SGM Journals
EVOLUTION, PHYLOGENY AND BIODIVERSITY

hsp60 and 16S rRNA gene sequence relationships among species of the genus Bacteroides with the finding that Bacteroides suis and Bacteroides tectus are heterotypic synonyms of Bacteroides pyogenes

Mitsuo Sakamoto, Natsuko Suzuki, Yoshimi Benno

  • 1Microbe Division/Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan
  • 2Benno Laboratory, Center for Intellectual Property Strategies, RIKEN, Wako, Saitama 351-0198, Japan
  • Correspondence
    Mitsuo Sakamoto
    sakamoto{at}jcm.riken.jp

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(12):2984–2990 · https://doi.org/10.1099/ijs.0.021154-0

    View at publisher PubMed

    Abstract

    hsp60 gene sequences were determined for members of the genus Bacteroides and sequence similarities were compared with those obtained for the 16S rRNA gene. Among the 29 Bacteroides type strains, the mean sequence similarity of the hsp60 gene (84.5 %) was significantly less than that of the 16S rRNA gene (90.7 %), indicating a high discriminatory power of the hsp60 gene. Species of the genus Bacteroides were differentiated well by hsp60 gene sequence analysis, except for Bacteroides pyogenes JCM 6294T, Bacteroides suis JCM 6292T and Bacteroides tectus JCM 10003T. The hsp60 gene sequence analysis and the levels of DNA–DNA relatedness observed demonstrated that these three type strains are a single species. Consequently, B. suis and B. tectus are heterotypic synonyms of B. pyogenes. This study suggests that the hsp60 gene is an alternative phylogenetic marker for the classification of species of the genus Bacteroides.

    • The GenBank/EMBL/DDBJ accession numbers for the partial hsp60 gene sequences of Bacteroides strains determined in this study are listed in Table 1⇑.

    • A table showing sequence similarity based on pairwise comparisons of the hsp60 and 16S rRNA gene sequences of species of the genus Bacteroides is available with the online version of this paper.

    INTRODUCTION

    Organisms of the genus Bacteroides are part of the indigenous microbiota of human and animal gastrointestinal tracts, but different species in this group are commonly associated with a variety of human and animal infections. The genus Bacteroides is important in clinical bacteriology. Although species of the genus Bacteroides are isolated frequently from hospitalized patients, it can be difficult to identify every clinical isolate to the species level. In the past, because of poor definition of the genus, more than 50 species of the genus Bacteroides have been included in this group. The taxonomy of the genus Bacteroides has undergone significant changes in the last two decades (Dewhirst et al., 1990; Moore & Moore, 1994; Rautio et al., 2003; Sakamoto et al., 2002, 2007; Sakamoto & Benno, 2006; Shah & Collins, 1988, 1989, 1990). It has also been reported that 16S rRNA gene sequencing is useful for the identification of isolates of clinically significant species of the genus Bacteroides (Song et al., 2005).

    To date, a multilocus sequence typing (MLST) approach has been widely used to infer the phylogeny of organisms (Alexandre et al., 2008; Itoh et al., 2006; Shah et al., 2007; Yamamoto & Harayama, 1995). However, only limited information has been accumulated for species of the genus Bacteroides. It has been reported that housekeeping gene sequences, e.g. rpoB, could be used to clarify interspecies phylogenetic relationships within the genus Bacteroides (Ko et al., 2007). The ad hoc committee for the re-evaluation of the species definition in bacteriology has recommended evaluation of protein-coding gene sequence analysis for its applicability to genomically circumscribe the taxon species and differentiate it from neighbouring species detected by 16S rRNA gene sequence analysis (Stackebrandt et al., 2002).

    Hsp60, also known as Cpn60 or GroEL, is a member of the heat-shock protein (Hsp) family. The hsp60 gene has been shown to be more discriminative than the 16S rRNA gene for the identification of Streptococcus suis serotypes (Brousseau et al., 2001) and species of the genus Prevotella (Sakamoto et al., 2010). The present study was designed to compare two methods, one based on the 16S rRNA gene and one based on the hsp60 gene, for the classification and establishment of phylogenetic relationships among species of the genus Bacteroides. Consequently, the description of Bacteroides pyogenes was emended.

