. merdae. Physiological reactions were determined in duplicate with an API 20A anaerobe test kit, as recommended by the manufacturer (bioMérieux). Fatty acid methyl esters (FAMEs) were obtained from about 40 mg wet cells by saponification, methylation and extraction, using minor modifications (Kuykendall et al., 1988
) of the method of Miller (1982)
. 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 in duplicate with a Rapid ID 32A anaerobe identification kit, as recommended by the manufacturer (bioMérieux). The 16S rRNA gene was analysed as described previously (Sakamoto et al., 2002
). Related sequences were aligned using 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. A 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 tree topologies.
Cells of strains JCM 1340013405 were obligately anaerobic, non-spore-forming, non-motile, Gram-negative rods. Strains JCM 1340013404 were identified as representing B. distasonis by using a multiplex-PCR assay; likewise, strain JCM 13405 was identified as representing B. merdae. The growth of these six clinical isolates and B. distasonis JCM 5825T, B. goldsteinii JCM 13446T and B. merdae JCM 9497T was not inhibited on medium containing 20 % bile. Cells on EG agar were 0.81.6x1.212 µm in size and occurred singly. Colonies on EG agar plates were 12 mm in diameter, grey to off-whitegrey, circular, entire, slightly convex and smooth. Phenotypic characteristics are given in the species descriptions.
The cellular fatty acid composition of Bacteroides species has been determined (Mayberry et al., 1982
; Miyagawa et al., 1979
; Shah & Collins, 1980
) and reviewed for the classification of the genus Bacteroides (Shah & Collins, 1983
). In this study, the cellular fatty acid compositions of the clinical isolates and B. distasonis JCM 5825T and B. merdae JCM 9497T were almost the same. The major cellular fatty acids of the above strains were anteiso-C15 : 0 and iso 3-OH-C17 : 0 (2532 and 2326 %, respectively). A significant amount of C18 : 1
9c (1416 %) was also present (Supplementary Table S1 in IJSEM Online). These findings are in agreement with that reported for B. goldsteinii, except for anteiso 3-OH-C17 : 0 (Song et al., 2005
). Song et al. (2005)
reported that the ratio iso 3-OH-C17 : 0/anteiso 3-OH-C17 : 0 in whole-cell methanolysates of B. goldsteinii was lower than that for B. merdae (ratio ranging from 1.4 to 2.2 for B. goldsteinii and 6.1 to 8.3 for B. merdae). In this study, the ratio iso 3-OH-C17 : 0/anteiso 3-OH-C17 : 0 in whole-cell methanolysates of B. distasonis and B. merdae was 5.79.4 and 5.27.6, respectively.
The major menaquinones of the clinical isolates and B. distasonis JCM 5825T, B. goldsteinii JCM 13446T and B. merdae JCM 9497T were MK-9 and MK-10 (Table 1
). The menaquinone compositions of B. distasonis JCM 5825T and B. goldsteinii JCM 13446T were almost the same. On the other hand, the major menaquinones of other Bacteroides species were MK-10 and MK-11, except for Bacteroides vulgatus JCM 5826T, which contained a small amount of MK-11 (Sakamoto et al., 2002
).
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Approximately 1500 bases of the 16S rRNA gene sequence were determined for the clinical isolates and B. distasonis JCM 5825T and B. merdae JCM 9497T. Strains JCM 1340013404 were closely related to B. distasonis JCM 5825T, with about 99 % similarity (>98.7 %). In addition, strain JCM 13405 was closely related to B. merdae JCM 9497T, with 99.9 % similarity. For the phylogenetic analysis, 1340 bp (positions 611375; Escherichia coli numbering system) sequences of each strain were used. 16S rRNA gene sequence analysis showed that B. distasonis JCM 5825T, B. goldsteinii JCM 13446T and B. merdae JCM 9497T were not species within the genus Bacteroides (Fig. 1
). These three species were phylogenetically closely related to each other (>92.3 %) and were related to T. forsythensis with about 90 % similarity. Other remotely related taxa included the genera Bacteroides (83.588.8 % sequence similarity), Dysgonomonas (85.989.4 %), Paludibacter (86.988.5 %; Ueki et al., 2006
), Porphyromonas (82.286.9 %), Prevotella (77.281.9 %) and Proteiniphilum (85.987.3 %; Chen & Dong, 2005
). In addition, a preliminary analysis of the 16S23S rRNA gene internal transcribed spacer (ITS) regions also showed that B. distasonis and B. merdae were phylogenetically distinct from species of the genus Bacteroides and were related to T. forsythensis (Supplementary Fig. S1 in IJSEM Online). The ITS regions have been used as an important tool for classification and differentiation of bacterial species (Conrads et al., 2005
).
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Based on the above-mentioned findings and the 16S rRNA gene sequence analysis, we propose a novel genus, Parabacteroides gen. nov. Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae are reclassified as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov., respectively. Differential characteristics of Parabacteroides gen. nov. and some related taxa are shown in Table 2
.
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| Description of Parabacteroides gen. nov. |
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Cells are Gram-negative, obligately anaerobic, non-spore-forming, non-motile and rod-shaped, and 0.81.6x1.212 µm in size. Colonies on EG agar plates are 12 mm in diameter, grey to off-whitegrey, circular, entire, slightly convex and smooth. Saccharolytic. Major end-products are acetic and succinic acids; lower levels of other acids may be produced. Grow on medium containing 20 % bile. Aesculin is hydrolysed. Indole is not produced. Glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malate dehydrogenase and glutamate dehydrogenase are present.
