Department of Microbiology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan1
Department of Oral Microbiology, St. Bartholomews and the Royal London School of Medicine and Dentistry, Turner Street, London E1 2AD, UK2
Author for correspondence: Yoshiaki Kawamura. Tel: +81 58 267 2240. Fax: +81 58 267 0156. e-mail: kawamura{at}cc.gifu-u.ac.jp
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ABSTRACT |
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The DDBJ accession numbers of the superoxide dismutase genes described in this paper are shown in Table 1.
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INTRODUCTION |
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Recently, the epithets of some species within the mitis group have been revised for grammatical reasons: Streptococcus sanguis was corrected to S. sanguinis, similarly, Streptococcus parasanguis was corrected to Streptococcus parasanguinis and Streptococcus crista was corrected to Streptococcus cristatus (Truper & De Clari, 1997 ). We have used the revised names throughout this report. In the 1990s, four new species were described within the mitis group, S. cristatus, S. parasanguinis, S. peroris and S. infantis, giving a total of nine species classified within this group (Handley et al., 1991
; Whiley et al., 1990
;Kawamura et al., 1998
). Due to the addition of these new species, we were strongly aware of the potential confusion surrounding the identification of members of the mitis group and were therefore motivated to establish a reliable, globally acceptable and easy practical identification method.
To this end, DNA probes were designed from 16S rRNA sequences. Even though some members of the mitis group share greater than 99% sequence similarities in their 16S rRNA gene, it was possible to design several DNA probes which could differentiate between the type strains of S. mitis, S. oralis, Streptococcus gordonii, S. cristatus, S. sanguinis and S. parasanguinis. When we applied these DNA probes to our clinical strains that had been identified, in advance, by DNADNA hybridization, all strains of S. gordonii, S. cristatus, S. sanguinis and S. parasanguinis could be correctly identified, although significantly, some clinical strains of S. oralis and S. mitis were mis-identified. From these results, we concluded that our 16S rRNA gene probe could not be applied unambiguously to the identification of S. mitis and S. oralis (unpublished data).
Identification on the basis of whole-cell protein profile comparison has also been investigated as a taxonomic tool. In a previous study, we found some clinical strains not included within the same cluster as their respective type strain (unpublished data). Similar data were published by Vandamme et al. (1998 ). According to their report, they observed that S. mitis biovar 2 strains were neither in the same cluster as S. mitis biovar 1 strains nor formed an independent cluster, and in fact migrated into several other species clusters, that included S. oralis, S. cristatus and S. parasanguinis. Furthermore, while the majority of strains of S. sanguinis formed a single cluster, this excluded the type strain of S. sanguinis, which was relatively close to the S. mitis biovar 1 cluster. From these results, it was apparent that applying protein profiles to the identification of the members of the mitis group would prove problematic.
Recently, some genetic methods for the identification of members of the mitis group at the species level have been proposed by other researchers, such as species-specific PCR primers based on the ddl gene and comparative analysis of the partial sequences of the manganese-dependent superoxide dismutase (sodA) gene (Garnier et al., 1997 ; Poyart et al., 1998
). In this study, we have investigated the reliability and usefulness of these genetic methods by using 96 human clinical strains isolated from different geographic areas. Finally, we confirm that the sodA partial sequence analysis method is a reliable and useful method for accurate identification of members of the mitis group.
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METHODS |
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DNADNA hybridization.
DNA from all strains was prepared by a standard procedure (Marmur, 1961 ) with minor modification (Ezaki et al., 1983
). DNA from each type strain was labelled with photobiotin (Sigma) and microplate quantitative DNADNA hybridization was carried out according to previously described methods (Ezaki et al., 1988
, 1989
). Briefly, purified DNA (100 µg ml-1) of each strain was heat denatured and then diluted to 10 µg ml-1 with ice cold PBS (pH 7·4) containing 0·1 M MgCl2. The diluted DNA solution was distributed into a microplate (Maxsorp; Inter Med) at 100 µl per well, and the plate incubated at 30 °C for 12 h. The solution was discarded and the plate dried at 60 °C. The plate was prehybridized for 30 min and then hybridized in the presence of 2x SSC and 50% formamide, with biotin-labelled DNA at 31 °C for 2 h. The plate was washed three times with 1x SSC and 100 µl streptavidinß-d-galactosidase (diluted 1:1000 with 0·5% BSA in PBS; Gibco-BRL) was added to each well. The plate was incubated at 37 °C for 30 min and washed three times with 1x SSC. Then 100 µl of the substrate (100 µg 4-methylumbelliferyl-ß-d-galactopyranoside ml-1; Sigma) was added to each well and the fluorescence intensity was measured by Cytofluor (model 2350; Millipore). The species name of an isolate was determined if the DNA strongly hybridized and showed greater than 70% similarity value with the DNA of only one type strain.