    METHODS

    Bacterial strains.

    A total of 29 Bacteroides type strains and two reference strains were obtained from the Japan Collection of Microorganisms (JCM), RIKEN BioResource Center, Wako, Japan, and are listed in Table 1⇓. The strains used in the present study were maintained on Eggerth Gagnon (EG) agar (Merck) supplemented with 5 % (v/v) horse blood for 2 to 5 days at 37 °C in an atmosphere containing 100 % CO2 [except for Bacteroides propionicifaciens JCM 14649T, which was grown on modified PY4S agar (Ueki et al., 2008) for 5 to 7 days at 30 °C in an atmosphere containing 10 % CO2 and 90 % N2].

    Table 1.

    Bacteroides strains investigated and accession numbers of the hsp60 and 16S rRNA gene sequences determined in this study

    API tests.

    Physiological and biochemical reactions were determined in duplicate with the API 20A anaerobe test kit and the Rapid ID 32A anaerobe identification kit, respectively (bioMérieux), according to the manufacturer's instructions.

    Cellular fatty acids.

    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 the MIDI microbial identification system (Microbial ID). Peaks were automatically integrated, fatty acids were identified by equivalent chain-length (ECL), and percentages of the total peak area were calculated. External calibration was done by using MIDI calibration mixture 1 (FAMEs of straight-chain saturated fatty acids from nine to 20 carbons in length and five hydroxy acids).

    DNA–DNA hybridization.

    Chromosomal DNA was isolated by previously described methods (Marmur, 1961; Saito & Miura, 1963), with some modifications. The DNA base composition was determined by 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 DNA–DNA relatedness experiment was carried out in microplate wells as described by Ezaki et al. (1989). Hybridization was performed at 44 °C for 16 h.

    PCR amplification and sequencing.

    The 16S rRNA gene was analysed as described previously (Sakamoto et al., 2002). The partial hsp60 gene was amplified by PCR using primers H729 (5′-CGC CAG GGT TTT CCC AGT CAC GAC GAI III GCI GGI GAY GGI ACI ACI AC-3′) and H730 (5′-AGC GGA TAA CAA TTT CAC ACA GGA YKI YKI TCI CCR AAI CCI GGI GCY TT-3′) (Brousseau et al., 2001); inosine was used to reduce the degeneracy of the sequences (Ohtsuka et al., 1985). These primers were derived from the previously described H279A and H280A primers (Goh et al., 1997) by addition of the sequences for commercially available M13 24 bp sequencing primers (underlined nucleotides). Primers were designed to amplify the ‘universal target’ (Hill et al., 2004), the region of the cpn60 gene, encoding 60 kDa chaperonin protein subunits (Cpn60, also known as Hsp60 or GroEL), corresponding to nucleotides 274–828 of the Escherichia coli cpn60 gene sequence. Amplification reactions were performed in a total volume of 50 μl containing 2.5 μl DNA (50 ng), 1.25 U TaKaRa Ex Taq (Takara Bio), 5 μl 10× Ex Taq buffer, 4 μl dNTP mixture (2.5 mM each), and 5 pmol each primer. hsp60 genes were amplified in a Biometra Thermocycler TGradient using the following programme: 94 °C for 5 min, followed by 40 cycles consisting of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 45 s, with a final extension period at 72 °C for 10 min. Sequencing of purified PCR products was performed with standard M13 sequencing primers, as indicated in the primer sequences above, and a BigDye Terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems) in an Applied Biosystems 3130xl Genetic Analyzer.

    Phylogenetic data analysis.

    Related sequences were aligned by using the clustal x 2.0.12 program (Larkin et al., 2007) and corrected by manual inspection. Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) after gaps and unknown bases were eliminated. The phylogenetic tree was reconstructed by the neighbour-joining method (Saitou & Nei, 1987). Bootstrap resampling analysis (Felsenstein, 1985) was performed to estimate the confidence of tree topologies. A maximum-likelihood tree was also generated by the dnaml program in the phylip software package v3.69.

