Parabacteroides chinchillae sp. nov., isolated from chinchilla (Chincilla lanigera) faeces
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International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3470–3474 DOI 10.1099/ijs.0.050146-0
Parabacteroides chinchillae sp. nov., isolated from
chinchilla (Chincilla lanigera) faeces
Maki Kitahara,1 Mitsuo Sakamoto,1 Sayaka Tsuchida,2 Koh Kawasumi,2
Hiromi Amao,2 Yoshimi Benno3 and Moriya Ohkuma1
Correspondence 1
Japan Collection of Microorganisms, RIKEN BioResource Center, Koyadai, Tsukuba,
Maki Kitahara Ibaraki 305-0074, Japan
kitahara@jcm.riken.jp 2
Laboratory of experimental animal science, Nippon Veterinary and Life Science University,
Musashino, Tokyo 180-8602, Japan
3
Benno Laboratory, RIKEN Innovation Center, Wako, Saitama 351-0198, Japan
Strains of Gram-stain-negative, anaerobic, rod-shaped bacteria were isolated from chinchilla
(Chinchilla lanigera) faeces, and strain ST166T was investigated taxonomically. Phylogenetic
analyses of 16S rRNA gene sequences revealed that strain ST166T belonged to the genus
Parabacteroides. Strain ST166T formed a distinct line of descent, and the highest sequence
similarity to ST166T was found with Parabacteroides merdae JCM 9497T (95.6 %) and
Parabacteroides johnsonii JCM 13406T (95.0 %). Analysis of hsp60 gene sequences also
supported these relationships. Based on the phenotypic and phylogenetic characteristics, the
novel species Parabacteroides chinchillae sp. nov. is proposed. The type strain of P. chinchillae
sp. nov. is ST166T (5JCM 17104T5CCUG 62154T).
Members of the genera Bacteroides and Parabacteroides Bacteroides (66 strains) and Parabacteroides (28 strains)
are important constituents of both human and animal (Kitahara et al., 2011, 2012) as shown in Table S1 (available
intestinal microbiota (Momose et al., 2011, Nakano et al., in IJSEM Online). The 16S rRNA gene sequences
2008). At the time of writing, six species with validly (approximately 800 bp) of the 94 strains were determined
published names exist in the genus Parabacteroides. as described previously (Kitahara et al., 2005). Nucleic acid
Macellibacteroides fermentans (Jabari et al., 2012) is also sequences were analysed by BLAST search and identified as
included in the Parabacteroides cluster (Fig. 1), although recognized species with .99 % sequence similarity.
16S rRNA gene sequence similarity between Parabac-
Strain ST166T was characterized in this study and
teroides chartae NS31-3T (Tan et al., 2012) and M.
incubated on Eggerth Gagnon (EG; Merck) agar plates
fermentans LIND7HT (Jabari et al., 2012) is 99.7 % and
supplemented with 5 % (v/v) horse blood for 2 days at
DNA–DNA hybridization experiments are required to
37 uC in an anaerobic jar (Hirayama Manufacturing) filled
confirm their taxonomic positions. In our studies on the
with 100 % CO2. Bile resistance was tested by growing the
microbial composition of chinchilla faeces, four novel
bacterium on GAM (Nissui) agar plates supplemented with
species, Bacteroides chinchillae, Bacteroides rodentium
2 % bacto-oxgall (Difco). Other physiological, biochemical
(Kitahara et al., 2011), Bacteroides stercorirosoris and
and enzymic activity tests were performed with API 20A
Bacteroides faecichinchillae (Kitahara et al., 2012) have
and API rapid ID 32 (bioMérieux) according to the
been reported. Additionally, we isolated one strain that
manufacturer’s instructions at 37 uC in an anaerobic jar. A
could not be clearly placed within any known species. Here
loopful of well-grown cells was harvested for fatty acid
we report the fifth novel species isolated from chinchilla
methyl ester analysis. Saponification, methylation, extrac-
faeces.
tion and determination of cellular fatty acid profiles were
A total of 94 strains of Gram-negative, anaerobic, rod- conducted as described by Sakamoto et al. (2002). Cellular
shaped bacteria were isolated from faeces of four healthy fatty acid profiles were determined by the Sherlock
chinchillas and identified as species in the genera Microbial Identification System (MIDI) version 2.99B with
database BHIBLA (version 3.80). Isoprenoid quinones were
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and extracted as described by Komagata & Suzuki (1987) and
partial hsp60 gene sequences of Parabacteroides chinchillae ST166T were analysed as described previously (Sakamoto et al.,
are AB574482 and AB576489, respectively. 2002). Near complete (1500 bases) 16S rRNA gene
Two supplementary tables are available with the online version of this sequences were analysed as described previously (Kitahara
paper. et al., 2005). The partial hsp60 gene sequences were
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On: Wed, 09 Jan 2019 07:37:37Parabacteroides chinchillae sp. nov.
