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Microorganism Bacteroides 박테로이데스 속 플라스미드벡터 Bacteroides plasmid vector, Bacteroides faecis sp. nov. 이엠생명과학연구원 서범구
2013-09-30 11:28:03

Bacteroides faecis sp. nov.

 

분열균류 진정세균목 박테로이데스과의 미생물로 그람음성균을 말하는데 인간을 포함하여 집토끼, 마못, 생쥐 등 포유류의 장관, 구강, 기도 내부에서 생활하며 포자을 형성하는 간균이다.


박테로이데스 [ Bacteroides ]는 혐기성의 그람음성간균. 다형태성을 보인다. 사람의 기도(氣道), 소화관, 성기(性器)에 상재(常在)하고 있다. 거의 모든 정상적인 치육이나 협점막, 편도에서 검출된다. 

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of
strain MAJ27T and MAJ26 are GQ496624 and GQ496623, respectively.

The results of biochemical tests (API 20A and API rapid ID 32A) and fatty acid
composition of strain MAJ27T and MAJ26 are available as Supplementary Table A and
B in IJSEM Online, respectively.

21 Abstract
22 Bacterial strains MAJ27T and MAJ26, which are anaerobic, Gram-negative, non23
motile and non-spore-forming bacteria, were isolated from human feces. The optimal
24 temperature for their growth was 37 °C. Tests for oxidase and catalase activities were
25 negative, but both strains were sensitive to bile. Acid production via glucose
26 fermentation of several substrates, including glucose, occurred. A comparative study
27 based on the 16S rRNA gene sequences showed that the two strains are closely related
28 to species of the genus Bacteroides, in particular B. thetaiotaomicron (98.6-98.7 %
29 similarity) and B. finegoldii (96.9-97.0 % similarity). Despite this high level of
30 similarity between the 16S rRNA gene sequences of the two strains and closely related
31 species, a DNA-DNA hybridization value of less than 22 % indicated that the two
32 strains differ significantly from closely related strains. The G+C content (42.7±1
33 mol%) and major fatty acid composition (C15:0 anteiso, 39.3-42.5 %) support the
34 assignment of the isolates into the genus Bacteroides. Based on phenotypic,
35 chemotaxonomic, genotypic and phylogenetic studies, we propose that the novel
36 species of bacteria isolated from human feces be classified in the genus Bacteroides as
37 Bacteroides faecis sp. nov., including MAJ27T as the type strain (= KCTC 5823T =
38 JCM 16478T).
39 Main text
40
41 Since the completion of the Human Genome Project, the contribution of symbiotic
42 human gastrointestinal tract (GI-tract) microbiota to normal physiology and
43 predisposition to disease has been the focus of many studies (Turnbaugh et al., 2007). In
44 intestinal microbiota, the phylum Bacteroidetes constitutes the dominant gut microbiota,
45 followed by the phylum Firmicutes. (Eckburg et al., 2005; Gill et al., 2006; Palmer et
46 al., 2007; Wang et al., 2005). Recently, it was reported that changes in the relative
47 abundance of the two dominant bacterial divisions, the Bacteroidetes and the Firmicutes,
48 are associated with obesity (Turnbaugh et al., 2006). The phylum Bacteroidetes is
49 composed of five major subgroups that constitute the Cytophaga-Flavobacter-
50 Bacteroides (CFB) group which includes the genus Bacteroides (Gherna & Woese,
51 1992). The phylum Bacteroidetes seem to account for about 23 % of the human
52 intestinal microbiota (Eckburg et al., 2005; Frank et al., 2007; Hattori & Taylor, 2009;
53 Ley et al., 2005), and the genus Bacteroides seem to account for up to 20 % of the
54 human intestinal microbiota (Matsuki et al., 2004; Rigottier-Gois et al., 2003). Some
55 Bacteroides species, including B. thethaiotaomicron, are known as decomposers in
56 carbohydrate fermentation and catabolism of polysaccharides (hemicellulose and xylan)