    RESULTS AND DISCUSSION

    hsp60 gene sequence analysis

    In this study, 558 bp nucleotide sequences (186 codons) of the hsp60 gene from 29 Bacteroides type strains were used for analysis and the sequences were deposited in the DDBJ and assigned accession numbers as listed in Table 1⇑. In addition, intra-species variations were determined for Bacteroides fragilis and B. propionicifaciens. High sequence similarities were observed among the hsp60 genes of B. fragilis (99.8 %) and B. propionicifaciens (100 %) as well as among the 16S rRNA genes of B. fragilis (99.7 %) and B. propionicifaciens (100 %). Mean hsp60 gene sequence similarity among the 29 Bacteroides type strains was 84.5 %, indicating a divergence of the hsp60 gene that is greater than that of the 16S rRNA gene (90.7 %) (Table 2⇓). Sequence similarity among the Bacteroides strains ranged from 72.9 to 100 % in the hsp60 gene (see Supplementary Table S1 available in IJSEM Online). On the other hand, a comparative nucleotide sequence analysis of the 16S rRNA gene revealed a tighter range of identities (83.5–100 %); most species of the genus Bacteroides showed more than 90 % similarity in their 16S rRNA gene sequences. These results indicated that the base substitution rate of the hsp60 gene sequence was much faster than that of the 16S rRNA gene sequence. Phylogenetic trees based on the hsp60 and 16S rRNA gene sequences were reconstructed by the neighbour-joining and maximum-likelihood methods. Whichever algorithm was used, most phylogenetic relationships among species of the genus Bacteroides were stably maintained, except for some minor differences at the deepest branching points (data not shown). The phylogenetic analysis indicated a higher evolutionary rate for the hsp60 gene sequence than for the 16S rRNA gene sequence (Fig. 1⇓). Phylogenetic analysis of species of the genus Bacteroides based on the hsp60 gene sequence suggested that the hsp60 gene would be able to differentiate related species, except for Bacteroides pyogenes JCM 6294T, Bacteroides suis JCM 6292T and Bacteroides tectus JCM 10003T. In this study, the hsp60 gene sequences were used, since the phylogenetic tree obtained from the Hsp60 amino acid sequences showed low resolution (data not shown). Nucleotide substitution of the hsp60 gene provoked synonymous amino acid substitution.

    Figure image not available in archive
    Fig. 1.

    Phylogenetic tree based on hsp60 (a) and 16S rRNA (b) gene sequences showing the relationships among the 29 species of the genus Bacteroides recognized at the time of writing. The tree was reconstructed by the neighbour-joining method. Numbers at nodes indicate percentage bootstrap values of 1000 replicates. Bars, 0.02 substitutions per nucleotide position. Accession numbers are given for each strain.

    Table 2.

    Percentage sequence similarity among the hsp60 and 16S rRNA genes of 29 Bacteroides type strains

    A range of similarity values of the hsp60 and 16S rRNA genes were compared for each group of the genus Bacteroides to evaluate discrimination powers (Table 2⇑). Among the 29 Bacteroides type strains, the mean sequence similarity between Bacteroides coprosuis JCM 13475T and the other strains for the hsp60 gene (76.5 %) was significantly less than that for the 16S rRNA gene (88.4 %), and the mean sequence similarity between B. propionicifaciens JCM 14649T and other Bacteroides strains was remarkably low (76.0 %). This comparison indicated that the hsp60 gene has a higher discriminatory power than the 16S rRNA gene, except for the B. pyogenes group.

    Similarity values for hsp60 gene sequences among 29 different species of the genus Staphylococcus ranged from 74 to 93 % (mean 82 %) (Kwok et al., 1999). In addition, pairwise sequence identity scores based on partial hsp60 gene sequences among the four Macrococcus species tested ranged from 82 to 87 % (mean 83 %) (Kwok & Chow, 2003). These values were in agreement with the present study (range 72.9–100 %, mean 84.5 %). Our results reconfirmed the usefulness of the hsp60 gene sequence for identification and phylogenetic analysis of micro-organisms.