0.01 Parabacteroides distasonis JCM 5825T (AB238922)
56/31
Parabacteroides merdae JCM 9497T (AB238928)
91/88 100/100
Parabacteroides johnsonii JCM 13406T (AB261128)
80/81 Parabacteroides chinchillae ST166T (AB574482)
Parabacteroides gordonii JCM 15724T (AB470343)
100/100
Parabacteroides goldsteinii JCM 13446T (AB547650)
Parabacteroides chartae NS31-3T (JN029805)
100/100
Macellibacteroides fermentans LIND 7HT (HQ020488)
Tannerella forsythia JCM 10827T (AB547708)
Fig. 1. Phylogenetic tree showing the relationship between Parabacteroides chinchillae sp. nov. ST166T and related species of
the genus Parabacteroides based on neighbour-joining analysis of the 16S rRNA gene sequences. Numbers at nodes indicate
the percentage bootstrap values of 1000 replicates (determined by neighbour-joining analysis and minimum-evolution method,
separated by /). Bar, 0.01 substitutions per nucleotide position. The accession number for the 16S rRNA gene sequence is
given for each strain.
analysed as described by Sakamoto et al. (2010). PCR performed with CLUSTAL X (version 1.83) (Thompson et al.,
products were sequenced using a BigDye Terminator cycle 1997) and a phylogenetic tree was reconstructed by the
sequencing kit (Applied Biosystems) and ABI PRISM 3130 neighbour-joining method (Saitou & Nei, 1987). The
Genetic Analyzer (Applied Biosystems). The closest topology of the tree was evaluated by bootstrap analysis
recognized relatives of the isolates were determined by with 1000 replicates using the CLUSTAL X software.
database searches, and sequences from closely related Minimum-evolution with the Kimura two-parameter
species were retrieved from DDBJ, EMBL and GenBank model (Kimura, 1980) was inferred using the software
nucleotide sequence databases. Phylogenetic analysis was package MEGA version 4.0 (Tamura et al., 2007). Bacterial
0.02 Parabacteroides distasonis JCM 5825T (AB547566)
Parabacteroides merdae JCM 9497T (AB547570)
100
Parabacteroides johnsonii JCM 13406T (AB547569)
71
Parabacteroides goldsteinii JCM 13446T (AB547567)
100
Parabacteroides gordonii JCM 15724T (AB547568)
Parabacteroides chinchillae ST166T (AB576489)
Macellibacteroides fermentans JCM 16313T (AB702690)
Tannerella forsythia JCM 10827T (AB547635)
Fig. 2. Phylogenetic tree showing the relationship between Parabacteroides chinchillae sp. nov. ST166T and related species of
the genus Parabacteroides based on neighbour-joining analysis of the hsp60 gene sequences. Numbers at nodes indicate the
percentage bootstrap values for 1000 replicates. Bootstrap values above 50 % are given at the branching points. Bar, 0.02
substitutions per nucleotide position. The accession number for the hsp60 gene sequence is given for each strain.
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M. Kitahara and others
Table 1. Differential characteristics of Parabacteroides chinchillae sp. nov. and related species of the genus Parabacteroides
Strains: 1, ST166T; 2, Parabacteroides gordonii JCM 15724T; 3, Parabacteroides goldsteinii JCM 13446T; 4, Parabacteroides distasonis JCM 5825T; 5, P. johnsonii JCM 13406T; 6, P. merdae JCM 9497T;
7, P. chartae JCM 17797T. All data for strain ST166T were taken from this study. The data in columns 2–6 are from Sakamoto et al. (2009) and the data in columns 7 are from Tan et al. (2012). +,
Positive; 2, negative; W, weakly positive; NT, not tested.
Characteristic 1 2 3 4 5 6 7
Isolation source Chinchilla faeces Human blood Human faeces Human faeces Human faeces Human faeces Wastewater of a paper mill
API 20A
Salicin 2 2 2 + 2 2 2
L-Arabinose 2 + 2 2 + 2 +
Cellobiose 2 2 W + 2 2 +
Melezitose 2 2 2 + 2 2 +
L-Rhamnose 2 2 + + + 2 +
Trehalose 2 2 + + + + +
Rapid ID 32A
a-Arabinosidase 2 + 2 + + + NT
+ + + +
International Journal of Systematic and Evolutionary Microbiology 63
b-Glucosidase 2 2 2
b-Glucuronidase 2 2 2 2 + + 2
Glutamic acid decarboxylase + 2 2 + + + NT
Phenylalanine arylamidase + 2 + + + + NT
Pyroglutamic acid arylamidase 2 2 2 2 2 + NT
Tyrosine arylamidase + 2 + + + + NT
Glycine arylamidase + 2 + + + + NT
Histidine arylamidase + 2 + + + + NT
Glutamyl glutamic acid arylamidase + 2 + + + + NT
Serine arylamidase + 2 + + + + NT
Distinctive major cellular fatty acids* C18 : 1v9c Summed C18 : 1v9c Summed C18 : 1v9c iso-C17 : 0 C18 : 1v9c iso-C17 : 0 iso-C17 : 0 3-OH C18 : 1v9c iso-C17 : 0 C15 : 0 iso-C17 : 0 3-OH
feature3 feature3 3-OH 3-OH 3-OH
DNA G+C content (mol%) 42.8 44.6 43 47.1 47.6 47.2 37.2
*Anteiso-C15 : 0 is a major component of cellular fatty acids in all strains.