-Fucosidase is negative. The principal respiratory quinones are menaquinones MK-9 and MK-10. Both non-hydroxylated and 3-hydroxylated long-chain fatty acids are present. The non-hydroxylated acids are predominantly of the saturated straight-chain and anteiso-methyl branched-chain types. The G+C content is 4346 mol%. Member of the Bacteroides subgroup of the phylum Bacteroidetes. The type species is Parabacteroides distasonis.
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Description of Parabacteroides distasonis (Eggerth and Gagnon 1933 |
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Basonym: Bacteroides distasonis Eggerth and Gagnon 1933
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The description of Parabacteroides distasonis is as those given by Eggerth & Gagnon (1933)
and Holdeman et al. (1977
, 1984)
. Urease is not produced. Catalase is produced. Gelatin is not liquefied. Acid is produced from D-cellobiose, glucose, lactose, D-mannose, D-melezitose, D-raffinose, L-rhamnose, salicin, sucrose, D-trehalose and D-xylose, but not from L-arabinose, glycerol, D-mannitol or D-sorbitol. Positive reactions are obtained using Rapid ID 32A for
-galactosidase,
-galactosidase,
-glucosidase,
-glucosidase, 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 and glutamyl glutamic acid arylamidase. Variable for
-arabinosidase and serine arylamidase. Mannose and raffinose are fermented. All the other tests are negative. Major cellular fatty acids are anteiso-C15 : 0 and iso 3-OH-C17 : 0. A significant amount of C18 : 1
9c is also present. The G+C content of the type strain is 44 mol%.
The type strain is JCM 5825T (=CCUG 4941T=DSM 20701T=ATCC 8503T), which was isolated from human faeces, where it is one of the most common species. Strains have been isolated occasionally from human clinical specimens.
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Description of Parabacteroides goldsteinii (Song et al. 2006 |
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Basonym: Bacteroides goldsteinii Song et al. 2006
.
The description of Parabacteroides goldsteinii is as that given by Song et al. (2005)
. Urease is not produced. Nitrate is not reduced. Acid is produced from cellobiose, glucose, rhamnose, sucrose, trehalose and xylose, but not from arabinose or xylan. In peptone yeast broth and peptone yeast glucose broth, major amounts of acetic and succinic acids and minor amounts of isovaleric acid, propionic acid and formic acid are produced. Using API ZYM, Rapid ID 32A and RapID ANA II systems, strains have the same profile. Positive reactions are obtained for
-glucosidase,
-galactosidase,
-galactosidase, N-acetyl-
-glucosaminidase, naphthol-AS-BI-phosphohydrolase, acid phosphatase, alkaline phosphatase, leucine arylamidase, p-nitrophenylphosphatase, arginine arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, leucyl glycine aminopeptidase, glycine aminopeptidase, phenylalanine aminopeptidase, arginine aminopeptidase and serine aminopeptidase. All the other tests are negative. Mannose and raffinose are fermented (Rapid ID 32A). Using Rosco diagnostic tablets (Rosco),
-xylosidase,
-glucuronidase,
-glucosidase,
-glucosidase,
-galactosidase,
-galactosidase (o-nitrophenol-
-D-galactopyranoside), N-acetyl-
-glucosaminidase, alkaline phosphatase and
-arabinosidase are present;
-arabinosidase is tested as positive only by Rosco tablets. Major cellular fatty acids are anteiso-C15 : 0 and iso 3-OH-C17 : 0 (2528 and 1823 %, respectively). Significant amounts of C18 : 1
9c and anteiso 3-OH-C17 : 0 (1116 and 915 %, respectively) are also present. Susceptible to metronidazole (MIC
2 µg ml1) and ertapenem (MIC
1 µg ml1). Some resistance is seen with clindamycin (MIC
8 µg ml1). Resistant to penicillin G (MIC
32 µg ml1), cefotetan (MIC
256 µg ml1) and vancomycin (MIC
32 µg ml1).
-Lactamase-positive. The G+C content of the type strain is 43 mol%.
The type strain is JCM 13446T (=CCUG 48944T), which was isolated from human clinical specimens of intestinal origin.
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Description of Parabacteroides merdae (Johnson et al. 1986 |
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Basonym: Bacteroides merdae Johnson et al. 1986
.
The description is the same as that given by Johnson et al. (1986)
. Urease and catalase are not produced. Gelatin is not liquefied. Acid is produced from glucose, lactose, maltose, D-mannose, D-raffinose, sucrose, D-trehalose and D-xylose, but not from L-arabinose, D-cellobiose, glycerol, D-mannitol, D-melezitose, L-rhamnose, salicin or D-sorbitol. Positive reactions are obtained using Rapid ID 32A for
-galactosidase,
-galactosidase,
-glucuronidase, N-acetyl-
-glucosaminidase, glutamic acid decarboxylase, alkaline phosphatase, arginine arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase and glutamyl glutamic acid arylamidase. Variable for
-glucosidase,
-arabinosidase and serine arylamidase. Mannose and raffinose are fermented. All the other tests are negative. Major cellular fatty acids are anteiso-C15 : 0 and iso 3-OH-C17 : 0. A significant amount of C18 : 1
9c is also present. The G+C content of the type strain is 44 mol%.
The type strain is JCM 9497T (=CCUG 38734T=ATCC 43184T), which was isolated from human faeces.
| ACKNOWLEDGEMENTS |
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