    B. pyogenes group

    B. pyogenes and B. suis were proposed by Benno et al. (1983) and deposited in several public culture collections. It has been reported that B. pyogenes and B. suis are genomically different species (Benno et al., 1983). In this study, the sequence similarity between B. pyogenes JCM 6294T and B. suis JCM 6292T was 100 % for the hsp60 gene and 100 % for the 16S rRNA gene. It is, therefore, considered that the depositors might have mistaken the strains. On the other hand, when B. tectus was proposed by Love et al. (1986) these authors did not use B. pyogenes and B. suis as reference strains. The similarity value of B. tectus JCM 10003T and B. pyogenes JCM 6294T or B. suis JCM 6292T was 99.6 % for the hsp60 gene and 98.7 % for the 16S rRNA gene. This finding suggests that B. tectus, B. pyogenes and B. suis may be the same species, with B. tectus a later heterotypic synonym of B. pyogenes or B. suis. To resolve these problems, further studies were performed.

    16S rRNA gene sequences of B. pyogenes and B. suis type strains obtained from other culture collections (ATCC, CCUG and DSMZ) (Table 1⇑) were analysed and compared with the JCM type strains. The levels of sequence similarity among the eight strains were 100 %. Consequently, other analyses were performed using only the JCM type strains. Physiological and biochemical characteristics of B. pyogenes JCM 6294T, B. suis JCM 6292T and B. tectus JCM 10003T are shown in Table 3⇓. These three strains were phenotypically similar. The cellular fatty acid compositions of B. pyogenes JCM 6294T, B. suis JCM 6292T and B. tectus JCM 10003T were almost the same. The major cellular fatty acid of the above strains was anteiso-15 : 0 (22–24 %) (Table 4⇓). The DNA G+C contents of B. pyogenes JCM 6294T, B. suis JCM 6292T and B. tectus JCM 10003T were 46.6, 46.7 and 46.9 mol%, respectively. The levels of DNA–DNA relatedness between B. pyogenes JCM 6294T, B. suis JCM 6292T and B. tectus JCM 10003T are shown in Table 5⇓ and demonstrate that these three type strains represent a single species. Consequently, B. suis and B. tectus are heterotypic synonyms of B. pyogenes.

    Table 3.

    Phenotypic and biochemical characteristics of Bacteroides pyogenes, Bacteroides suis and Bacteroides tectus

    Strains: 1, B. pyogenes JCM 6294T; 2, B. suis JCM 6292T; 3, B. tectus JCM 10003T. All strains produced acid from glucose, lactose and maltose. All strains failed to produce acid from l-arabinose, d-mannitol, melezitose, d-rhamnose, salicin, trehalose and d-xylose. All strains were negative for indole production, gelatin digestion, aesculin hydrolysis and urease and catalase activities. All strains were positive in Rapid ID 32A tests for β-N-acetylglucosaminidase, alkaline phosphatase, α-fucosidase, β-galactosidase, α-glucosidase, leucyl glycine arylamidase and alanine arylamidase activities. All strains were negative in Rapid ID 32A tests for indole production, nitrate reduction, urease, arginine dihydrolase, α-arabinosidase, α-galactosidase, β-galactosidase 6-phosphate, β-glucuronidase, glutamic acid decarboxylase, arginine arylamidase, glycine arylamidase, histidine arylamidase, leucine arylamidase, phenylalanine arylamidase, proline arylamidase, pyroglutamic acid arylamidase, serine arylamidase and tyrosine arylamidase activities. +, Positive; w, weakly positive; −, negative.

    Table 4.

    Cellular fatty acid compositions of Bacteroides pyogenes, Bacteroides suis and Bacteroides tectus

    Strains: 1, B. pyogenes JCM 6294T; 2, B. suis JCM 6292T; 3, B. tectus JCM 10003T. Values are percentages of total fatty acids. tr, Trace amount (<1 %).

    Table 5.