57 in the colon (Falony et al., 2009; Flint, 2006; Salyers, 1995; Van Meulen et al., 2006).
58 Bacteria belonging to the genus Bacteroides are Gram-negative, non-spore-forming,
59 non-motile, anaerobic rods, and are generally isolated from the gastrointestinal tract
60 environment (Smith et al., 2005). Unknown novel strains of Bacteroides have been
61 identified from the human intestine and feces using culture-independent techniques
62 based on the 16S rRNA gene sequence (Dore et al., 1998; Eckburg et al., 2005; Gill et
63 al., 2006; Hayashi et al., 2003; Li et al., 2009; Palmer et al., 2007). Of unknown strains,
64 several novel Bacteroides species from human feces and intestinal organs have recently
65 been identified and characterized (Bakir et al., 2006a; Bakir et al., 2006b; Bakir et al.,
66 2006c; Chassard et al., 2008; Hayashi et al., 2007; Kitahara et al., 2005; Robert et al.,
67 2007; Song et al., 2004). In this study, we determined the taxonomic position of two
68 anaerobic Bacteroides-like strains isolated from human feces using polyphasic
69 approaches.
70
71 The strains, designated MAJ27T and MAJ26 (= KCTC 5822 = JCM 16477), were
72 isolated from feces collected from a healthy, 26-year-old male during a Korean study on
73 the diversity of cultivable intestinal microbiota. The strains were discovered on
74 supplemented brain heart diffusion medium (BHIS) containing 50 mg/L kanamycin

75 (Sigma) after a two days culture period at 37 °C in an anaerobic chamber (Bactron II
76 SHEL LAB Anaerobic chamber) containing a mixture of gases (N2:H2:CO2=90:5:5)
77 (Bacic & Smith, 2008; Hecht, 2006; Smith et al., 2006). Then, the isolates were
78 transferred and subcultured onto Eggerth-Gagnon (EG) medium supplemented with 5 %
79 horse blood under anaerobic conditions, respectively. The isolates were suspended in
80 10 % skim milk (BBL) containing 10 % glycerol and stored at − 80 °C. Two references,
81 Bacteroides thetaiotaomicron VPI-5482T and Bacteroides finegoldii 199T, obtained
82 from DSMZ (German Collection of Microorganisms and Cell Cultures) and KCTC
83 (Korean Collection for Type Cultures), were cultured under the same conditions. The
84 growth of the isolates was observed in PYG medium (DSMZ 104) at various
85 temperatures (12, 15, 25, 30, 37, 43 and 47 ˚C) and under aerobic conditions. To
86 determine bile resistance, the isolates were cultivated in PYG medium supplemented
87 with bile salts (Sigma) (0.1-0.4 %, w/v). The entire set of experiments was performed
88 with the strains cultivated and maintained in EG medium at 37 °C and at a pH of 7.6-7.8
89 for two or three days, unless stated otherwise. Gram staining was performed with a
90 Gram Staining Kit (bioMérieux). Spore staining was determined with malachite green
91 dye. Phase-contrast microscopy (ECLIPSE 50i, Nikon) was performed to observe the
92 morphology of cells, as well as Gram and spore staining. The enzyme activities of

93 catalase and oxidase were investigated with a 3 % (v/v) hydrogen peroxide solution and
94 a 1 % (w/v) p-tetramethyl phenylenediamine solution (bioMérieux), respectively. The
95 motility of the isolate was determined by stabbing the center of the column of PYG
96 medium containing 0.4 % agar. To determine other phenotypic characteristics, API 20A
97 and rapid ID 32A were applied, according to the manufacturer’s instructions
98 (bioMérieux), which is the same method as described by Bakir et al. (2006a). Results of
99 biochemical analyses are given in Table 1 and Supplementary Table A in IJSEM Online.
100
101 The fatty acid composition of the isolates and two references was detected via gas
102 chromatography (Hewlett Packard 6890) and identified using the Microbial
103 Identification software package (Sasser, 1990) after performing the saponification,
104 methylation and extraction steps described by Sherlock Microbial Identification
105 Systems (MIDI, 1999). The isolates and B. thetaiotaomicron VPI-5482T were grown on
106 EG medium supplemented 5 % horse blood at 37 °C and pH 7.6-7.8 for three days. The
107 predominant patterns of fatty acids of strains MAJ27T and MAJ26 were similar to that
108 of B. thetaiotaomicron, which is the phylogenetically closest species. In the Cytophaga-
109 Flavobacter-Bacteroides (CFB) group, high amounts of different branched fatty acids of
110 the iso and/or anteiso type are found (Brondz et al., 1991; Paster et al., 1994), and the