PCR amplification of part of the sodA gene.
An internal portion of the sodA gene was amplified by PCR using our designed primer set (SOD-UP; 5'-biotin-TRCAYCATGAYAARCACCAT-3' and SOD-DOWN; 5'-ARRTARTAMGCRTGYTCCCARACRTC-3'). Each PCR reaction (100 µl) contained 1x PCR buffer (Pharmacia Biotech), 100 µM dNTPs, 0·1 µM and 0·2 µM biotin-labelled (SOD-UP) primer and non-labelled (SOD-DOWN) primer, respectively, 20 ng template DNA and 1 U Taq polymerase (Pharmacia Biotech). PCR was carried out on a thermal cycler (model 2400, Perkin Elmer) as follows: 35 cycles of 30 sec at 94 °C, 1 min at 50 °C and 1 min at 72 °C, and a final cycle of 1 min at 50 °C followed by 10 min at 72 °C.
Determination and analysis of sodA partial sequences.
After confirming the presence of a single 435 bp amplification product of the sodA gene on 1% agarose gels, the sequence was determined using a Pharmacia automatic sequencer (ALF express) with an auto load sequencing kit (Pharmacia Biotech). Briefly, the avidin-conjugate comb was soaked in the PCRed solution to capture the biotin-labelled PCR product. Then the comb was washed in 0·1 M NaOH solution to denature the amplicon and to remove other substances of the PCR reaction mixture. The sequencing reaction was carried out on the comb using T7 polymerase and Cy5-labelled primer (the sequence was the same as for the SOD-DOWN primer). The reacted comb was applied directly onto the sequencing gel and the sequencer run for 10 h.
Phylogenetic analysis of sodA partial sequences.
clustal w software originally described by Thompson et al. (1994 ) was used to align the sequences, and the phylogenetic distances were calculated using the neighbour-joining method. The phylogenetic tree was drawn using TreeView software (Page, 1996
). The sodA partial sequence of Streptococcus agalactiae was obtained from the DNA Database of Japan (DDBJ, accession number is Z95893) and used as the outgroup.
D-Alanine:D-alanine ligase gene specific PCR.
Species-specific PCR for S. mitis, S. oralis, S. gordonii and S. sanguinis based on the d-alanine:d-alanine ligase gene (ddl gene) was carried out according to Garnier et al. (1997 ). Ten microlitres of each PCR reaction was applied to a 1% agarose gel and electrophoresed.
Autolysin gene (lytA) specific PCR.
lytA-specific PCR for the identification of S. pneumoniae was carried out according to Gillespie et al. (1994 ). PCR amplification was confirmed by electrophoresis through 1% agarose gels.
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RESULTS |
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ddl gene amplification
We confirmed the specificity of each species-specific primer set based on the ddl gene by using the type strains of members of the mitis group, except S. oralis (Table 1). We also applied this method to some of the S. mitis, S. oralis, S. gordonii and S. sanguinis strains. All strains of S. gordonii (three strains including the type strain) and S. sanguinis (five strains including the type strain) were positive. However, the S. oralis type strain and one of our clinical strains were negative. The type strain of S. mitis gave a positive result. However, one reference strain (NCTC 10712) and four of six clinical strains tested were negative (Fig. 1
, Table 1
).
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Phylogenetic analysis of sodA partial sequences
The phylogenetic tree of partial sequences of sodA was compared with that of 16S rRNA sequences for the type strains. It can be seen that the evolutionary rate of the sodA partial gene is much faster than that of the 16S rRNA gene (Fig. 2). We took this to indicate that partial sodA sequencing would be useful for differentiating genetically closely related organisms, and decided to apply sodA partial sequencing to the main collection of isolates. We determined the partial sequences of the sodA gene from 96 strains including all species currently within the mitis group and constructed a phylogenetic tree using the neighbour-joining method. Eight clusters were clearly generated corresponding to recognized species as confirmed by DNADNA hybridization (Fig. 3
). With respect to the Japanese and UK isolates of S. mitis and S. oralis, each species formed its own cluster with no evidence of geographic variation. S. pneumoniae strains did not form an independent cluster but formed a sub-cluster within the S. mitis cluster on the sodA phylogenetic tree.