    DNA base compositions and levels of DNA–DNA relatedness between Bacteroides pyogenes, Bacteroides suis and Bacteroides tectus

    Emended description of Bacteroides pyogenes Benno et al. 1983

    The description is as given by Benno et al. (1983) and Love et al. (1986) with the following modification: the major cellular fatty acid is anteiso-15 : 0.

    In conclusion, this study suggests that the hsp60 gene is an alternative phylogenetic marker for the classification of species of the genus Bacteroides. Analysis based on the hsp60 gene sequences in combination with other housekeeping gene sequences may provide a highly reliable identification system for species of the genus Bacteroides. In addition, the greater variability of the hsp60 gene sequences should be useful in designing specific primers for PCRs capable of assisting in the rapid identification of species of the genus Bacteroides. We are currently planning the analysis of multiple genes (dnaJ, gyrB, recA, rpoB, etc.) other than hsp60 to generate more useful data.

    Acknowledgments

    The authors thank Dr Kwang-Deuk An for phylogenetic analysis. This work was supported in part by a research grant from the IFO (Institute for Fermentation, Osaka, Japan; 2009-2011) to M. S.

    References

    1. Alexandre, A., Laranjo, M., Young, J. P. W. & Oliveira, S. (2008). dnaJ is a useful phylogenetic marker for alphaproteobacteria. Int J Syst Evol Microbiol 58, 2839–2849.
    2. Benno, Y., Watabe, J. & Mitsuoka, T. (1983). Bacteroides pyogenes sp. nov., Bacteroides suis sp. nov., and Bacteroides helcogenes sp. nov., new species from abscesses and feces of pigs. Syst Appl Microbiol 4, 396–407.
    3. Brousseau, R., Hill, J. E., Préfontaine, G., Goh, S.-H., Harel, J. & Hemmingsen, S. M. (2001). Streptococcus suis serotypes characterized by analysis of chaperonin 60 gene sequences. Appl Environ Microbiol 67, 4828–4833.
    4. Dewhirst, F. E., Paster, B. J., La Fontaine, S. & Rood, J. I. (1990). Transfer of Kingella indologenes (Snell and Lapage 1976) to the genus Suttonella gen. nov. as Suttonella indologenes comb. nov.; transfer of Bacteroides nodosus (Beveridge 1941) to the genus Dichelobacter gen. nov. as Dichelobacter nodosus comb. nov.; and assignment of the genera Cardiobacterium, Dichelobacter, and Suttonella to Cardiobacteriaceae fam. nov. in the gamma division of Proteobacteria on the basis of 16S rRNA sequence comparisons. Int J Syst Bacteriol 40, 426–433.
    5. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
    6. Felsenstein, J. (1985). Confidence limits of phylogenies: an approach using the bootstrap. Evolution 39, 783–791.
    7. Goh, S. H., Santucci, Z., Kloos, W. E., Faltyn, M., George, C. G., Driedger, D. & Hemmingsen, S. M. (1997). Identification of Staphylococcus species and subspecies by the chaperonin 60 gene identification method and reverse checkerboard hybridization. J Clin Microbiol 35, 3116–3121.
    8. Hill, J. E., Penny, S. L., Crowell, K. G., Goh, S. H. & Hemmingsen, S. M. (2004). cpnDB: a chaperonin sequence database. Genome Res 14, 1669–1675.
    9. Itoh, Y., Kawamura, Y., Kasai, H., Shah, M. M., Nhung, P. H., Yamada, M., Sun, X., Koyana, T., Hayashi, M. & other authors (2006). dnaJ and gyrB gene sequence relationship among species and strains of genus Streptococcus. Syst Appl Microbiol 29, 368–374.
    10. Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.
    11. Ko, K. S., Kuwahara, T., Haehwa, L., Yoon, Y.-J., Kim, B.-J., Lee, K.-H., Ohnishi, Y. & Kook, Y.-H. (2007). RNA polymerase β-subunit gene (rpoB) sequence analysis for the identification of Bacteroides spp. Clin Microbiol Infect 13, 48–54.
    12. Kuykendall, L. D., Roy, M. A., O'neill, J. J. & Devine, T. E. (1988). Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361.
    13. Kwok, A. Y. C. & Chow, A. W. (2003). Phylogenetic study of Staphylococcus and Macrococcus species based on partial hsp60 gene sequences. Int J Syst Evol Microbiol 53, 87–92.