111 branched 15-carbon non-hydroxy acids, followed by the branched 17-carbon 3-hydroxy
112 acids, are the predominant fatty acids of Bacteroides species (Mayberry et al., 1982).
113 Anteiso-branched 15-carbon (39.3 and 42.5 %, respectively), followed by iso-branched
114 17-carbon 3-hydroxy acid (14.2 and 14.8 %, respectively) was mainly found in strains
115 MAJ27T and MAJ26. The fatty acid composition of strains MAJ27T and MAJ26 was a
116 key for the identification of Bacteroides species. The results of chemotaxonomic
117 analyses are given in Supplementary Table B in IJSEM Online.
118
119 To compare the 16S rRNA gene sequences of the isolates with those of references in
120 the Genbank database, 16S rRNA gene sequences of the isolates were amplified by
121 colony PCR using four bacteria-specific primers (8F, 968F, 518R and 1492R) (Baker
122 et al., 2003). After purification of the PCR product using the QIAquick® PCR
123 Purification Kit, the PCR product was sequenced using the BigDye Terminator Cycle
124 Sequencing Ready Reaction Kit (Applied Biosystems), according to the
125 manufacturer’s instructions. The reaction mixtures were analyzed by an automated
126 DNA analyzer system (PRISM 3730XL DNA analyzer, Applied Biosystems). The
127 partial fragments of 16S rRNA gene sequences were subsequently assembled using
128 SeqMan software (DNASTAR). Assembled 16S rRNA gene sequences of the isolates

129 were compared with other sequences in the Genbank database (NCBI database). In a
130 comparative study, both isolates, MAJ27T and MAJ26, were found to be closely
131 related to species belonging to the genus Bacteroides in the phylum Bacteroidetes.
132 There was 99.9 % similarity between the 16S rRNA gene sequences of strain MAJ27T
133 and strain MAJ26, while the 16S rRNA gene sequence of MAJ27T was 98.8 % similar
134 to that of Bacteroides thetaiotaomicron VPI-5482T and 97.0 % similar to that of
135 Bacteroides finegoldii 199T. The 16S rRNA gene sequence of strain MAJ26 was found
136 to be 98.7 % similar to that of B. thetaiotaomicron VPI-5482T and 96.9 % similar to
137 that of B. finegoldii 199T. A total of 30 16S rRNA gene sequences of Bacteroides
138 species collected from the Genbank database were aligned with those of the isolates
139 using the multiple sequence alignment program CLUSTAL X (1.83) (Thompson et al.,
140 1997). The trimmed alignment was converted to MEGA format for phylogenetic
141 analyses. Phylogenetic consensus trees were constructed using neighbor-joining and
142 maximum-parsimony methods with MEGA 4 (Tamura et al., 2007). The phylogenetic
143 trees were tested by randomly selecting 1000 bootstrap replicates for the algorithm of
144 neighbor-joining and maximum-parsimony (Kluge & Farris, 1969; Saitou & Nei,
145 1987). This analysis also demonstrated that the 16S rRNA gene sequence of the
146 isolates was closely related to that of B. thetaiotaomicron and B. finegoldii, and the

147 phylogenetic analysis positioned the two strains within the Bacteroides group (Fig. 1).

149 The genomic DNAs of the isolates and two references were extracted using the G150
spinTM Genomic DNA Extraction Kit (iNtRON Biotechnology). For DNA-DNA
151 hybridization, a fluorometric method described by Ezaki et al. (1989) was performed,
152 with modifications (Hirayama et al., 1996). As previously reported by Wayne et al.
153 (1987), strains are generally considered as belonging to the same species unless their
154 DNA-DNA relatedness is 70 % or less. In a DNA-DNA hybridization experiment, the
155 homology between strain MAJ27T and MAJ26 was determined to be 97 %. However,
156 DNA-DNA hybridization levels between strain MAJ27T and the two reference strains
157 were 22 % or less (22 % in the case of B. thetaiotaomicron and 21 % in the case of B.
158 finegoldii). This indicates that the two strains, MAJ27T and MAJ26, belong to a single
159 species, and we confirmed that strain MAJ27T is a novel species belonging to the
160 genus Bacteroides. The G+C content of the isolates was determined using a
161 fluorimetric method employing SYBR Green I and real-time PCR (Gonzalez & Saiz-
162 Jimenez, 2002). The genomic DNA of Escherichia coli K12 was used as the
163 calibration reference (Gonzalez & Saiz-Jimenez, 2002). The G+C content of the
164 isolates was estimated as being 42.7±1 mol%, which falls within the limits of the G+C

165 content range (40-48 mol%) of the genus Bacteroides (Shah, 1992).
166
167 On the basis of phenotypic, chemotaxonomic, genotypic, and phylogenetic studies, we
168 propose that strain MAJ27T and strain MAJ26 be classified as a novel species of the
169 genus Bacteroides, and we propose to name this species Bacteroides faecis sp. nov.