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DISCUSSION |
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After developing lytA-specific PCR (Rudolph et al., 1993 ), many researchers have successfully detected or identified S. pneumoniae by this method using purified DNA as a template (Gillespie et al., 1994
; Hassan-King et al., 1994
; Ubukata et al., 1996
). Recently, Whatmore et al. (1996
) reported that a subset of S. mitis and S. oralis strains isolated were positive for this gene by PCR. However we have applied this method to more than 20 strains of S. pneumoniae and more than 70 strains of S. mitis or S. oralis, and did not observe any positive results from S. mitis nor S. oralis strains. From these results, we thought that the lytA-specific PCR might be helpful, in many cases, for identification of S. pneumoniae.
Recently, several identification methods using gene sequence data have been established, such as DNA gyrase B subunit (Yamamoto & Hirayama, 1995 , 1996
), heat-shock protein 60 (Goh et al., 1996
, 1998
) and the sodA gene (Smith & Doolittle, 1992
; Zolg & Philippi-Schulz, 1994
;Poyart et al., 1995
, 1998
). Of these, the sodA partial gene sequencing seemed to be relatively useful, because less than 400 bp is enough to determine the phylogenetic position of a strain. Therefore, we selected this gene for the identification of members of the mitis group.
When the sodA phylogenetic tree from each species type strain within the mitis group was constructed, it became apparent that the evolutionary rate of the sodA gene was much faster than that of 16S rRNA sequence (Fig. 2). Thus, we suspected that this gene would be useful in differentiating genetically closely related organisms. However, it was possible that there might be too much variation within the same species, possibly due to geographic differences, for this approach to be practicable. To test the method, clinical strains from different areas, mainly from Japan and the UK, were examined but failed to reveal any obvious geographic variation (Fig. 3
). For these reasons, we believe that this method could be applied globally.
Some bootstrap values of the branching point of each species on the sodA phylogenetic tree were not very high (the lowest is 66%, at the S. gordonii and S. sanguinis branching point, Fig. 3). However, before making the final phylogenetic tree (Fig. 3
), we remade the tree each time we collected new sequence data, and each time the species separated in the same way as Fig. 3
. From this experience, we consider that the branching of each species on the sodA tree is reproducible.
All strains of each member species of the mitis group formed a single cluster. However, many strains of the same species showed several base substitutions (maximum 31 bases) within the 366 bp region of the sodA gene examined, while the gene of S. pneumoniae, by comparison, was very conserved. Eleven of 16 S. pneumoniae strains showed completely identical sequences, and only two strains (strains 1510 and 3203) and three strains (strains 1639, 1293 and 661) showed two and one base substitutions, respectively. Similar data have been published by Poyart et al. (1998 ). These authors used eight isolates of S. pneumoniae of which only two showed a single base substitution. We cannot explain why the sodA gene of S. pneumoniae is so conserved, but are encouraged that this conservation is helpful for the identification of S. pneumoniae (Table 2
).
In this study, we determined the sequences direct from PCR amplicons using a Pharmacia sequencer with an auto-load sequencing kit. By using this method, we could apply the sample onto the sequencer within a day from the bacterial culture without doing any troublesome manipulation, such as ethanol precipitation and prolonged centrifugation of the sample, and could obtain the sequence data the next morning. There are many internet home pages providing a service to enable calculation of phylogenetic relationships, e.g. clustal w could be carried out on the home page of the DNA Databank of Japan (http://www.ddbj.nig.ac.jp). By using these easy practical methods, we believe that this method could be carried out rapidly in many laboratories.
In view of the data presented above, we believe that the sodA partial sequence analysis method could be applied globally as a reliable and practical method for the accurate identification of all species currently within the mitis group.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bentley, R. W., Leigh, J. A. & Collins, M. D. (1991). Intragenetic structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. Int J Syst Bacteriol 41, 487-494.[Abstract]
Ezaki, T., Yamamoto, N., Ninomiya, K., Suzuki, S. & Yabuuchi, E. (1983). Transfer of Peptococcus indolicus, Peptococcus asaccharolyticus, Peptococcus prevotii, and Peptococcus magnus to the genus Peptostreptococcus and proposal of Peptostreptococcus tetradius sp. nov. Int J Syst Bacteriol 33, 683-698.
Ezaki, T., Hashimoto, Y., Takeuchi, N., Yamamoto, H., Liu, S., Miura, H., Matsui, K. & Yabuuchi, E. (1988). Simple genetic method to identify viridans group streptococci by colorimetric dot hybridization and fluorometric hybridization in microdilution wells. J Clin Microbiol 26, 1708-1713.[Medline]
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.