    14. Kwok, A. Y. C., Su, S.-C., Reynolds, R. P., Bay, S. J., Av-Gay, Y., Dovichi, N. J. & Chow, A. W. (1999). Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus. Int J Syst Bacteriol 49, 1181–1192.
    15. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A. & other authors (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948.
    16. Love, D. N., Johnson, J. L., Jones, R. F., Bailey, M. & Calverley, A. (1986). Bacteroides tectum sp. nov. and characteristics of other nonpigmented Bacteroides isolates from soft-tissue infections from cats and dogs. Int J Syst Bacteriol 36, 123–128.
    17. Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.
    18. Miller, L. T. (1982). Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J Clin Microbiol 16, 584–586.
    19. Moore, L. V. H. & Moore, W. E. C. (1994). Oribaculum catoniae gen. nov., sp. nov.; Catonella morbi gen. nov., sp. nov.; Hallella seregens gen. nov., sp. nov.; Johnsonella ignava gen. nov., sp. nov.; and Dialister pneumosintes gen. nov., comb. nov., nom. rev., anaerobic gram-negative bacilli from the human gingival crevice. Int J Syst Bacteriol 44, 187–192.
    20. Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y. & Matsubara, K. (1985). An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J Biol Chem 260, 2605–2608.
    21. Rautio, M., Eerola, E., Väisänen-Tunkelrott, M. L., Molitoris, D., Lawson, P., Collins, M. D. & Jousimies-Somer, H. (2003). Reclassification of Bacteroides putredinis (Weinberg et al. 1937) in a new genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp. nov., from human sources. Syst Appl Microbiol 26, 182–188.
    22. Saito, H. & Miura, K. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629.
    23. Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
    24. Sakamoto, M. & Benno, Y. (2006). Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. Int J Syst Evol Microbiol 56, 1599–1605.
    25. 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.
    26. Sakamoto, M., Lan, P. T. N. & Benno, Y. (2007). Barnesiella viscericola gen. nov., sp. nov., a novel member of the family Porphyromonadaceae isolated from chicken caecum. Int J Syst Evol Microbiol 57, 342–346.
    27. Sakamoto, M., Suzuki, N. & Okamoto, M. (2010). Prevotella aurantiaca sp. nov., isolated from the human oral cavity. Int J Syst Evol Microbiol 60, 500-–503.
    28. Shah, H. N. & Collins, M. D. (1988). Proposal for reclassification of Bacteroides asaccharolyticus, Bacteroides gingivalis, and Bacteroides endodontalis in a new genus, Porphyromonas. Int J Syst Bacteriol 38, 128–131.
    29. Shah, H. N. & Collins, M. D. (1989). Proposal to restrict the genus Bacteroides (Castellani and Chalmers) to Bacteroides fragilis and closely related species. Int J Syst Bacteriol 39, 85–87.
    30. Shah, H. N. & Collins, M. D. (1990). Prevotella, a new genus to include Bacteroides melaninogenicus and related species formerly classified in the genus Bacteroides. Int J Syst Bacteriol 40, 205–208.
    31. Shah, M. M., Iihara, H., Noda, M., Song, S. X., Nhung, P. H., Ohkusu, K., Kawamura, Y. & Ezaki, T. (2007). dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus. Int J Syst Evol Microbiol 57, 25–30.
    32. Song, Y., Liu, C., Bolanos, M., Lee, J., McTeague, M. & Finegold, S. M. (2005). Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species. J Clin Microbiol 43, 1531–1537.
    33. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, M. C. J., Nesme, X., Rosselló-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.
    34. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125–128.
    35. Ueki, A., Abe, K., Kaku, N., Watanabe, K. & Ueki, K. (2008). Bacteroides propionicifaciens sp. nov., isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms. Int J Syst Evol Microbiol 58, 346–352.
    36. Yamamoto, S. & Harayama, S. (1995). PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61, 1104–1109.