170 Description of Bacteroides faecis sp. nov.
171 Bacteroides faecis (fa.e'cis. L. gen. n. faecis, of dregs, of feces, referring to fecal origin).
172
173 The novel strains presented here are anaerobic, Gram-negative, non-motile and non174
spore-forming bacteria. The cells are rod-shaped, 1.5–2.0 μm in length, 1.0 μm in width,
175 and generally observed singly. Bacterial colonies cultured on PYG medium for four
176 days at 37°C are 1.0-1.5 mm in diameter and have a pale yellow color, circular form,
177 glistening surface, convex side view, and buttery texture. The optimal temperature for
178 growth is 37 °C, and the growth range is from 25 °C to 43 °C. These strains test negative
179 for oxidase and catalase activities. Cells are sensitive to bile for growth. They are
180 indole-positive, but urease-negative. The strains are gelatin hydrolysis-negative, but
181 esculin hydrolysis-positive. In API 20A strips, acid production occurs from D-glucose,
182 D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-cellobiose, D-mannose,
183 D-raffinose and L-rhamnose, but not from D-mannitol, salicin, glycerol, D-melezitose,
184 D-sorbitol, or D-trehalose. In API rapid ID 32A strips, it is positive for α-galactosidase,
185 β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, D-mannose
186 fermentation, D-raffinose fermentation, glutamic acid decarboxylase, α-fucosidase,
187 indole production, alkaline phosphatase, leucyl glycine arylamidase, alanine

188 arylamidase, and glutamyl glutaminsäure arylamidase, but negative for urease, arginine
189 dihydrolase, β-galactosidase-6-phosphate, α-arabinosidase, β-glucuronidase, reduction
190 of nitrate, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucin
191 arylamidase, pyroglutaminsäure arylamidase, tyrosine arylamidase, glycine arylamidase,
192 histidine arylamidase, and serine arylamidase. The major fatty acids present in these
193 strains are C15:0 anteiso, C17:0 iso 3-OH and C16:0 3-OH. The G+C content is 42.7±1
194 mol%.
195
196 The type strain MAJ27T (= KCTC 5823T = JCM 16478T), was isolated from human
197 feces, and we conclude that strain MAJ26 (= KCTC 5822 = JCM 16477) and strain
198 MAJ27T belong to the same species.

199 Acknowledgements
200
201 This research was supported by the grant (09172KFDA996) from Korea Food & Drug
202 Administration in 2009.

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23
337 Table 1. Comparative characteristics of Bacteroides faecis sp. nov. with closely related species of the genus Bacteroides
338 Strains: 1, Bacteroides sp. nov. (data from current study); 2, B. thetaiotaomicron VPI-5482T (Bakir et al., 2006a); 3, B. finegoldii 199T
339 (Bakir et al., 2006a); 4, B. ovatus ATCC 8483T (Bakir et al., 2006a; Chassard et al., 2008); 5, B. xylanisolvens XB1AT (Chassard et al.,
340 2008); 6, B. caccae ATCC 43185T (Johnson et al., 1986); 7, B. nordii WAL 10018T (Song et al., 2004); 8, B. salyersiae WAL 10018T
341 (Song et al., 2004). All strains are positive for acid production from D-xylose, D-cellobiose, L-rhamnose and esculin hydrolysis.
342 Symbols: +, positive; -, negative; −+, most strain negative; +w, weakly positive; ND, not determined.

344 Figure Legend
345
346 Fig. 1. Phylogenetic consensus tree based on the 16S rRNA gene sequences. Filled
347 diamonds indicate generic branches that were present in phylogenetic consensus trees
348 generated by neighbor-joining and maximum-parsimony methods. The numbers at the
349 nodes indicate bootstrap values as percentages of 1000 and 1000 replicates, respectively
350 (neighbor-joining probability / maximum-parsimony probability). Values below 50 %
351 are not indicated at the branch points. Bar, 0.02 substitutions per site.

 




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