Garnier, F., Gerbaud, G., Courvalin, P. & Galimand, M. (1997). Identification of clinically relevant viridans group streptococci to the species level by PCR. J Clin Microbiol 35, 2337-2341.[Abstract]
Gillespie, S. H., Ullman, C., Smith, M. D. & Emery, V. (1994). Detection of Streptococcus pneumoniae in sputum sample by PCR. J Clin Microbiol 32, 1308-1311.[Abstract]
Goh, S. H., Potter, S., Wood, J. O., Hemmingsen, S. M., Reynolds, R. P. & Chow, A. W. (1996). HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J Clin Microbiol 34, 818-823.[Abstract]
Goh, S. H., Driedger, D., Gillett, S. & 8 other authors (1998). Streptococcus iniae, a human and animal pathogen: specific identification by the chaperonin 60 gene identification method. J Clin Microbiol 36, 21642166.
Handley, P., Coykendall, A., Beighton, D., Hardie, J. M. & Whiley, R. A. (1991). Streptococcus crista sp. nov. a viridans streptococcus with tufted fibrils, isolated from the human oral cavity and throat. Int J Syst Bacteriol 41, 543-547.[Abstract]
Hassan-King, M., Baldeh, I., Secka, O., Falade, A. & Greenwood, B. (1994). Detection of Streptococcus pneumoniae DNA in blood cultures by PCR. J Clin Microbiol 32, 1721-1724.[Abstract]
Kawamura, Y. (1996). Evaluation and comparison of newly available identification kits for streptococci. J Med Tech 40, 409416 (in Japanese).
Kawamura, Y. (1998). Recent classification of the Genus Streptococcus. Jpn J Bacteriol 53, 493507 (in Japanese).
Kawamura, Y., Hou, X. G., Sultana, F., Miura, H. & Ezaki, T. (1995). Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 45, 406-408.[Abstract]
Kawamura, Y., Hou, X. G., Todome, Y., Sultana, F., Hirose, K., Shu, S., Ezaki, T. & Ohkuni, H. (1998). Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov., new members of the Streptococcus mitis group, isolated from human clinical specimens. Int J Syst Bacteriol 48, 921-927.
Kilian, M., Mikkelsen, L. & Henrichsen, J. (1989). Taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended descriptions of Streptococcus sanguis (White and Niven 1946), Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrewes and Horder 1906). Int J Syst Bacteriol 39, 471-484.
Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3, 208-218.
Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comp Appl Biosci 12, 357-358.[Medline]
Poyart, C., Berche, P. & Trieu-Cuot, P. (1995). Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers. FEMS Microbiol Lett 131, 41-45.[Medline]
Poyart, C., Quesne, G., Coulon, S., Berche, P. & Trieu-Cuot, P. (1998). Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase. J Clin Microbiol 36, 41-47.
Rudolph, K. M., Parkinson, A. J., Black, C. M. & Mayer, L. W. (1993). Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia. J Clin Microbiol 31, 2661-2666.[Abstract]
Smith, M. W. & Doolittle, R. F. (1992). A comparison of evolutionary rates of the two major kinds of superoxide dismutase. J Mol Evol 34, 175-184.[Medline]
Thompson, J. D., Higgins D. G. & Gibson, T. J. (1994). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Trüper, H. G. & De Clari, L. (1997). Taxonomic note: necessary correction of specific epithets formed as substantives (nouns) in apposition. Int J Syst Bacteriol 47, 908-909.
Ubukata, K., Asahi, Y., Yamane, A. & Konno, M. (1996). Combinational detection of autolysin and penicillin-binding protein 2B genes of Streptococcus pneumoniae by PCR. J Clin Microbiol 34, 592-596.[Abstract]
Vandamme, P., Torck, U., Falsen, E., Pot, B., Goossens, H. & Kersters, K. (1998). Whole-cell protein electrophoretic analysis of viridans streptococci: evidence for heterogeneity among Streptococcus mitis biovars. Int J Syst Bacteriol 48, 117-125.
Whatmore, A. M., Pickerill, A. P., Woodward, G. E. & Dowson, C. G. (1996) Allelic variation of the lytA gene of S. pneumoniae and related species. Abstracts of the Lancefield International Symposium on Streptococci and Streptococcal Diseases, Paris.
Whiley, R. A., Fraser, H. Y., Douglas, C. W. I., Hardie, J. M., Williams, A. M. & Collins, M. D. (1990). Streptococcus parasanguis sp. nov. an atypical viridans Streptococcus from human clinical specimens. FEMS Microbiol Lett 68, 115-122.
Yamamoto, S. & Hirayama, 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.[Abstract]
Yamamoto, S. & Hirayama, S. (1996). Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products. Int J Syst Bacteriol 46, 506-511.[Abstract]
Zolg, J. W. & Philippi-Schulz, S. (1994). The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR. J Clin Microbiol 32, 2801-2812.[Abstract]
Received 12 February 1999;
revised 14 May 1999;
accepted 20 May 1999.