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DNA of the isolated strain was extracted from the cells Cells cultivated on EG agar plates are strictly anaerobic,
harvested from GAM broth after 12 h at 37 uC as described non-spore-forming, non-motile and Gram-negative. The
previously (Kitahara et al., 2005) and purified by the short rods or rod-shaped cells are 1.2 mm in width and
methods of Saito & Miura (1963). The DNA G+C content variable in length, mostly ranging from 2 to 15 mm.
was determined as described previously (Kitahara et al., Colonies on EG blood agar plates are 1–2 mm in diameter,
2005). disc shaped, white–greyish and translucent. The optimum
Strain ST166T was isolated from an 11-year-old male temperature for growth is around 37 uC. Grows in the
chinchilla (using EG agar). Cells of strain ST166T were presence of bile. Produces acid from glucose, lactose,
obligately anaerobic, non-spore-forming, non-motile, maltose, D-mannose, raffinose and sucrose. Does not
Gram-negative short rods. Approximately 1500 bases of produce acid from L-arabinose, cellobiose, glycerol, D-
the 16S rRNA gene sequence were determined. The mannitol, melezitose, L-rhamnose, salicin, D-sorbitol,
phylogenetic analyses clearly indicated that this isolate trehalose and D-xylose. Aesculin is hydrolysed. Gelatin is
was related to the strains of species in the genus not hydrolysed. Indole is not produced. Catalase and
Parabacteroides as shown in Fig. 1. Strain ST166T formed urease are not produced. Using Rapid ID 32A, displays
a distinct line of descent, and the highest sequence positive reactions for a-galactosidase, b-galactosidase,
similarity to ST166T was found with Parabacteroides a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase,
merdae JCM 9497T (95.6 %) and Parabacteroides johnsonii glutamic acid decarboxylase, a-fucosidase, alkaline phos-
JCM 13406T (95.0 %). These sequence similarity values phatase, arginine arylamidase, leucyl glycine arylamidase,
were low enough to allow the proposal of a novel species phenylalanine arylamidase, leucine arylamidase, tyrosine
without DNA–DNA hybridization experiments (Stackebrandt arylamidase, alanine arylamidase, glycine arylamidase,
& Ebers, 2006). These results showed that ST166T represents a histidine arylamidase, glutamyl glutamic acid arylamidase
novel species. The phylogenetic analysis of the hsp60 gene and serine arylamidase. Negative reactions for urease,
sequences also supported classification of ST166T as a repre- arginine dihydrolase, b-galactoside-6-phosphatase, a-ara-
sentative of a novel species with low sequence similarities binosidase, b-glucuronidase, nitrate reduction, indole
(,87.1 %) to six closely related species (Fig. 2). production, proline arylamidase and pyroglutamic acid
arylamidase. The major fatty acids are anteiso-C15 : 0, C16 : 0
Physiological and biochemical properties of strain ST166T and C16 : 0v9c. The main respiratory quinones are mena-
were determined with API 20A and rapid ID 32A kits and quinones MK-9 and MK-10.
the results are given in the species description. The G+C
content of strain ST166T was 42.8 mol%, which is slightly The type strain is strain ST166T (5JCM 17104T5CCUG
lower than the contents for the members of the genus 62154T), which was isolated from faeces of chinchilla. The
Parabacteroides (43–46 mol%) as it is currently defined DNA G+C content of the type strain is 42.8 %.
(Sakamoto & Benno, 2006).
The major cellular fatty acid of strain ST166T was anteiso- Acknowledgements
C15 : 0 (28.9 % of the total). Significant amounts of C18 : 1v9c
We are grateful to Professor Jean P. Euzéby for his suggestions regarding
(13.4 %) and summed feature 3 (comprising iso-C15 : 0 ALDE nomenclature. This work was supported in part by a research grant
and/or an unknown fatty acid of equivalent chain length) (2009–2011) from the Institute for Fermentation, Osaka, Japan to M. S.,
(12.5 %) were also present in strain ST166T (Table S2). a Grant-in-Aid for Scientific Research (C) from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) (no.
The major quinones of members of the genus
24570117) to M. K. and the Japan Science and Technology Agency/Japan
Parabacteroides are menaquinones MK-9 and MK-10 International Cooperation Agency (JST/JICA), Science and Technology
(Sakamoto & Benno, 2006). Strain ST166T had a high Research Partnership for Sustainable Development (SATREPS).
level of MK-9 (56 %) and MK-10 (23 %) but a low level of
MK-7 (6 %) and MK-8 (13 %).
On the basis of the results shown in this study, strain
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